EP4669656A1 - Micropeptides for improving plant immunity and their application - Google Patents

Micropeptides for improving plant immunity and their application

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Publication number
EP4669656A1
EP4669656A1 EP24707495.8A EP24707495A EP4669656A1 EP 4669656 A1 EP4669656 A1 EP 4669656A1 EP 24707495 A EP24707495 A EP 24707495A EP 4669656 A1 EP4669656 A1 EP 4669656A1
Authority
EP
European Patent Office
Prior art keywords
seq
plant
mipep
virus
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24707495.8A
Other languages
German (de)
French (fr)
Inventor
Martina Susanne BECK
Mikaël Stéphane COURBOT
Jean-Claude HAW-KING-CHON
Stéphanie Léa ROUCAL
Amélie Suzanne Renée BAROZET
Clémence MEDINA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Micropep Technologies SA
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Micropep Technologies SA
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Publication date
Application filed by Micropep Technologies SA filed Critical Micropep Technologies SA
Publication of EP4669656A1 publication Critical patent/EP4669656A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P15/00—Biocides for specific purposes not provided for in groups A01P1/00 - A01P13/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y406/00—Phosphorus-oxygen lyases (4.6)
    • C12Y406/01—Phosphorus-oxygen lyases (4.6.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to micropeptides and their use for increasing plant immunity.
  • MicroRNAs modulate the abundance and spatial-temporal accumulation of target mRNAs and indirectly regulate several plant processes, including response to pathogens.
  • Transcriptional regulation of the genes encoding miRNAs can be activated by numerous transcription factors, which in turn can be regulated by other miRNAs. Modulation of miR gene expression, or of the levels of miRNAs in a cell, can lead to improve tolerance to abiotic or biotic stresses in crops of economic importance.
  • miRNAs modulate the abundance and spatial-temporal accumulation of target mRNAs and indirectly regulate several plant processes, including response to pathogens.
  • Transcriptional regulation of the genes encoding miRNAs miR genes
  • Modulation of miR gene expression, or of the levels of miRNAs in a cell can lead to improve tolerance to abiotic or biotic stresses in crops of economic importance.
  • There are currently more than 28000 identified miRNAs and increasing evidence indicates that several miRNAs families are involved in fine-tuning plant immunity against pathogen invasion (Padmanabhan et al.
  • miPEPs micropeptides encoded by one or several short open reading frames contained in pri-miRNA sequences have been identified (Chen, et al. 2020, Sharma et al, 2019). These miPEPs have also been found to modulate the expression of the miRNA produced from their encoding pri-miRNA (Lauressergues, et al., 2005), thus modulating the levels of the mature miRNAs in the cell.
  • MiPEPs may present and alternative to GMO-based technologies and chemical peptides, as they regulate specific plant processes transiently and specifically by targeting expression of miRNAs.
  • Embodiment A A method for identifying and synthesizing (i) an immunity enhancing micropeptide (miPEP) encoded by a nucleotide sequence contained in the sequence of the primary transcript of a microRNA (miRNA), or (ii) a nucleic acid sequence encoding the miPEP that does not comprise the mature miRNA, comprising: a) selecting an miRNA from a miR169 family or a miR396 family; b) identifying an open reading frame (ORF) from 12 to 303 nucleotides in length contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; d) exogenously adding to the first plant (i) a peptide encoded by the ORF or (ii) a nucleic acid sequence encoding the peptide; e) comparing immunity in the
  • Embodiment Bl A method of increasing immunity in a plant comprising providing to the plant a miPEP that regulates an immunity enhancing miR169 or an immunity enhancing miR396.
  • Embodiment B2 The method of Embodiment Bl, where the miPEP is selected from the group consisting of d /mi P EP 169c, A /mi P EP 169h, A /PE P396b, PvmiPEP 169e, P vmi P EP 169kl, PvmiPEP 169pl, FvmiPEP169hI, FvmiPEP1691I, FvmiPEP369a, S7miPEP169a, S7miPEP169d, S7miPEP169k, S7miPEP396c, MP19043, MP19042, S7miPEP169kDlD10, MP19051, MP19048, and MP19049, wherein the miPEP regulates an immunity enhancing miRNA.
  • the miPEP regulates an immunity enhancing miRNA.
  • Embodiment B3 A method of increasing immunity in a plant comprising providing to the plant a miPEP comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:
  • Embodiment B4 A method of increasing resistance to a plant pathogen or pest in a plant comprising providing to the plant a miPEP comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,
  • Embodiment B6 The method of Embodiment B4, wherein the plant pathogen or pest is a fungal pathogen or pest selected from Cercospora spp., Mycosphaerella spp., Glomerella spp., Cladosporium spp., Diplodia maydis, Fusarium oxysporum, Fusarium graminearum, Fusarium monilforme, Fusarium verticillioides, Cochliobolus sativus, Collectotrichum graminicola, Stagonospora nodorum, Stagonospora avenae, Stenocarpella maydis, Sclerotinia minor, Sclerotinia sclerotiorum, Sclerotinia sp., Alternaria spp., Phytophthora spp., Botrytis spp., Pyrenophora tritici-repentis, Phytophthora
  • phaseoli Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Glomerella glycines, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Fusarium solani; Canola: Albugo Candida, Altenaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassicicola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter michiganese subsp.
  • alfalfae Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulina briosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium botryosum, Leptotrichila medicaginis; Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v.
  • translucens Pseudomonas syringae p.v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis fsp. tritici, Puccinia graminis fsp. tritici, Puccinia recondite fsp.
  • Embodiment B7 The method of claim Embodiment B4, wherein the plant pathogen or pest is a bacterial pathogen or pest selected from Pseudomonas spp., Pantoua spp., and Erwinia spp.
  • Embodiment B9 The method of Embodiment B4, wherein the plant pathogen or pest is selected from the group consisting of Botrytis cinerea, Altemaria solani, and Phytophthora infestans.
  • Embodiment B12 The method of any one of Embodiments Al and B1-B9, wherein the miPEP comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19, with zero, one, two, three, or four amino acid substitutions, insertions, or deletions, and the plant is a Phaseolus vulgaris plant.
  • Embodiment B13 The method of any one of Embodiments Al and B1-B9, wherein the miPEP comprises the amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, with zero, one, two, three, or four amino acid substitutions, insertions, or deletions, and the plant is a tomato plant.
  • Embodiment B14 The method of any one of Embodiments Al and Bl -Bl 3, wherein the oligopeptide is provided by expressing a nucleic acid encoding the miPEP.
  • Embodiment B 15 The method of Embodiment Bl 4, wherein the nucleic acid is operably linked to a heterologous promoter.
  • Embodiment B16 The method of Embodiment B 14 or Bl 5, wherein the heterologous promoter is a constitutive promoter, a tissue specific promoter, or an inducible promoter.
  • Embodiment Bl 7 The method of Embodiment Bl 6, wherein the inducible promoter is induced by fungal infection, optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., phenylalanine ammonia lyase, chaicone synthase promoters), a gene that modifies plant cell walls (e.g., hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase promoters), a gene encoding an enzyme that degrade fungal cell walls (e.g., chitinase or glucanase promoters), or a gene encoding a thaumatin-like protein, or or optionally a maize Misl promoter or a flax Fisl promoter.
  • a promoter associated with a gene involved in phenylpropanoid metabolism e.g., phenylalanine ammonia lyase,
  • Embodiment Bl 8 The method of any one of Embodiments Al and B1-B13, wherein the miPEP is provided by application to the plant or a part thereof.
  • Embodiment B 19 The method of Embodiment B 18, wherein the plant part is a leaf, a bud, a root, a shoot, a floral part, or a seed.
  • Embodiment B20 The method of Embodiment B18 or Bl 9, wherein the miPEP is applied as a coating to the seed prior to planting.
  • Embodiment B21 The method of any one of Embodiments Al and B1-B13, wherein the miPEP is provided by application to the soil in which the plant is planted.
  • Embodiment B22 The method of any one of Embodiments Al and B1-B13, wherein the miPEP is provided by addition to water provided to the plant.
  • Embodiment B23 The method of any one of Embodiments Al and B1-B13, wherein the oligopeptide is provided following harvest to seeds, fruits, and/or plant parts.
  • Embodiment B24 The method of any one of Embodiment B18-B23, wherein the application is by a spreader, a power duster, a boom sprayer, a hand sprayers, a spray dusters, or a granular applicator.
  • Embodiment B25 The method of any one of Embodiments Al and B1-B24, wherein the miPEP comprises a secretion tag, a tag that promotes entry into a plant cell, or a nuclear localization tag.
  • Embodiment B26 The method of Embodiment B25, wherein the tag that promotes entry into a plant cell comprises a cell penetrating peptide linked to the amino acid sequence.
  • Embodiment C4 The oligopeptide of Embodiment Cl or C2, wherein the oligopeptide enhances resistance in a plant to a plant pathogen or pest.
  • Embodiment C5 The oligopeptide of any one of Embodiments C1-C4, comprising the one, two, three, or four amino acid substitutions, insertions, or deletions.
  • Embodiment C6 The oligopeptide of any one of Embodiments C1-C5, wherein the oligopeptide comprises a proline at its N-terminus, an aspartic acid at the C-terminus, or both.
  • Embodiment C7 The oligopeptide of any one of Embodiments C1-C6, wherein the oligopeptide comprises one or more D-amino acids.
  • Embodiment C8 The oligopeptide of Embodiment C7, wherein the one or more D-amino acids enhance stability of the oligopeptide as compared to an oligopeptide of the same amino acid sequence comprising all L-amino acids.
  • Embodiment C9 The oligopeptide of any one of Embodiments C1-C8, wherein the oligopeptide comprises a secretion tag, a tag that promotes entry into a plant cell, or a nuclear localization tag.
  • Embodiment CIO The oligopeptide of Embodiment C9, wherein the tag that promotes entry into a plant cell comprises a cell penetrating peptide linked to the amino acid sequence.
  • Embodiment Cl 1 The oligopeptide of Embodiment CIO, wherein the cell penetrating peptide comprises a protein transduction domain, an amphipathic peptide, a synthetic cationic polypeptide, optionally polylysine, polyhistidine, or polyarginine, a dendrimeric polycationic molecule, a peptide vascular endothelial-cadherin cell penetrating peptide, a transportan cell penetrating peptide, a monomer or dimer of HIV- 1 TAT basic domain cell penetrating peptide, a penetratin cell penetrating peptide, a synthetic cationic homoarginine oligopeptide cell penetrating peptide, a gamma zein cell penetrating peptide, a Zea mays knottedl cell penetrating peptide, a Saccharomyces pombe TP10 cell penetrating peptide,
  • Embodiment DI A composition comprising the oligopeptide of any one of Embodiments Cl- C11 with an agriculturally acceptable formulant that can include water, organic solvents, paraffinic oils, vegetable oils, dispersants, emulsifiers, wetting agents, buffering agents, hydrotrope agents, rheology modifiers, antifoam agents and defoamers, antifreeze agents, biocides, dyes, polymer walls, catalysts, thermosetting materials, cross-polymerizing agents, UV protectants, antioxidants, and chelating agents.
  • an agriculturally acceptable formulant can include water, organic solvents, paraffinic oils, vegetable oils, dispersants, emulsifiers, wetting agents, buffering agents, hydrotrope agents, rheology modifiers, antifoam agents and defoamers, antifreeze agents, biocides, dyes, polymer walls, catalysts, thermosetting materials, cross-polymerizing agents, UV protectants, antioxidants, and chelating agents.
  • Embodiment D2 A composition comprising the oligopeptide of any one of Embodiments Cl- C11 with an agriculturally acceptable carrier, diluent, or excipient.
  • Embodiment D3 The composition of Embodiment D2, wherein the agriculturally acceptable carrier, diluent, or excipient comprises a buffer and the pH is in the range of from about 3.0 to about 9.0 or about 4.5 to about 8.0.
  • Embodiment D4 The composition of Embodiments D1-D3, wherein the oligopeptide is in a concentration range of from about 0. 1 pg/ml to about 100 mg/ml, or from about 5 pg/ml to about 5 mg/ml.
  • Embodiment D5 The composition of any one of Embodiments D 1-D4, wherein the composition is in the form of a finely-divided particulate solid, granules, pellets, wettable powders, dust, an aqueous suspension, a dispersion, a gel, or an emulsion.
  • Embodiment E A plant immunity enhancing peptide identified by: a) selecting an miRNA from a miR169 family or a miR396 family; b) identifying an open reading frame (ORF) from 12 to 303 nucleotides in length contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; d) exogenously adding to the first plant (i) a peptide encoded by the ORF, or (ii) a nucleic acid sequence encoding the peptide; e) comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing immunity enhancing miRNA and the peptide is an immunity enhancing micropeptide.
  • ORF open reading frame
  • Embodiment F 1 A nucleic acid encoding the oligopeptide of any one of Embodiments Cl-Cl 1.
  • Embodiment F2 A nucleic acid construct comprising the nucleic acid of Embodiment Fl, operably linked to a promoter.
  • Embodiment F5 The nucleic acid construct of claim Embodiment F4, wherein the inducible promoter is induced by fungal infection, optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., phenylalanine ammonia lyase, chaicone synthase promoters), a gene that modifies plant cell walls (e.g., hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase promoters), a gene encoding an enzyme that degrade fungal cell walls (e.g., chitinase or glucanase promoters), or a gene encoding a thaumatin-like protein, or a maize Mis 1 promoter or a flax Fis 1 promoter.
  • Embodiment G A cell comprising the nucleic acid of Embodiment Fl or the nucleic acid construct of any one of Embodiments
  • Embodiment H The method of any one of Embodiments A-B27, the oligopeptide of any one of Embodiments Cl-Cl 1, the composition of any one of Embodiments D1-D6, the immunity enhancing peptide of Embodiment E, the nucleic acid of Embodiment F 1, the nucleic acid construct of Embodiments F2-F5, or the cell of Embodiment G, wherein: (a) the miPEP is selected from the group consisting of G/wmiR I 69g. G/wmiR I69m.
  • the miPEP regulates an immunity enhancing miRNA and the plant is a soybean plant
  • the miPEP, the oligopeptide, or the immunity enhancing peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 54-59, wherein the miPEP, the oligopeptide, or the immunity enhancing peptide regulates miR169 and the plant is a soybean plant
  • the miPEP, the oligopeptide, or the immunity enhancing peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 54- 59 with one, two, three, or four amino acid substitutions, insertions, or deletions, wherein the miPEP, the oligopeptide, or the immunity enhancing peptide regulates miR169 and the plant is a soybean plant
  • the miPEP, the oligopeptide, or the immunity enhancing peptide comprises the
  • Embodiment I A method of increasing immunity in a plant comprising providing to the plant an oligopeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79 and/or providing to the plant an oligopeptide comprising an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions compared to the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79.
  • Embodiment J A method of reducing fungal growth on a plant comprising providing to the plant an oligopeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79 and/or providing to the plant an oligopeptide comprising an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions compared to the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79.
  • Embodiment K 1 A method of inhibiting a plant pathogen or pest on a plant comprising providing to the plant an oligopeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79 and/or providing to the plant an oligopeptide comprising an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions compared to the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79.
  • Embodiment K2 The method of Embodiment J or KI, wherein the plant pathogen or pest is selected from fungal pathogens or pests, bacterial pathogens or pests, and viral pathogens or pests.
  • Embodiment K3 The method of Embodiment J or KI, wherein the plant pathogen or pest is a fungal pathogen or pest selected from Cercospora spp., Mycosphaerella spp., Glomerella spp., Cladosporium spp., Diplodia maydis, Fusarium oxysporum, Fusarium graminearum, Fusarium monilforme, Fusarium verticillioides, Cochliobolus sativus, Collectotrichum graminicola, Stagonospora nodorum, Stagonospora avenae, Stenocarpella maydis, Sclerotinia minor, Sclerotinia sclerotiorum, Sclerotinia sp., Alternaria spp., Phytophthora spp., Botrytis spp., Pyrenophora tritici-repentis
  • phaseoli Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Glomerella glycines, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium deharyanum, Fusarium solani; Canola: Albugo Candida, Altenaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassicicola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter michiganese subsp.
  • alfalfae Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulina briosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium botryosum, Leptotrichila medicaginis; Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v.
  • translucens Pseudomonas syringae p.v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis fsp. tritici, Puccinia graminis fsp. tritici, Puccinia recondite fsp.
  • carotovora Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis; Com: Colletotrichum graminicola, Fusarium verticillioides var. subglutinans, Erwinia stewartii, F.
  • nebraskense Trichoderma viride, Claviceps sorghi, Pseudomonas avenae, Erwinia chrysanthemi pv. zea, Erwinia carotovora, Corn stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium acremonium, Exserohilum turcicum, C.
  • holcicola Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium verticillioides, Altemaria altemata, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philip
  • Embodiment K4 The method of Embodiment KI or K2, wherein the plant pathogen or pest is a bacterial pathogen or pest selected from Pseudomonas spp., Pantoua spp., and Erwinia spp.
  • Embodiment K5 The method of Embodiment KI or K2, wherein the plant pathogen or pest is a viral pathogen or pest selected from cucumber mosaic, tobacco mosaic, and barley yellow dwarf virus, alfalfa mosaic virus (Alfamovirus), Apple chlorotic leaf spot virus (Trichovirus), Apple scar skin viroid (Viroids), Arabis mosaic virus (Nepovirus), Barley mild mosaic virus (Bymovirus), Barley stripe mosaic virus (Hordeivirus), Barley yellow mosaic virus (Bymovirus), Bean common mosaic virus (Potyvirus), Bean yellow mosaic virus (Potyvirus), Beet necrotic yellow vein virus (Furovirus), Blackeye cowpea mosaic virus (Potyvirus), Bean common mosaic virus (Potyvirus), Broad bean wilt virus (Fabavirus), Butterbur mosaic virus (Carlavirus), Carnation mottle virus (Carmovirus), Carnation vein mottle virus (Potyvirus), Cauliflower mosaic virus (Caulim
  • Embodiment K6 The method of Embodiment KI or K2, wherein the plant pathogen or pest is selected from the group consisting of Septoria tritici, Botrytis cinerea, Phytophthora cactorum, and Phakopsora pachyrhizi s.
  • Embodiment K7 The method of any one of Embodiments I-K6, wherein the oligopeptide is provided by expressing a nucleic acid encoding the oligopeptide.
  • Embodiment K9 The method of Embodiment K7 or K8, wherein the heterologous promoter is a constitutive promoter, a tissue specific promoter, or an inducible promoter.
  • Embodiment K10 The method of Embodiment K9, wherein the inducible promoter is induced by fungal infection, optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., phenylalanine ammonia lyase, chaicone synthase promoters), a gene that modifies plant cell walls (e.g., hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase promoters), a gene encoding an enzyme that degrade fungal cell walls (e.g., chitinase or glucanase promoters), or a gene encoding a thaumatin-like protein, or optionally a maize Misl promoter or a flax Fisl promoter.
  • Embodiment K10 The method of any one of Embodiments I-K6, wherein the oligopeptide is provided by application to the plant or
  • Embodiment KI 1 The method ofEmbodimentKIO, wherein the plant part is a leaf, a bud, a root, a shoot, a floral part, or a seed.
  • Embodiment K 12 The method of Embodiment K 11 or K12, wherein the oligopeptide is applied as a coating to the seed prior to planting.
  • Embodiment K13 The method of any one of Embodiments I-K6, wherein the oligopeptide is provided by application to the soil in which the plant is planted.
  • Embodiment K14 The method of any one of Embodiments I-K7, wherein the oligopeptide is provided by addition to water provided to the plant.
  • Embodiments K15 The method of any one of Embodiments I-K7, wherein the oligopeptide is provided following harvest to seeds, fruits, and/or plant parts.
  • Embodiments K16 The method of any one of Embodiments K10-K15, wherein the application is by a spreader, a power duster, a boom sprayer, a hand sprayers, a spray dusters, or a granular applicator.
  • Embodiments KI 7 The method of any one of Embodiments I-K16, wherein the miPEP comprises a secretion tag, a tag that promotes entry into a plant cell, or a nuclear localization tag.
  • Embodiments B28 The method of Embodiment B21, wherein the tag that promotes entry into a plant cell comprises a cell penetrating peptide linked to the amino acid sequence.
  • Embodiments B29 The method of Embodiments B28, wherein the cell penetrating peptide comprises a protein transduction domain, an amphipathic peptide, a synthetic cationic polypeptide, optionally polylysine, polyhistidine, or polyarginine, a dendrimeric polycationic molecule, a peptide vascular endothelial-cadherin cell penetrating peptide, a fransportan cell penetrating peptide, a monomer or dimer of HIV- 1 TAT basic domain cell penetrating peptide, a penefratin cell penetrating peptide, a synthetic cationic homoarginine oligopeptide cell penetrating peptide, a gamma zein cell penetrating peptide, a Zea mays knottedl cell penetrating peptide, a Saccharomyces pombe TP10 cell penetrating peptide, a
  • Embodiment LI An isolated oligopeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 54, 58, 59, and 79 or the amino acid sequence set forth in one of SEQ ID NOs: 54, 58, 59, and 79 comprising one, two, three, or four amino acid substitutions, insertions, or deletions.
  • Embodiment L2 The oligopeptide of Embodiment L, wherein the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.
  • Embodiment L3 The oligopeptide of Embodiment LI or L2, comprising the one, two, three, or four amino acid substitutions, insertions, or deletions.
  • Embodiment L4 The oligopeptide of any one of Embodiments L1-L3, wherein the oligopeptide comprises a proline at its N-terminus, an aspartic acid at the C-terminus, or both.
  • Embodiment L5 The oligopeptide of any one of Embodiments L1-L4, wherein the oligopeptide comprises one or more D-amino acids.
  • Embodiment L6 The oligopeptide of Embodiment L5, wherein the one or more D-amino acids enhance stability of the oligopeptide as compared to an oligopeptide of the same amino acid sequence comprising all L-amino acids.
  • Embodiment L7 The oligopeptide of any one of Embodiments L1-L6, wherein the oligopeptide comprises a secretion tag, a tag that promotes entry into a plant cell, or a nuclear localization tag.
  • Embodiment L8 The oligopeptide of Embodiment L7, wherein the tag that promotes entry into a plant cell comprises a cell penetrating peptide linked to the amino acid sequence.
  • Embodiment L9 The oligopeptide of Embodiment L8, wherein the cell penetrating peptide comprises a protein transduction domain, an amphipathic peptide, a synthetic cationic polypeptide, optionally polylysine, polyhistidine, or polyarginine, a dendrimeric polycationic molecule, a peptide vascular endothelial-cadherin cell penetrating peptide, a transportan cell penetrating peptide, a monomer or dimer of HIV- 1 TAT basic domain cell penetrating peptide, a penetratin cell penetrating peptide, a synthetic cationic homoarginine oligopeptide cell penetrating peptide, a gamma zein cell penetrating peptide, a Zea mays knottedl cell penetrating peptide, a Saccharomyces pombe TP10 cell penetrating peptide, a
  • Embodiment Ml A composition comprising the oligopeptide of any one of Embodiments L1-L9 with an agriculturally acceptable formulant that can include water, organic solvents, paraffinic oils, vegetable oils, dispersants, emulsifiers, wetting agents, buffering agents, hydrotrope agents, rheology modifiers, antifoam agents and defoamers, antifreeze agents, biocides, dyes, polymer walls, catalysts, thermosetting materials, cross-polymerizing agents, UV protectants, antioxidants, and chelating agents.
  • Embodiment M2 A composition comprising the oligopeptide of any one of Embodiments L1-L9 75-83 with an agriculturally acceptable carrier, diluent, or excipient.
  • Embodiment M3 The composition of Embodiment M2, wherein the agriculturally acceptable carrier, diluent, or excipient comprises a buffer and the pH is in the range of from about 3.0 to about 9.0, or from about 4.5 to about 8.0.
  • Embodiment M4 The composition of any one of Embodiments Ml -M3, wherein the oligopeptide is in a concentration range of from about 0. 1 pg/ml to about 100 mg/ml, or from about 5 pg/ml to about 5 mg/ml.
  • Embodiment M5 The composition of any one of Embodiments M1-M4, wherein the composition is in the form of a finely-divided particulate solid, granules, pellets, wettable powders, dust, an aqueous suspension, a dispersion, a gel, or an emulsion.
  • Embodiment M6 The composition of any one of Embodiments M1-M5, further comprising one or more other active agents selected from the group consisting of a pesticide, a fertilizer, an insecticide, an attractant, a sterilizing agent, an acaricide, a nematocide, a herbicide, a biostimulant, a biological, and a growth regulator.
  • a pesticide a fertilizer, an insecticide, an attractant, a sterilizing agent, an acaricide, a nematocide, a herbicide, a biostimulant, a biological, and a growth regulator.
  • Embodiment N A nucleic acid encoding the oligopeptide of any one of Embodiments L1-L9.
  • Embodiment 01 A nucleic acid construct comprising the nucleic acid of Embodiment N, operably linked to a promoter.
  • Embodiment 02 The nucleic acid construct of Embodiment 01, wherein the promoter is a heterologous promoter.
  • Embodiment 03 The nucleic acid construct of Embodiment 01 or Embodiment 02, wherein the promoter is a constitutive promoter, a tissue specific promoter, a development stage specific promoter, or an inducible promoter.
  • Embodiment 04 The nucleic acid construct of Embodiment 03, wherein the inducible promoter is induced by fungal infection, optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., phenylalanine ammonia lyase, chaicone synthase promoters), a gene that modifies plant cell walls (e.g., hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase promoters), a gene encoding an enzyme that degrade fungal cell walls (e.g., chitinase or glucanase promoters), or a gene encoding a thaumatin-like protein, or a maize Misl promotor or a flax Fisl promoter.
  • a promoter associated with a gene involved in phenylpropanoid metabolism e.g., phenylalanine ammonia lyase, chaic
  • Embodiment P A cell comprising the nucleic acid of Embodiment N or the nucleic acid construct of any one of Embodiments O1-O4. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows the experimental screening approach used to determine micropeptides (miPEPs) involved in plant defense.
  • Peptides were applied as a foliar spray on three consecutive days prior to infection, followed by droplet infection with Botrytis cinerea spores incubated in peptide solution (Panel A). At the last day, leaves were harvested and pictures of isolated leaves were acquired (Panel B). Lesion sizes were measured at all infection points (encircled in Panel B), and were compared between control infected and peptide-treated plants.
  • FIG. 2 shows the measurements of Botrytis cinerea-inflicted lesions on Arabidopsis thaliana leaves after peptide application of miPEPs targeting members of respective microRNA (miR) families (miR169, miR168, miR396, and miR858).
  • miR microRNA
  • the black arrows indicate miPEPs that significantly decreased lesion size compared to control plants (black dotted line).
  • FIG. 3 shows the relative induction of d/pri-miR I69h and d/pri-miR 169c after respective miPEP treatment in A. thaliana.
  • FIG. 4 shows the relative induction of 4/pri-miR396b after miPEP treatment in A. benthamiana expressing 35.S'A/pri-miR396b.
  • FIG. 5 shows the measurements of Botrytis cinerea-inflicted lesions on strawberry leaves after peptide application of respective miPEPs.
  • the black arrows indicate miPEPs that significantly decreased lesion size compared to control plants (black dotted line). Significance was determined as
  • FIG. 6 depicts the strawberry fruit infection phenotypes after miPEP application.
  • FIG. 7 shows the measurements of Botrytis cinerea-infficted lesions on tomato plants after peptide application of respective miPEPs.
  • the black arrows indicate miPEPs that significantly decreased lesion size compared to control plants (black dotted line). Significance was determined as
  • FIG. 8 shows the phenotype of lesion development on tomato leaves after peptide application with respective miPEPs. The phenotypes 48 hours after Botrytis cinerea infection are shown.
  • FIG. 9 shows the relative fold induction of S/pri-miR169d after miPEP treatments in N. benthamiana expressing 35.8':.S7pri-miR 169d.
  • FIGS. 10A-10B show the results of convex hull analysis of tomato plants after Botrytis cinerea spray infection. Tomato plants were sprayed with isolated Botrytis spores and phenotyped every day for five consecutive days.
  • FIG. 10A depicts the convex hull analysis. The convex hull was analyzed with an automated analysis software using the top RGB image.
  • FIGS. 11A-11B show the results of plant fitness analysis of Botrytis czfterea-infected tomato plants after treatment with miPEPs. Tomato plants were sprayed with isolated Botrytis spores, and sprayed with either miPEPs or buffer control after 24 hours.
  • FIG. 11 A shows the phenotype of plants treated with .S7miPEP I69k or buffer control.
  • FIG. 11B shows disease control level in tomato plants treated with either miPEP or buffer control. The quantification of the convex hull in control and miPEP -treated tomato plants was used to calculate a disease control index.
  • FIG. 12A shows the disease control after S/miPEP 169k application on tomato plants inoculated with Alternaria solani.
  • FIG. 12B shows the disease control after S/miPEP169k application on tomato plants inoculated with Phytophthora infestans.
  • FIGS. 13A-13B show the comparison of a .S7mi PEP 169k sequence variant.
  • FIG. 13A shows the peptide purity of S7miPEP169k and the S7miPEP169kDlD10 variant. The peptide purity of S7miPEP169k and S7miPEP169kDlD10 was compared after incubation for 40 hours in a tomato wash-off solution.
  • FIGS. 14A-14B show the analysis of S7miPEP169k and S/miPEP169a sequence variants and other miPEPs encoded in pri-miR169k or pri-miR169a. Different peptides targeting pri-miR169k or pri- miR169a were tested for their biological activity against tomato grey mold (Botrytis cinerea).
  • FIG. 14A shows the analysis of biological activity of S/miPEP169k sequence variants and other miPEPs encoded in pri-miR169k.
  • FIG. 14B shows the analysis of biological activity of S/miPEP169a sequence variants and other miPEPs encoded in pri-miR169a.
  • FIGS. 15A-15B show bar graph results of disease control by spraying MP19114 or MP19127 peptides (left bars in FIGS. 15A and 15B, respectively) or a reference antifungal peptide (right bars in FIGS. 15A and 15B) onto soybean plants infected with Asian Soybean Rust (ASR).
  • ASR Asian Soybean Rust
  • FIG. 15A shows the plotted results of percentage of contaminated leaf area for plants sprayed with peptide MP 19114 (medium grey boxes, “MP19114”) compared to plants sprayed with reference peptide MP18279 (dark grey boxes, “Reference”, “*”). Error bars represent standard error, with 18 total measurements (2 leaves on each of 3 plants in each of 3 pots).
  • FIG. 15B shows the plotted results for plants sprayed with peptide MP19127 (medium grey boxes, “MP19127”) compared to plants sprayed with reference peptide MP18279 (dark grey boxes, “Reference”, “*”).
  • FIG. 16 shows a schematic of amino acids comprising peptideMP19114.
  • N-ter represents the N-terminus of the protein
  • C-ter represents the C-terminus of the peptide.
  • the two brackets under the schematic indicate the lengths of the peptide’s pair of hydrophobic blocks (“2 Hydrophobic blocks”).
  • the “M” amino acid at the N-terminus end of the peptide represents amino acid methionine. Types of amino acid are categorized by polarity, hydrophobicity, and ionicity, wherein “aa” represents “amino acid”.
  • Cationic amino acids are denoted by positive signs in the schematic
  • hydrophobic amino acids are denoted by medium grey circles labeled “h” in the schematic
  • polar amino acids are denoted by dark grey circles labeled “p” in the schematic
  • nonpolar amino acids are denoted by light grey circles labeled “np” in the schematic
  • polar hydrophobic amino acids are denoted by striped circles in the schematic.
  • micropeptides targeting miR169 or miR396 led to modulation of Botrytis infection, with some micropeptides producing increased resistance to infection with this fungal pathogen in four different plant species (Arabidopsis thaliana, Phaseolus vulgaris, Fragaria vesca, Solarium lycopersicum).
  • Micropeptides targeting the same miRNA families also led to increased resistance to other common fungal pathogens, Pseudomonas and Alternaria, indicating a role for these miRNAs in immunity and resistance to fungal pathogens in plants.
  • amino acid or “amino acid residue” refers to any naturally occurring amino acid, any non-naturally occurring amino acid, any modified amino acid, including derivatized amino acids, or any amino acid mimetic known in the art.
  • the amino acids are D-amino acids.
  • the amino acids are L-amino acids.
  • the amino acid may be referred by both their common three letter abbreviation and single letter abbreviation.
  • microRNA As used herein, “microRNA”, “non-coding microRNA” and “miRNA” are equivalent and may be used interchangeably to refer to small molecules of RNA of about 21 nucleotides, which regulate certain genes via post-transcriptional mechanisms, for example by means of the RISC complex.
  • the primary transcript of a microRNA or “pri-miRNA” corresponds to the RNA molecule obtained directly 1 from transcription of the miRNA gene.
  • this primary transcript undergoes one or more post- transcriptional modifications, involving for example a particular structure of the RNA or cleavage of certain portions of the RNA by splicing phenomena, and which lead to the precursor form of the microRNA or “pre-miRNA”, then to the mature form of the microRNA or “miRNA”.
  • the pri-miRNA contains an open reading frame in addition to the miRNA sequence.
  • micropeptides and “miPEPs” (microRNA encoded PEPtides) are equivalent and may be used interchangeably to refer to a peptide that is encoded by an open reading frame present in the primary transcript of a microRNA, and which is capable of modulating the accumulation of said microRNA.
  • the miPEPs are 4 to 50 or 6-30 amino acids in length.
  • open reading frame or “ORF” are equivalent and may be used interchangeably to refer to a nucleotide sequence in a DNA or RNA molecule that may potentially encode a peptide or a protein.
  • the ORF may be identified by having a plant recognized start codon (the start codon generally encoding a methionine), followed by a series of codons (each codon encoding an amino acid), and ends with a stop codon (the stop codon not being translated).
  • the ORF is present in the primary transcript of a miRNA.
  • the ORF is 12 to 303 nucleotides in length.
  • the ORF encodes a peptide of 3 to 100 amino acids in length.
  • the term “peptide” refers to any peptide structure comprising or consisting of two or more amino acids, including chemical modifications and derivatives of amino acids.
  • the peptide is a short peptide (e.g., 4 to 50 amino acids in length).
  • the ORF is located in a primary transcription of an miRNA.
  • an isolated nucleic acid molecule includes the nucleic acid molecule introduced into the genome of a cell in a position where it is not found in nature or when it is resident in progeny of cells into which the nucleic acid molecule has been introduced into its genome in a position where it is not found in nature.
  • sequence identity refers to the degree of similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, and otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
  • immunogenity enhancing peptide refers to an amino acid sequence capable of inducing an increased immune response in living cells and/or organisms that are exposed to said amino acid sequence.
  • the immunity enhancing peptide increases immunity against one or more pathogens or pests.
  • the immunity enhancing peptide is a plant immunity enhancing peptide.
  • miPEP immunity enhancing micropeptide
  • a nucleic acid encoding the miPEP e.g., a nucleic acid encoding the miPEP.
  • miPEPs are thought to be transcriptional regulators, in particular transcriptional activators that can operate at the transcription level to modulate the accumulation of a pri-miR or downstream miRNA.
  • the method for identifying and synthesizing comprises: a) selecting an miRNA from a miR169 family or a miR396 family; b) identifying an open reading frame (ORF) from 12 to 303 nucleotides in length contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; exogenously adding to the first plant (i) a peptide encoded by the ORF or (ii) a nucleic acid sequence encoding the peptide; comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the
  • the method comprises selecting a miRNA.
  • the miRNA is a plant miRNA.
  • the miRNA is a miRNA from a miR169 or miR396.
  • MicroRNA families can consist of different number of members, with different plant species having a different number of members of these families. Each microRNA family defined by the mature microRNA produced from miRNA genes in these families, which has a high degree of homology amongst members of a particular family. The mature miRNA sequence dictates the subset of genes regulated by a particular family.
  • the miR169 family is known to target HAPLESS2 (HAP2) genes
  • the miR396 family targets growth regulating factors (GRFs) genes in plant.
  • the secondary mRNA structure of each member of a particular family can vary.
  • the miRNA (such as miR169 or miR396) controls genes involved in plant immunity or resistance to plant pathogens or pests.
  • the miR169 or miR396 genes contain an ORF encoding a micropeptide.
  • the miRNA is a miR169 or miR396 from a dicot or monocot plant.
  • the miR169 or miR396 from are from an Arabidopsis spp., Phaseolus spp., Fragaria spp., or Solarium spp..
  • the method comprises identifying an ORF contained in the primary transcript sequence of the miRNA.
  • the ORF has a size of 12 to 303 nucleotides.
  • the ORF has a size of 12, 15, 18, 21, 24, 27 , 30, 33, 36, 39, 42, 45, 47, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 2
  • the ORF has a size of 15 to 153 nucleotides. In some embodiments, the ORF has a size of 21 to 93 nucleotides. In some embodiments, the ORF encodes a peptide of 3 to 100 amino acids in length.
  • the ORF encodes a peptide of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 20 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 amino acids in length.
  • the ORF encodes a miPEP of 4 to 50 amino acids in length. In some embodiments, the ORF encodes a miPEP of 6 to 30 amino acids in length. In some embodiments, the ORF is contained in a pri-miR169 or pri-miR396. In some embodiments, the ORF encodes a miPEP that regulates a miR169 or a miR396. In some embodiments, the ORF encodes a miPEP having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • ORFs contained in miRNA genes may be identified by any method known to those in the art, including, without limitation, bioinformatics, RNA sequencing, DNA sequencing, or mass spectrometry- based approaches. For example, protein extract from cells may be analyzed through mass spectrometry to identify novel short peptides and then identify nucleic acid sequences corresponding to a sequence encoding the peptide. Methods that detect translation of peptides encoded by ORFs contained in noncoding RNAs, such as ribosome profiling, may also be used to identify ORFs.
  • identifying an ORF contained in the primary transcript sequence of the miRNA comprises analysis of genomic databases and/or genomic sequences.
  • the genomic database is analyzed to identify genes or transcript sequences comprising a start codon, at least three codons coding an amino acid, and a stop codon.
  • the gene or transcript may also be analyzed to further identify sequences that regulate translation.
  • the analysis may further comprise comparing the identified ORF to a known ORF or amino acid sequence encoded therein.
  • identifying an ORF contained in the primary transcript sequence of the miRNA comprises searching for ORFs on a genomic database.
  • the genomic database is a plant genomic database.
  • identifying an ORF contained in the primary transcript sequence of the miRNA comprises searching for ORFs on a pri-miR database.
  • the method comprises providing a first plant cell and second plant cell that both express the miRNA.
  • the first and second plant cells are from the same type of plant.
  • the first and second plant cell express a miR169 or miR396.
  • the first and second plant cell express a miPEP having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • the first and second plant cell are from a dicot or monocot plant. In some embodiments, the first and second plant are from a crop plant. In some embodiments, the first and second cell are from a row crop plant, a fruit-producing plant, a free, a vine, a vegetable, or an ornamental plants (e.g. ornamental flowers, trees, shrubs, groundcovers, and turf grasses). In some embodiments, the first and second plant cell are from an Arabidopsis spp., Phaseolus spp., Fragaria spp., or Solanum spp.
  • the first and second plant cell are from Arabidopsis thaliana, Phaseolus vulgaris, Fragaria vesca, or Solanum lycopersicum.
  • Other Exemplary types of types of plants include, without limitation, Medicago sativa, Prunus dulcis, Malus domestica, Prunus spp., Asparagus officinalis, Arabidopsis spp., Musa spp., Hordeum vulgare, Phaseolus spp., Vaccinium spp., Theobroma cacao, Brassica spp.,, Dianthus caryophyllus, Daucus carota sativus, Manihot esculentum, Prunus avium, Cicer arietinum, Cichorium intybus, Capsicum spp., Chrysanthemum spp., Cocos nucifera, Coffea spp., Gossypium hirsutum L.,
  • the plant is a wild plant variety. In some embodiments, the plant is a domesticated plant variety. In some embodiments, the plant is a hybrid plant variety. In some embodiments, the plant is a genetically modified plant and/or a gene- edited plant.
  • the method comprises exogenously adding to the first plant (i) a peptide encoded by the ORF or (ii) a nucleic acid sequence encoding the peptide.
  • the method comprises exogenously adding to the first plant a peptide encoded by the ORF.
  • the peptide may be added by any method known in the art. For instance, the peptide may be added by foliar spray, foliar drench, drip irrigation, coating, mixing, pouring, dusting, atomizing, soil irrigation, fumigation, soil injection, seepage irrigation, sprinklers or manual irrigation, or a combination thereof.
  • exogenously adding to the first plant a peptide encoded by the ORF comprises applying the peptide by foliar spray.
  • exogenously adding to the first plant a peptide encoded by the ORF comprises adding a cell that expresses the peptide encoded by the ORF.
  • the peptide encoded by the ORF is a miPEP.
  • the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • the miPEP is provided as a composition.
  • the composition is in the form of a finely-divided particulate solid, granules, pellets, wettable powders, dust, an aqueous suspension, a dispersion, a gel, or an emulsion.
  • the composition comprises a miPEP at a concentration range of from about 0. 1 pg/ml to about 100 mg/ml, or from about 5 pg/ml to about 5 mg/ml.
  • the composition comprises a miPEP at a concentration of about 0.1 pg/ml, 0.5 pg/ml, 1 pg/ml, 5 pg/ml, 10 pg/ml, 20 pg/ml, 30 pg/ml, 40 pg/ml, 50 pg/ml, 60 pg/ml, 70 pg/ml, 80 pg/ml, 90 pg/ml, 1 mg/ml, 5 mg/ml, 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, or 100 mg/ml.
  • the miPEP regulates a miR169 or a miR396.
  • the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • the miPEP is provided by application to a plant or part thereof.
  • the plant part is a leaf, a bud, a root, a shoot, a floral part, or a seed.
  • the miPEP is provided to a plant or part thereof after harvest.
  • the miPEP is provided to a plant or part thereof prior to harvest.
  • the miPEP is provided to a plant by application to the soil in which the plant is planted.
  • the miPEP is provided to a plant by addition of water provided to the plant.
  • the miPEP is applied to a plant by a spreader, a power duster, a boom sprayer, a hand sprayers, a spray dusters, or a granular applicator.
  • the miPEP regulates a miR169 or a miR396.
  • the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • the method comprises exogenously adding to the first plant a nucleic acid sequence encoding the peptide encoded by the ORF.
  • nucleic acids encoding the peptide are added to a plant by transformation.
  • transformation comprises Agrobacterium-mediated transformation, micro-projectile-mediated transformation, sonication, electroporation, or liposome- or spheroplast-mediated vector delivery.
  • the nucleic acids encoding the peptide are added to a plant by genome editing.
  • Gene editing may be performed by any method known in the art, including, but not limited to using zinc fmger-nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), oligonucleotide-directed mutagenesis (ODM), a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, or by gene writing (see, e.g., PCT Patent Application Publication W02020/047124).
  • ZFNs zinc fmger-nucleases
  • TALENs transcription activator-like effector nucleases
  • ODM oligonucleotide-directed mutagenesis
  • CRISPR clustered regularly interspaced short palindromic repeats
  • nucleic acids encoding the peptide are added to a plant by crossing a first plant comprising the nucleic acids encoding the peptide to a second plant.
  • the method comprises comparing immunity in the first plant cell and the second plant cell after exogenously adding to the first plant a peptide encoded by the ORF, or a nucleic acid sequence encoding the peptide, wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing miRNA and the peptide is the miPEP.
  • the miPEP regulates a miR169 or a miR396.
  • the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • plant immunity may be measure as a reduction in pest number on the plant when compared to an untreated plant, or as a reduction in physical damage to the plant when compared to an untreated plant.
  • Physical damage includes feeding damage and boring damage, and may manifest in a variety of plant phenotypes, including, but not limited to, chewed or ragged leaves, missing leaves, tunnels in leaves, holes in stems, leaf distortion, leaf discoloration, leaf spotting, wilting, stunted growth, girdled or dead stems, yellowing, breakage damage, or root damage.
  • Convex hull analysis may be used to examine the physical damage to an infected plant. In some embodiments, plant immunity is determined by convex hull analysis.
  • disease control In some embodiments, an increase of disease control of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% as compared to an untreated infected plant indicates enhanced immunity. In some embodiments, a 2-to 20-fold increase in disease control of as compared to an untreated infected plant indicates enhanced immunity.
  • a 2-folf, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, or 20-fold increase in disease control as compared to an untreated infected plant indicates enhanced immunity.
  • the method comprises synthetizing a miPEP, or a nucleic acid encoding the miPEP.
  • the miPEP regulates a miR169 or a miR396.
  • the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • the method comprises synthetizing a miPEP, or a nucleic acid encoding the miPEP by chemical synthesis.
  • Various approaches been developed for chemical synthesis of peptides such as for example, by solid-phase methods.
  • Solid phase peptide synthesis is a method for chemically synthesizing peptides on a solid support.
  • amino acids or peptides are typically attached to a solid support via the C-terminus. The new amino acid is added to the bound amino acid or peptide by a coupling reaction.
  • Translation-based approaches for peptide synthesis have also been developed, whereby a peptide is produced from an encoding transcript by in vitro translation.
  • Synthetic nucleic acids may be prepared using column-based synthesizers, or produced from an existing nucleic acid by PCR or in vitro transcription. Other methods of nucleic synthesis are described for example in U.S. Pat. No. 6,586,211 Bl, in PCT/EP2004/013131, in WO 00/13017 A2, in S. Rayner et al., PCR Methods and Applications 8 (7), 741-747, 1998, in WO 90/00626 Al, in EP 385 410 A2, in WO 94/12632 Al, in WO 95/17413 Al, in EP 316 018 A2, in EP 022 242 A2, in L. E. Sindelar and J. M.
  • the method comprises synthetizing a miPEP, or a nucleic acid encoding the miPEP by biological synthesis.
  • Any suitable method of biological synthesis of peptides or nucleic acids may be used.
  • Biological synthesis may comprise expressing a peptide in a cell and purifying the peptide from the cell.
  • Nucleic acids may introduced into a cell, such as a bacteria, and allowed to be replicated before isolating them.
  • synthetizing a miPEP comprises expressing a fusion polypeptide comprising a polypeptide that forms inclusion bodies in a cell operably linked to two or more miPEPs. In some embodiments, synthetizing a miPEP comprises expressing a fusion polypeptide comprising an carrier polypeptide operably linked to two or more miPEPs by a peptide bond. In some embodiments, synthetizing a miPEP comprises expressing a fusion polypeptide comprising an carrier polypeptide operably linked to two or more miPEPs by a peptide bond, and releasing the two or more miPEPs from the carrier polypeptide.
  • synthetizing a miPEP comprises expressing a fusion polypeptide comprising an carrier polypeptide operably linked to two or more miPEPs by a peptide bond, and releasing the two or more miPEPs from the carrier polypeptide.
  • the two or more miPEPs in the fusion polypeptide are released from the carrier polypeptide and/or from each other by sequence-specific chemical cleavage of the peptide bond.
  • the peptide bond is an Asp-Pro bond and the sequence-specific cleavage is performed using acetic acid.
  • releasing the two or more miPEPs from the carrier polypeptide is performed in the absence of a chaotropic agent.
  • releasing the two or more miPEPs from the carrier polypeptide is performed without a column-based purification step.
  • the miPEP is 4-50 amino acids long.
  • the miPEP comprises an N-terminal proline and a C-terminal aspartic acid.
  • the miPEP regulates a miR169 or miR396.
  • the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • the method comprises providing to the plant a miPEP that regulates an immunity enhancing miR169 or an immunity enhancing miR396.
  • the miPEP may be provided to a plant by any suitable method known in the art.
  • the miPEP is provided to a plant by foliar spray, foliar drench, drip irrigation, coating, mixing, pouring, dusting, atomizing, soil irrigation, fiimigation, soil injection, seepage irrigation, sprinklers or manual irrigation.
  • the miPEP is applied to a plant by a spreader, a power duster, a boom sprayer, a hand sprayers, a spray dusters, or a granular applicator.
  • the miPEP is provided to a plant by foliar spray. In some embodiments, the miPEP is provided to the plant at a concentration of 2 g/ha to 200 g/ha. In some embodiments, the miPEP is provided to the plant at a concentration of 2 g/ha, 5 g/ha, 10 g/ha , 15 g/ha, 20 g/ha, 25 g/ha, 30 g/ha, 35 g/ha, 40 g/ha, 45 g/ha, 50 g/ha, 55 g/ha, 60 g/ha, 65 g/ha, 70 g/ha, 75 g/ha, 80 g/ha, 85 g/ha, 90 g/ha, 95 g/ha, 100 g/ha, 110 g/ha, 120 g/ha, 130 g/ha, 140 g/ha, 150 g/ha, 160 g/ha, 170 g/ha, 180 g/ha, 190 g/ha, or
  • the miPEP is provided to the plant by foliar spray at a concentration of 100 g/ha. In some embodiments, the miPEP is provided to one or more of a leaf, a bud, a root, a shoot, a floral part, or a seed. In some embodiments, the miPEP is provided to a plant or part thereof after harvest. In some embodiments, the miPEP is provided to a plant or part thereof prior to harvest. In some embodiments, the miPEP is provided to the plant prior, during, or after an infection with a pathogen or pest. In some embodiments, the miPEP is provided to a plant by application to the soil in which the plant is planted. In some embodiments, the miPEP is provided to a plant by addition of water provided to the plant. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • the method comprises preparing a composition comprising the miPEP.
  • the composition is an agriculturally acceptable composition.
  • the composition comprises a miPEP and an agriculturally acceptable formulant.
  • the agriculturally acceptable formulant comprises one or more of a water, organic solvents, paraffinic oils, vegetable oils, dispersants, emuiers, wetting agents, buffering agents, hydrotrope agents, rheology modifiers, antifoam agents and defoamers, antifreeze agents, biocides, dyes, polymer walls, catalysts, thermosetting materials, cross-polymerizing agents, UV protectants, antioxidants, and chelating agents.
  • the composition comprises a miPEP at a concentration range of from about 0. 1 pg/ml to about 100 mg/ml, or from about 5 pg/ml to about 5 mg/ml.
  • the composition fiirther comprises one or more other active agents selected from the group consisting of a pesticide, a fertilizer, an insecticide, an attractant, a sterilizing agent, an acaricide, a nematocide, a herbicide, a biostimulant, a biological, and a growth regulator.
  • the composition is formulated as a liquid, a gel, an emulsion, a suspension, an encapsulation, a solid, a powder, an aerosol, a paste, a coating, a spray, a soil drench, a microcapsule, an emulsifiable concentrate, or as granules.
  • the composition is in the form of a finely-divided particulate solid, granules, pellets, wettable powders, dust, an aqueous suspension, a dispersion, a gel, or an emulsion.
  • the agriculturally acceptable composition is formulated as a seed treatment, a foliar spray, a foliar drench, a Ready-To-Use (RTU) formulation, a produce coating, a suspension concentrate, a tank-mix, an aerosol, a root dip, a soil treatment, a dipping formulation, an irrigation formulation, or a sprinkler formulation.
  • the agriculturally acceptable composition comprising a miPEP is a foliar spray composition.
  • the composition comprises a miPEP that regulates a miR169 or a miR396.
  • the composition comprises a miPEP having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • the method comprises preparing composition comprising a miPEP and an agriculturally acceptable carrier, diluent or excipient.
  • the composition comprises a miPEP and an agriculturally acceptable carrier, diluent or excipient.
  • the agriculturally acceptable carrier, diluent, or excipient comprises a buffer and the pH is in the range of from about 3.0 to about 9.0 or about 4.5 to about 8.0.
  • the agriculturally acceptable carrier comprises a solid carrier, a liquid carrier, a gel carrier, a suspension, or an emulsion.
  • the agriculturally acceptable carrier comprises an adjuvant, an inert component, a dispersant, a surfactant, a humectant, an emulsifier, a thickener, a wetting agent, a fertilizer, a mineral, a solvent, a tackifier, a binder, or a stabilizer.
  • the agriculturally acceptable composition comprising a miPEP and a surfactant or humectant.
  • the method comprises providing a composition comprising an miPEP to a plant or part thereof.
  • the plant part is a leaf, a bud, a root, a shoot, a floral part, or a seed.
  • the composition is provided to a plant or part thereof after harvest.
  • the miPEP is provided to a plant or part thereof prior to harvest.
  • the miPEP is provided to the plant prior, during, or after an infection with a pathogen or pest.
  • the miPEP is provided to a plant by application to the soil in which the plant is planted.
  • the miPEP is provided to a plant by addition of water provided to the plant.
  • the composition comprises a miPEP that regulates a miR169 or a miR396.
  • the composition comprises a miPEP having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • the method comprises increasing plant immunity.
  • increasing plant immunity comprises inducing an increase in disease control.
  • increasing plant immunity comprises inducing an increase in of 10% to 90% as compared to an untreated infected plant indicates enhanced immunity.
  • disease control In some embodiments, disease control.
  • increasing plant immunity comprises inducing an increase in at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% as compared to an untreated infected plant indicates enhanced immunity.
  • increasing plant immunity comprises inducing an increase in 2-to 20-fold increase in disease control as compared to an untreated infected plant indicates enhanced immunity.
  • increasing plant immunity comprises inducing an increase in a 2-folf, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, or 20-fold increase in disease control as compared to an untreated infected plant indicates enhanced immunity.
  • the plant pathogen or pest is a fungus, bacteria, virus, or an eukaryote.
  • the plant pathogen or pest is a fungus.
  • plant pathogenic fungi include, without limitation, Cercospora spp., Mycosphaerellasp ., Glomerella spp., Cladosporium spp., Fusarium oxysporum, Fusarium graminearum, Fusarium monilforme, Cochliobolus sativus, Collectotrichum graminicola, Stagonospora nodorum, Stagonospora avenae, Stenocarpella maydis, Sclerotinia minor, Sclerotinia sclerotiorum, Sclerotinia sp., Alternaria spp., Phytophthora spp., Botrytis spp., Pyrenophora tritici-repentis, and Phytophthora parasitica.
  • Plant fungal pathogens or pests for which resistance may be provided include, Collelolrichum graminicola, Diplodia maydis, Fusarium graminearum, and Fusarium verticillioides.
  • Specific pathogens for major crops include: Soybeans: Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var.
  • phaseoli Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Glomerella glycines, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Fusarium solani; Canola: Albugo Candida, Altenaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassicicola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter michiganese subsp.
  • alfalfae Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulina briosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium botryosum, Leptotrichila medicaginis; Whca/: Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v.
  • translucens Pseudomonas syringae p.v. syringae, Alternaria altemata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis fsp. tritici, Puccinia graminis fsp. tritici, Puccinia recondite fsp.
  • holcicola Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium verticillioides, Alternaria altemata, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas civenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philip
  • the plant pathogen or pest is a bacteria.
  • plant pathogenic bacteria include those belonging to Pseudomonas spp., Pantoua spp., and Erwinia spp. Additional plant pathogenic bacteria are described in Robert W. Jackson, Plant Pathogenic Bacteria: Genomics and Molecular Biology, published by Horizon Scientific Press, 2009, ISBN 1904455379, 9781904455370; Samuel S.
  • the plant pathogen or pest is a virus.
  • viruses that may cause infections in plants include, without limitation, cucumber mosaic, tobacco mosaic, and barley yellow dwarf virus, alfalfa mosaic virus (Alfamovirus), Apple chlorotic leaf spot virus (Tricho virus), Apple scar skin viroid (Viroids), Arabis mosaic virus (Nepovirus), Barley mild mosaic virus (Bymovirus), Barley stripe mosaic virus (Hordeivirus), Barley yellow mosaic virus (Bymovirus), Bean common mosaic virus (Potyvirus), Bean yellow mosaic virus (Potyvirus), Beet necrotic yellow vein virus (Furovirus), Blackeye cowpea mosaic virus (Potyvirus), Bean common mosaic virus (Potyvirus), Broad bean wilt virus (Fabavirus), Butterbur mosaic virus (Carlavirus), Carnation mottle virus (Carmovirus), Carnation vein mottle virus (Potyvirus), Cauliflower mosaic virus (Caulimovirus), Chrysant
  • viruses that may cause Odontoglossum ringspot virus Tobamovirus
  • Papaya ringspot virus Pierisavirus
  • Peach latent mosaic viroid Peanut mottle virus (Potyvirus), Peanut stripe virus (Potyvirus), Bean common mosaic virus (Potyvirus), Peanut stunt virus (Cucumovirus)
  • Potato virus A Pieris virus
  • Potato virus M Carlavirus
  • Potato virus S Carlavirus
  • Potato virus X Piervirus
  • Potato virus Y Pierune dwarf virus
  • Ilarvirus Primus necrotic ringspot virus
  • Radish mosaic virus Comovirus
  • Rice black streaked dwarf virus Fijivirus
  • Rice dwarf virus Reovirus
  • Rice grassy stunt virus Tenuivirus
  • Rice stripe virus Tuttle virus
  • Rice tungro spherical virus Sequivirus
  • Rice waika virus Rice tungro spherical virus (Sequivirus)
  • Ryegrass mottle virus Satsuma dwarf virus
  • the plant pathogen or pest is an eukaryote.
  • the eukaryote is an insect or a nematode.
  • Agricultural insect pests can be classified into: chewing insects, sucking insects, and soil insects. Common chewing insects are, for example, beet armyworm (Spodoptera exigua), diamondback moth ( lulella xylostella), com earworm (He I loth is zea. a.k.a.
  • bollworm and tomato fruitworm blister beetles (Epicauta and others), carrot weevils (Listronotus oregonensis, Hyperodes texana), cabbage looper (Trichopulsia m), grasshopper (several species), flea beetles (e.g., tobacco fleabeetle (Epitrix hirtipennis), eggplant fleabeetle (E. fuscula), potato fleabeetle (E.
  • psyllid e.g. Asian citrus psyllid
  • squash bug e.g. Asian citrus psyllid
  • leaffooted bugs Leafptoglossus spp.
  • leafhoppers e.g., bean leafhopper, Empoasca solana, aster leafhopper, Macrosteles fascifrons, western potato leafhopper, Empoasca abrupta, grape leafhopper, variegated leafhopper, beet leafhopper, Circulifer tenellus
  • aphids e.g.
  • Common rasping insects include, but are not limited to, thrips (e.g. citrus thrips, western flower thrips (Frankliniella occidentalis), onion thrips (Thrips tabaci), melon thrips, chili thrips).
  • thrips e.g. citrus thrips, western flower thrips (Frankliniella occidentalis), onion thrips (Thrips tabaci), melon thrips, chili thrips.
  • Common soil insects are, for example, granulate cutworm (Feltia subterranea), mole crickets (e.g. northern mole cricket, Neocurtilla hexadactyla, southern moire cricket Scapteriscus acletus), com rootworm (e.g.
  • Exemplary nematodes that act as plant pathogens or pest can be classified into parasitic nematodes such as root-knot nematodes, including Heterodera spp., Meloidogyne spp., and Globodera spp.; cyst nematodes, which include Heterodera spp. Meloidogyne spp. (cotton cyst nematode) and Globodera spp.; and lesion neamtodes, which include Pratylenchus spp.
  • parasitic nematodes such as root-knot nematodes, including Heterodera spp., Meloidogyne spp., and Globodera spp.
  • cyst nematodes which include Heterodera spp. Meloidogyne spp. (cotton cyst nematode) and Globodera spp.
  • lesion neamtodes which include Pratylenchus spp.
  • the pathogen is a pathogen of Arabidopsis thaliana, Phaseolus vulgaris, Fragaria vesca, or Solanum lycopersicum.
  • the plant pathogen or pest is a fungi.
  • the fungi is Botrytis spp., Phytophthora spp. or Alternaria spp.
  • the fungi is Botrytis cinerea, Phytophthora infestans or Alternaria solani.
  • the method comprises providing to a plant a miPEP.
  • the miPEP is selected from the group consisting of A/miPEP169c, A/miPEP169h, AtPEP396b, PvmiPEP169e, PvmiPEP169kI, PvmiPEP169pI, FvmiPEP169hI, FvmiPEP1691I, FvmiPEP369a, S7miPEP169a, S7miPEP169d, S7miPEP169k, S7miPEP396c, MP19043, MP19042, S7miPEP169kDlD10, MP19051, MP19048, and MP19049.
  • the miPEP regulates an immunity enhancing miRNA.
  • the immunity enhancing miRNA is a miR169 or a miR396.
  • the method comprises providing to the a plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NOs: 1-13.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ IDNO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the miPEP further comprises an N-terminal proline and a C-terminal aspartate.
  • the miPEP regulates miR169 or miR396.
  • the method comprises providing to a plant a miPEP comprising a proline at the N-terminus and an aspartate at the C-terminus.
  • the method comprises providing to a plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartate at the C- terminus result from fusion polypeptide expression of the peptide.
  • the miPEP regulates miR169 or miR396.
  • the method comprises providing to miPEP to an Arabidopsis thaliana plant.
  • the method comprises providing .4 /mi PEP 169c to an Arabidopsis thaliana plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 1 to an Arabidopsis thaliana plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 1.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus.
  • the method comprises providing to an Arabidopsis thaliana plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 14.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 14. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
  • the method comprises providing 4/miPEP I 69h to an Arabidopsis thaliana plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 2 to an Arabidopsis thaliana plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 2.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus.
  • the method comprises providing to an Arabidopsis thaliana plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 15.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 15. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus.
  • the method comprises providing to an Arabidopsis thaliana plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 16.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 4.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus.
  • the method comprises providing to an Phaseolus vulgaris plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 17.
  • the method comprises providing PvmiPEP 169kl to a Phaseolus vulgaris plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 5 to a Phaseolus vulgaris plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 5.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 18. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO18. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miRI69.
  • the method comprises providing PvmiPEP 169pl to a Phaseolus vulgaris plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 6 to a Phaseolus vulgaris plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 6.
  • the method comprises providing to miPEP to a Fragaria vesca plant. [0177] In some embodiment, the method comprises providing FvmiPEP 169hl to a Fragaria vesca plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 7 to a Fragaria vesca plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 7.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C-terminus.
  • the method comprises providing to a Fragaria vesca plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 20.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 20.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 20.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide.
  • the miPEP regulates miR169.
  • the method comprises providing FvmiPEP 16911 to a Fragaria vesca plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 8 to a Fragaria vesca plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 8.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C-terminus.
  • the method comprises providing to a Fragaria vesca plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 21.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 21.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 21.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide.
  • the miPEP regulates miR169.
  • the method comprises providing FvmiPEP369a to a Fragaria vesca plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 9 to a Fragaria vesca plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 9.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C-terminus.
  • the method comprises providing to a Fragaria vesca plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 22.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 22.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 22.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide.
  • the miPEP regulates miR396.
  • the method comprises providing to miPEP to a Solarium lycopersicum plant.
  • the method comprises providing S7miPEP169a to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 10 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 10.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 10.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus.
  • the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide.
  • the miPEP regulates miR169.
  • the method comprises providing S7miPEP169d to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 11 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 11.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 11.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus.
  • the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth SEQ ID NO: 28.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 28.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 28.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide.
  • the miPEP regulates miR169.
  • the method comprises providing MP 19043 to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 26 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 26.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 26.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus.
  • the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 24.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 24.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 24.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide.
  • the miPEP regulates miR169.
  • the method comprises providing MP 19042 to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 1 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 1.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 27.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus.
  • the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 25.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 25.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 25.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide.
  • the miPEP regulates miR169.
  • the method comprises providing S7miPEP169k to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 12 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 12.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 12.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus.
  • the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide.
  • the miPEP regulates miR169.
  • the method comprises providing S/miPEP169kDlD10 to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 33 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 33.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 33.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the miPEP regulates miR169.
  • the method comprises providing MP 19051 to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 34 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 34.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 34.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus.
  • the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 30.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 30.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 30.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide.
  • the miPEP regulates miR169.
  • the method comprises providing MP 19048 to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 35 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 35.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 35.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus.
  • the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth SEQ ID NO: 31.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 31.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 31.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide.
  • the miPEP regulates miR169.
  • the method comprises providing MP 19049 to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 36 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 36.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 36.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus.
  • the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 32.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 32.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 32.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide.
  • the miPEP regulates miR169.
  • the method comprises providing S7miPEP396c to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 13 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 13.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13.
  • the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus.
  • the method comprises providing to a Solanum lycopersicum plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 37.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 37.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 37.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide.
  • the miPEP regulates miR396.
  • immunity enhancing peptides are antimicrobial or antifungal oligopeptides.
  • the immunity enhancing peptides are miPEPs.
  • the miPEP regulates a miR169 or miR396.
  • the miPEP comprises an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ IDNO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 1.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 4.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 5.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 6.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 10.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 11.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 12.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the miPEP fiirther comprises an N-terminal proline and a C-terminal aspartate.
  • the miPEP regulates miRI69 or miR396.
  • the miPEP comprises a proline at the N-terminus and an aspartic acid at the C-terminus.
  • the miPEP comprises an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 36, or SEQ ID NO: 37.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 14.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 15.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 16.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 17.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 18.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 19.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 20.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 21.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 22. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 23.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 24. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 25.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 26. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 28. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 29.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 30. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 31.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 32.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 33.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 34.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 35.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 36.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 37.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • proline at the N-terminus and the aspartic acid at the C-terminus result from fusion polypeptide expression of the miPEP.
  • the miPEP regulates miR169 or miR396.
  • the miPEP comprises a protease-resistant amino acid sequence.
  • the miPEP comprise one or more D-amino acids.
  • the one or more D-amino acids enhance the stability of the miPEP in a cell.
  • the one or more D- amino acids enhance stability of the oligopeptide as compared to an oligopeptide of the same amino acid sequence comprising all L-amino acids.
  • the miPEP comprising one or more D- amino acids regulates a miR169 or a miR396.
  • the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37, with one or more D-amino acids.
  • the immunity enhancing peptide is identified by a) selecting an miRNA from a miR169 family or a miR396 family; b) identifying an ORF contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; d) exogenously adding to the first plant (i) a peptide encoded by the ORF, or (ii) a nucleic acid sequence encoding the peptide; e) comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing immunity enhancing miRNA and the peptide is an immunity enhancing miPEP.
  • the ORF is 12 to 303 nucleotides in length.
  • the plant is an Arabidopsis spp., Phaseolus spp., Fragaria spp., or Solarium spp.
  • the plant is Arabidopsis thaliana, Phaseolus vulgaris, Fragaria vesca, or Solarium lycopersicum.
  • the immunity enhancing peptide is a miPEP that regulates a plant miR169 or miR396.
  • the plant miR169 or miR396 are from Arabidopsis thaliana, Phaseolus vulgaris, Fragaria vesca, or Solanum lycopersicum.
  • the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • the nucleic acids of the disclosure may be prepared using any technique known in the art. Without limitation, these may include cloning, DNA isolation, amplification and purification, enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques, such as gel electrophoresis and chromatography. A number of standard techniques are described in Ausubel et al. (1992) Current Protocols in Molecular Biology, Green/Wiley, New York, N. Y.; Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al.
  • vectors comprising nucleic acids encoding any of the fusion polypeptides, carrier polypeptides or oligopeptides disclosed herein.
  • a “vector” is a nucleic acid that is capable of transporting another nucleic acid.
  • Vectors may be, for example, plasmids, viruses, cosmids or phage.
  • An “expression vector” is a vector that is capable of directing expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment. Examples of vectors are those that can autonomously replicate and express structural gene products present in the DNA segments to which they are operatively linked. Vectors, therefore, can contain the replicons and selectable markers described earlier. Vectors include, but are not necessarily limited to, expression vectors.
  • the vector is a bacterial vector.
  • the vector is a yeast vector.
  • expression vectors that can be used in prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pET plasmids (Novagen, Madison, Wis., USA) or pBR322 (ATCC 37017).
  • the pBR322 vector contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells.
  • an appropriate promoter and a DNA sequence encoding one or more of the polypeptides of the invention are inserted into the pBR322 vector.
  • vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM-1 (Promega Biotec, Madison, Wis., USA).
  • Other commercially available vectors include those that are specifically designed for the expression of proteins; these would include pMAL-p2 and pMAL-c2 vectors that are used for the expression of proteins fused to maltose binding protein (New England Biolabs, Beverly, Mass., USA).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the variant squalene synthase enzyme expressed, and whether it is desired to isolate the enzyme and in what state of purity. For example, when large quantities are to be produced, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • yeast a number of vectors containing constitutive or inducible promoters may be used.
  • Current Protocols in Molecular Biology Vol. 2, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13 (1988); Bitter et al., Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp. 516-544 (1987); Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch.
  • yeast promoter such as ADH1 or LEU2 or an inducible promoter such as GAL4 may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol. 11, A Practical Approach, Ed. D M Glover, IRL Press, Wash., D.C. (1986)).
  • vectors may be used which promote integration of foreign DNA sequences into the yeast or bacterial chromosome.
  • Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include the bacteriophage T7 promoter (Studier and Moffatt, J. Mol. Biol. 189: 113 (1986)), [3-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; Goeddel et al., Nature 281:544 (1979)), tryptophan (tap) promoter system (Goeddel et al., Nucl. Acids Res.
  • a particularly useful prokaryotic host cell expression system employs a phage PL promoter and a cl857ts thermolabile repressor sequence.
  • Plasmid vectors available from the American Type Culture Collection (ATCC), which incorporate derivatives of the PL promoter, include plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).
  • the nucleic acid encoding the immunity enhancing peptide is operably linked to a heterologous promoter.
  • the heterologous promoter is a is a constitutive promoter, a tissue specific promoter, or an inducible promoter.
  • the inducible promoter is induced by fungal infection, optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., phenylalanine ammonia lyase, chaicone synthase promoters), a gene that modifies plant cell walls (e.g., hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase promoters), a gene encoding an enzyme that degrade fungal cell walls (e.g., chitinase or glucanase promoters), a gene encoding a thaumatin-like protein, or a maize or flax promoters, designated as Mis 1 and Fisl, respectively, are also induced by fungal infections in plants and can be used (US Patent Appl. Pub. No. 20020115849).
  • a promoter associated with a gene involved in phenylpropanoid metabolism e.g., phenylalanine am
  • the nucleic acid encodes a fusion polypeptide comprising a polypeptide that forms inclusion bodies in a cell operably linked to two or more miPEPs. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an carrier polypeptide operably linked to two or more miPEPs. In some embodiments, the operable linkage comprises a peptide bond susceptible to sequence-specific chemical cleavage. In some embodiments, the operable linkage is an Asp-Pro bond and the sequence-specific cleavage is acetic acid cleavage. In some embodiments, the miPEP is 4-50 amino acids long.
  • the miPEP comprises an N-terminal proline and a C-terminal aspartic acid. In some embodiments, the miPEP regulates a miR169 or miR396. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • the nucleic acids encode an miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ IDNO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the miPEP further comprises an N-terminal proline and a C-terminal aspartate.
  • the miPEP regulates miR169 or miR396.
  • the nucleic acid encodes a miPEP comprising a proline at the N-terminus and an aspartic acid at the C-terminus.
  • the nucleic acid encodes an miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartic acid at the C-terminus result from fiision polypeptide expression of the miPEP.
  • the miPEP regulates a miRI69 or miR396.
  • the nucleic acid encodes a miPEP identified by: a) selecting an miRNA from a miRI69 family or a miR396 family; b) identifying an ORF contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; d) exogenously adding to the first plant (i) a peptide encoded by the ORF, or (ii) a nucleic acid sequence encoding the peptide; e) comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing immunity enhancing miRNA and the peptide is an immunity enhancing miPEP.
  • the ORF is from 12 to 303 nucleotides in length.
  • the nucleic acid encodes an miPEP further comprising a tag.
  • the tag is a secretion tag, a tag that promotes entry into a plant cell, or a nuclear localization tag.
  • the cell comprises an immunity enhancing peptide or a nucleic acid encoding the immunity enhancing peptide.
  • the immunity enhancing peptide is a miPEP that regulates a miRI69 or miR396.
  • the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • the cell is a host cell.
  • the cell is a plant cell. In some embodiments, the cell is a cell of the plant. In some embodiments, the plant cell is a dicot or monocot plant cell. In some embodiments, the plant cell is a cell from an Arabidopsis spp., Phaseolus spp., Fragaria spp., or Solarium spp. plant. In some embodiments, the cell is a transgenic cell. In some embodiments, the cell expresses a miRI69 or miR396. In some embodiments, the cell has increased immunity to a fiingal pathogen.
  • the cell is a bacterial cell.
  • the bacteria is an E. colt strain.
  • the cells is a cell that is capable of forming inclusion bodies.
  • the cell is a prokaryotic cell.
  • the cell is a eukaryotic cell.
  • the cell is a fiingal cell.
  • the cell is an algal cell.
  • the cell is an animal cell. In some embodiments, the animal cell is a mammalian cell or an insect cell.
  • Microorganism host cells usefill in the present invention may include, but are not limited to, bacteria, such as the enteric bacteria ⁇ Escherichia and Salmonella for example) as well as Bacillus, Acinetobacter, Streptomyces, Methylobacter, Rhodococcus and Pseudomonas,' Cyanobacteria, such as Rhodobacter and Synechocystis,' yeasts, such as Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia, Yarrowia, and Torulopsis,' and filamentous fungi such as Aspergillus and Arthrobotrys, and algae for example.
  • bacteria such as the enteric bacteria ⁇ Escherichia and Salmonella for example
  • Bacillus Acinetobacter, Streptomyces, Methylobacter, Rhodococcus and Pseudomonas
  • the host cell comprises at least one copy of a nucleic acid sequence encoding an immunity enhancing miPEP.
  • the at least one copy of the nucleic acid sequence encoding an immunity enhancing miPEP can be present in the chromosome of a prokaryotic (bacterial) cell or in one chromosome of a eukaryotic cell.
  • the at least one copy of the nucleic acid sequence encoding an immunity enhancing miPEP can be present in a vector or plasmid that is present in the cell.
  • the host cell as described above, can be a prokaryotic or eukaryotic cell. If it is a prokaryotic cell, it can be a bacterial cell. If it is a eukaryotic cell, it can be a yeast cell, a plant cell, or an animal cell. Suitable host cells are described herein.
  • the cell comprises a miPEP or a nucleic acid encoding an miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ IDNO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the miPEP fiirther comprises an N-terminal proline and a C-terminal aspartate.
  • the miPEP regulates miRI69 or miR396.
  • the cell comprises a miPEP or a nucleic acid encoding a miPEP comprising a proline at the N-terminus and an aspartic acid at the C-terminus.
  • the cell comprises an miPEP or a nucleic acid encoding a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
  • the miPEP is 4-50 or
  • the cell comprises an miPEP, or a nucleic acid encoding an miPEP, identified by: a) selecting an miRNA from a miRI69 family or a miR396 family; b) identifying an ORF contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; d) exogenously adding to the first plant (i) a peptide encoded by the ORF, or (ii) a nucleic acid sequence encoding the peptide; e) comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing immunity enhancing miRNA and the peptide is an immunity enhancing miPEP.
  • the ORF is from 12 to 303 nucleotides in length.
  • kits comprising an immunity enhancing peptide and a package insert comprising instructions for the application of the immunity enhancing peptide.
  • the immunity enhancing peptide is a miPEP.
  • the kit comprises a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NOV, SEQ ID NO:8, SEQ ID NOV, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, and instructions for the application of the miPEP.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NOV, SEQ ID NO:8, SEQ ID NOV, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 1, SEQ ID NOV, SEQ ID NOV, SEQ ID NO:4, SEQ ID NOV, SEQ ID NOV, SEQ ID NOV, SEQ IDNO:8, SEQ ID NOV, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the miPEP further comprises an N-terminal proline and a C-terminal aspartate.
  • the miPEP regulates miR169 or miR396.
  • the kit comprises a miPEP comprising a proline at the N-terminus and an aspartic acid at the C-terminus.
  • the kit comprises a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NOVO, SEQ ID NO:21, SEQ ID NOV2, SEQ ID NO:23, SEQ ID NOV4, SEQ ID NOV5, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NOV8, SEQ ID NOV9, SEQ ID NOVO, SEQ ID NO:31, SEQ ID NOV2, SEQ ID NOV3, SEQ ID NOV4, SEQ ID NO:35, SEQ ID NOV6, or SEQ ID NO:37, and instructions for the application of the miPEP.
  • the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NOVO, SEQ ID NO:21, SEQ ID NOV2, SEQ ID NOV3, SEQ ID NOV4, SEQ NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
  • the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.
  • the miPEP is 4-50 or 5-30 amino acids in length.
  • the proline at the N-terminus and the aspartic acid at the C- terminus result from fiision polypeptide expression of the miPEP.
  • the miPEP regulates miRI69 or miR396.
  • the kit comprises instructions and a miPEP identified by: a) selecting an miRNA from a miRI69 family or a miR396 family; b) identifying an ORF contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; d) exogenously adding to the first plant (i) a peptide encoded by the ORF, or (ii) a nucleic acid sequence encoding the peptide; e) comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing immunity enhancing miRNA and the peptide is an immunity enhancing miPEP.
  • the ORF is from 12 to 303 nucleotides in length.
  • the kit comprises a composition comprising a miPEP and, and a package insert comprising instructions for application of the composition.
  • the composition comprises a miPEP and an agriculturally acceptable formulant.
  • the agriculturally acceptable formulant comprises one or more of a water, organic solvents, paraffinic oils, vegetable oils, dispersants, emulsifiers, wetting agents, buffering agents, hydrotrope agents, rheology modifiers, antifoam agents and defoamers, antifreeze agents, biocides, dyes, polymer walls, catalysts, thermosetting materials, cross-polymerizing agents, UV protectants, antioxidants, and chelating agents.
  • the composition is in the form of a finely-divided particulate solid, granules, pellets, wettable powders, dust, an aqueous suspension, a dispersion, a gel, or an emulsion.
  • the composition further comprises one or more other active agents selected from the group consisting of a pesticide, a fertilizer, an insecticide, an attractant, a sterilizing agent, an acaricide, a nematocide, a herbicide, a biostimulant, a biological, and a growth regulator.
  • the kit comprises a composition comprising a miPEP that regulates a miRI69 or a miR396.
  • the kit comprises a composition comprising a miPEP comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • the kit comprises a composition comprising a miPEP and a agriculturally acceptable carrier, diluent or excipient.
  • the kit comprises a composition comprising a miPEP and a package insert comprising instructions for applying the miPEP to a plant or part thereof.
  • the plant part is a leaf, a bud, a root, a shoot, a floral part, or a seed.
  • the package insert comprises instructions for providing the composition to a plant by application to the soil in which the plant is planted.
  • the package insert comprises instructions for providing the composition by addition of water provided to the plant.
  • the package insert comprises instructions for applying the composition by a spreader, a power duster, a boom sprayer, a hand sprayers, a spray dusters, or a granular applicator.
  • the miPEP regulates a miRI69 or a miR396.
  • the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • kits comprising cells comprising a miPEP, or a nucleic acid encoding the miPEP, and a package insert comprising instructions for application of the cells.
  • the cells comprise a miPEP that regulates a miRI69 or a miR396.
  • the cells comprises a miPEP having s an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
  • Example 1 miPEP candidate screening on Arabidopsis thaliana to determine microRNA families involved in defense against Botrytis cinerea
  • Micropeptides are characterized by short peptide sequences (7-44 amino acids in length) defined by one or several short open reading frames (ORFs) located in the pri-miR sequence of a specific miR family member.
  • ORFs short open reading frames
  • miRs shortened micropeptides targeting various microRNAs (miRs) were designed and tested to identify miR families involved in the defense against grey mold in Arabidopsis thaliana.
  • miR68, miRI69, miR396 and miR848 families were chosen for screening of miR families involved in the defense against Botrytis cinerea (grey mold) in Arabidopsis thaliana.
  • Open reading frames (ORFs) in pri-miRs from these families were analyzed to design 10 amino acid-long miPEP sequences to screen.
  • a total of seven miPEPs for four different miR families were designed and chemically synthesized.
  • AtaiiPEP168a did not show any difference to control treatment, suggesting that general peptide application does not interfere with Botrytis infection (FIG. 2).
  • AtaiiR396b was not detectable in Arabidopsis leaves.
  • the pri-miR sequence was amplified from cDNA, sequenced and cloned into a plant transformation vector for transient expression of in Nicotiana benthamiana leaves. Nicotiana benthamiana leaves were transformed with the pri-AtaiiR396b sequence construct and then sprayed with AtmiPEP396b. RNA was extracted and pri-miR transcript expression was analyzed. A 2-fold induction of pri-miR transcript after peptide application compared to control plants (FIG. 4).
  • Example 3 miPEP screening on Phaseolus vulgaris (bean) to analyze the effect of family members of PvmiR169 and PvmiR396 involved in defense against Botrytis cinerea.
  • This example describes the screening of miPEPs targeting the miR169 and miR396 families in bean plant defense against Botrytis infection.
  • Example 4 miPEP screening on Fragaria vesca (strawberry) to analyze the effect of family members of FvmiR169 and FvmiR396 involved in defense against Botrytis cinerea.
  • This example describes the screening of miPEPs targeting the miR169 and miR396 families in strawberry plant defense against Botrytis infection.
  • Fragaria vesca (strawberry) pri-miR sequences were analyzed, and miPEPs for twelve members of theFvmiR169 family and six members of the FvmiR396 family were designed and tested. Peptide application and infection were then performed as described in Example 1. Of 18 miPEPs tested, four (FvmiPEP169hI, FvmiPEP1691I, FvmiPEP396a, FvmiPEP396f) significantly reduced Botrytis- induced lesions size on strawberry leaves after peptide application (FIG. 5).
  • Example 5 miPEP screening on Solanum lycopersicum (tomato) to analyze the effect of family members of SlvmiR169 and SlmiR396 involved in defense against Botrytis cinerea.
  • This example describes the screening of miPEPs targeting the miR169 and miR396 families in tomato plant defense against Botrytis infection.
  • Example 6 Induction of SLmiR169a transcript by the SlmiPEP169a miPEP in tomato.
  • the pri-miR S/miR169a sequence was synthesized, sequenced and cloned into a plant transformation vector for transient expression in Nicotiana benthamiana leaves. Nicotiana benthamiana leaves were transformed with the pri-S/miR169a sequence construct, and the plants were then sprayed with S/miPEP169a. RNA was then extracted and pri-miR transcript expression was analyzed. A 2-3 fold induction of pri-miR transcript was observed after peptide application as compared to control plants (FIG. 9).
  • Example 7 Foliar spray application of SlmiPEP169a and SlmiPEP169k controls Botrytis growth on tomato leaves.
  • Botrytis spores were applied as droplets in high concentrations to assess quantitatively the lesion sizes, with non-formulated miPEPs sprayed preventively or applied curatively as droplets on the site of infection.
  • Botrytis spores were applied at low concentrations and sprayed on tomato plants in order to mimic the infection occurring in field conditions.
  • the effect of pre- and post-spray application of miPEPs was then evaluated using S/miPEP169a and S/miPEP169k miPEPs.
  • formulated buffers with spreader and humectant ingredients were used to enhance the peptide effect on leaves.
  • Example 8 Foliar spray application of SlmiPEP169k increases disease control in tomato plants against Alternaria solani and Phytophthora inf estans.
  • This example describes the evaluation of a miPEP targeting a miR169 family member in defense against Alternaria solani (fungi) and Phytophthora infestans (oomycetes) infection in tomato plants.
  • S7miPEP169k tomato plants infected with Phytophthora infestans were sprayed with either S7miPEP169k or S7miPEP169kDlD10 at 30 g/ha 2 hours pre-inoculation. Similar disease control (18-27%) was observed for both peptides (FIG. 13B), and the S/miPEP169kDlD10 variant was still biologically active, showing 27% disease control. The S7miPEP169kDlD10 maintains its full biological activity while appearing to be less prone to degradation.
  • Example 10 Identification of miPEPs targeting miR169 family members with increased biological activity or stability.
  • This example describes the identification of additional miPEPs targeting miR169i and miR169d that regulate defense against Botrytis cinerea in tomato plants.
  • Example 11 Identification of miPEPs boosting soybean immunity against Asian Soybean Rust
  • This example describes the identification of additional miPEPs targeting miR169g and miR169m that regulate defense against Phakopsora pachyrhizi (commonly known as Asian Soybean Rust, or ASR) in Glycine max (soybean) plants.
  • ASR Asian Soybean Rust
  • the miR169 family in Glycine max (referred to herein as gma-MIR169 and G/wmiR 169, with family members indicated by a letter suffixed thereon), is known to be involved in plant defense pathways.
  • 44 miPEPs collectively targeting 22 members of the G/wmiR 169 family (including both miR169g and miR169m) were designed, with two peptides designed per G/wmiR 169 family member.
  • the 44 miPEP peptides were chemically synthetized and screened on ASR-infected soybean plants. Peptides solutions were foliar sprayed at 24 hours pre-infection and at 24 hours post-infection, both at 200 g/ha. Peptides that displayed more than 30% disease control compared to blank controls (comprising reference antifungal peptide MP18279; SEQ ID NO: 60) were tested a second time. Peptides that displayed more than 30% disease control compared to blank controls in the second replicate were tested a third time. These tests validated two miPEP peptides 3 times each with > 30% disease control: MP19114 and MP19127 (FIGS. 15A-15B and SEQ ID NOs: 54-55, respectively), each of which were found to be as efficient as the MP 18279 reference in controlling ASR disease progression.
  • Disease control following peptide application can be due to a boost in plant immunity or by antifungal properties of the peptide (e.g., wherein the peptide itself has a negative impact on, e.g., fungal spore development).
  • peptides were designed as miPEPs to boost plant immunity only.
  • In vitro tests were designed in order to efficiently check for potential antifungal activities against Septoria tritici, Phytophthora cactorum, and Botrytis cinerea. In vitro tests could not be run on ASR, however, because it is an obligate parasite that cannot survive outside a plant.
  • a coding nucleic acid including one copy, 3 copies, or 6 copies of the nucleotide sequence encoding the peptide sequence ofMP19114 or MP19127 (Table 4) were integrated into the E. coli genome.
  • the copies of the nucleic acid sequences encoding the peptide were linked by nucleic acids encoding a cleavable linker peptide, such that individual copies of MP19114 or MP19127 could be cleaved from the inclusion bodies.
  • Example 14 In planta activity of MP19114 and MP19127 variants with P and DP additions
  • This example describes experiments testing the in planta activities of variants of MP 19114 and MP 19127 having additions of P or DP amino acids.
  • Concatemeric expression strategies such as those described in Example 13 involve adding, for example, up to 6 copies of a peptide sequence into a bacterial genome in order to increase yield.
  • P and/or D amino acids remain appended to one or both ends of the peptide.
  • the MP19114 and MP19127 peptides expressed as concatemers necessarily had the amino acids DP present between each copy.
  • Acidic cleavage of the cleavable linker then cut 2 peptide copies between D and P and released copies of the peptide flanked by D and P (e.g., with P on the N-terminus and/or D on the C-terminus).
  • the sequence of the released peptides was not the original MP 19114 or MP 19127 sequence, but had 2 additional amino acids: D and P.
  • Table 6 The relationship between each of these peptides and their names and SEQ ID NOs herein are summarized in Table 6.
  • MP 19114 was found to be bioproducible, while MP 19127 was not (Table 5). However, the multiple chromatogram peaks seen for MP 19127 (Table 5) could potentially be overcome through optimization. Because it retained activity with the DP addition, MP 19127 was chosen as the most promising candidate for field tests and optimization, while MP19114 was chosen as the second-most- promising candidate.
  • This example describes additional synthetic variants of MP 19127 designed based on potential acidic deamination of MP 19127.
  • SEQ ID NO: 61 is a 2021-nt-long nucleic acid sequence that corresponds to an extended version of the hairpin sequence gma-MIR169g referenced in miRBase 22. 1 under ID MI0017837, extended based on genomic positions from assembly Wm82.v4.4PTR (soybase.org) glyma.Wm82.gnm4. Gm 17 (4593256-4595276)-.
  • SEQ ID NO: 62 is a 2021-nt-long nucleic acid sequence that corresponds to an extended version of the hairpin sequence gma-MIR169m referenced in miRBase 22. 1 under ID MI0018665, extended based on genomic positions from assembly Wm82.v4.4PTR (soybase.org) glyma.Wm82.gnm4. Gm 13 (20517719-20519739)+.

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Abstract

The present invention relates to micropeptides (miPEPs) and their use for enhancing plant immunity and increasing resistance to pathogens and pests. Also provided herein are kits comprising miPEPs.

Description

MICROPEPTIDES TO IMPROVE PLANT IMMUNITY AND APPLICATION THEREOF
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[0001] The contents of the electronic sequence listing (185952000840SEQLIST.xml; Size: 100,513 bytes; and Date of Creation: February 9, 2024) is herein incorporated by reference in its entirety.
FIELD OF INVENTION
[0002] The present invention relates to micropeptides and their use for increasing plant immunity.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0003] This application claims the benefit of U. S. Provisional Patent Application Nos. 63/486,652, 63/486,653, and 63/486,654, all of which were filed on February 23, 2023, and the entire contents of each of which are incorporated herein by reference.
BACKGROUND
[0004] Fungal plant pathogens are major threats to food security worldwide. For example, infection by the fungus Botrytis cinerea causes yearly several 100 millions of US dollars crop losses worldwide (Bolton et al., 2006; Dean et al., 2012). Genetic modification approaches, such as the use of genetically-modified organisms (GMOs), and chemical pesticides are widely used to control wide range of plant pathogens, including fungal infections. However, these approaches have limited applicability and can have a significant environmental impact. As such, there is a need for natural and sustainable solutions for controlling plant pathogens.
[0005] MicroRNAs (miRNAs) modulate the abundance and spatial-temporal accumulation of target mRNAs and indirectly regulate several plant processes, including response to pathogens. Transcriptional regulation of the genes encoding miRNAs (miR genes) can be activated by numerous transcription factors, which in turn can be regulated by other miRNAs. Modulation of miR gene expression, or of the levels of miRNAs in a cell, can lead to improve tolerance to abiotic or biotic stresses in crops of economic importance. There are currently more than 28000 identified miRNAs, and increasing evidence indicates that several miRNAs families are involved in fine-tuning plant immunity against pathogen invasion (Padmanabhan et al. , 2009; Katiyar-Agarwal and Jin, 2010, Chen et al 2017). Differential expression of miRs after pathogen infection can negatively or positively impact plant immunity (Chen, et al., 2017). For instance, upon infection with a fungal pathogen (Botrytis cinerea) several miRNAs are upregulated in affected plants (Jin and Wu, 2015). Of these miRNAs, the miR169 family was identified as a Botrytis responsive miR, which is upregulated in leaves after Botrytis infection. (Jin and Wu, 2015).
[0006] More recently, micropeptides (miPEPs) encoded by one or several short open reading frames contained in pri-miRNA sequences have been identified (Chen, et al. 2020, Sharma et al, 2019). These miPEPs have also been found to modulate the expression of the miRNA produced from their encoding pri-miRNA (Lauressergues, et al., 2005), thus modulating the levels of the mature miRNAs in the cell. MiPEPs may present and alternative to GMO-based technologies and chemical peptides, as they regulate specific plant processes transiently and specifically by targeting expression of miRNAs.
[0007] Accordingly, there is a need to identify and produce micropeptides that regulate plant immunity to control infection by common plant pathogens, such as bacteria and fungi.
SUMMARY OF INVENTION
[0008] The disclosure is directed to multiple aspects including, without limitation, Embodiment A: A method for identifying and synthesizing (i) an immunity enhancing micropeptide (miPEP) encoded by a nucleotide sequence contained in the sequence of the primary transcript of a microRNA (miRNA), or (ii) a nucleic acid sequence encoding the miPEP that does not comprise the mature miRNA, comprising: a) selecting an miRNA from a miR169 family or a miR396 family; b) identifying an open reading frame (ORF) from 12 to 303 nucleotides in length contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; d) exogenously adding to the first plant (i) a peptide encoded by the ORF or (ii) a nucleic acid sequence encoding the peptide; e) comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing immunity enhancing miRNA and the peptide is the miPEP; and f) synthesizing the miPEP or the nucleic acid sequence encoding the miPEP.
[0009] Embodiment Bl. A method of increasing immunity in a plant comprising providing to the plant a miPEP that regulates an immunity enhancing miR169 or an immunity enhancing miR396.
[0010] Embodiment B2: The method of Embodiment Bl, where the miPEP is selected from the group consisting of d /mi P EP 169c, A /mi P EP 169h, A /PE P396b, PvmiPEP 169e, P vmi P EP 169kl, PvmiPEP 169pl, FvmiPEP169hI, FvmiPEP1691I, FvmiPEP369a, S7miPEP169a, S7miPEP169d, S7miPEP169k, S7miPEP396c, MP19043, MP19042, S7miPEP169kDlD10, MP19051, MP19048, and MP19049, wherein the miPEP regulates an immunity enhancing miRNA.
[0011] Embodiment B3: A method of increasing immunity in a plant comprising providing to the plant a miPEP comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, or the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ IDNO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37 with one, two, three, or four amino acid substitutions, insertions, or deletions, wherein the miPEP regulates miR169 or miR396.
[0012] Embodiment B4: A method of increasing resistance to a plant pathogen or pest in a plant comprising providing to the plant a miPEP comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, or the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37 with one, two, three, or four amino acid substitutions, insertions, or deletions, wherein the miPEP regulates miR169 or miR396. [0013] Embodiment B5 : The method of Embodiment B4, wherein the plant pathogen or pest is selected from fungal pathogens or pests, bacterial pathogens or pests, and viral pathogens or pests.
[0014] Embodiment B6: The method of Embodiment B4, wherein the plant pathogen or pest is a fungal pathogen or pest selected from Cercospora spp., Mycosphaerella spp., Glomerella spp., Cladosporium spp., Diplodia maydis, Fusarium oxysporum, Fusarium graminearum, Fusarium monilforme, Fusarium verticillioides, Cochliobolus sativus, Collectotrichum graminicola, Stagonospora nodorum, Stagonospora avenae, Stenocarpella maydis, Sclerotinia minor, Sclerotinia sclerotiorum, Sclerotinia sp., Alternaria spp., Phytophthora spp., Botrytis spp., Pyrenophora tritici-repentis, Phytophthora parasitica, Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichum dematium (Colletotichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v. glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Glomerella glycines, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Fusarium solani; Canola: Albugo Candida, Altenaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassicicola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter michiganese subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium oxysporum, Verticillium albo-atrum, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulina briosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium botryosum, Leptotrichila medicaginis; Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis fsp. tritici, Puccinia graminis fsp. tritici, Puccinia recondite fsp. tritici, Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae, P seudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola, Pythium aphanidermatum, Plasmopora
[0015] Embodiment B7: The method of claim Embodiment B4, wherein the plant pathogen or pest is a bacterial pathogen or pest selected from Pseudomonas spp., Pantoua spp., and Erwinia spp.
[0016] Embodiment B8: The method of Embodiment B4, wherein the plant pathogen or pest is a viral pathogen or pest selected from cucumber mosaic, tobacco mosaic, and barley yellow dwarf vims, alfalfa mosaic vims (Alfamovims), Apple chlorotic leaf spot vims (Trichovims), Apple scar skin viroid (Viroids), Arabis mosaic vims (Nepovims), Barley mild mosaic vims (Bymovirus), Barley stripe mosaic vims (Hordeivims), Barley yellow mosaic vims (Bymovirus), Bean common mosaic vims (Potyvims), Bean yellow mosaic virus (Potyvirus), Beet necrotic yellow vein virus (Furovirus), Blackeye cowpea mosaic virus (Potyvirus), Bean common mosaic virus (Potyvirus), Broad bean wilt virus (Fabavirus), Butterbur mosaic virus (Carlavirus), Carnation mottle virus (Carmovirus), Carnation vein mottle virus (Potyvirus), Cauliflower mosaic virus (Caulimovirus), Chrysanthemum mild mottle virus (Cucumovirus), Tomato aspermy virus (Cucumovirus), Chrysanthemum stunt viroid (Viroids), Citrus mosaic virus, Citrus tristeza virus (Closterovirus), Clover yellow vein virus (Potyvirus), Cocksfoot mottle virus (Sobemovirus), Cucumber green mottle mosaic virus (Tobamovirus), Cucumber mosaic virus (Cucumovirus), Cycas necrotic stunt virus (Nepovirus), Dasheen mosaic virus (Potyvirus), Grapevine Algerian latent virus (Tombusvirus), Konjac mosaic virus (Potyvirus), Melon necrotic spot virus (Carmovirus), Mulberry ringspot virus (Nepovirus), and Narcissus mosaic virus (Potexvirus). Plant viruses are viruses affecting plants. Additional examples of viruses affecting plants include Odontoglossum ringspot virus (Tobamovirus), Papaya ringspot virus (Potyvirus), Peach latent mosaic viroid, Peanut mottle virus (Potyvirus), Peanut stripe virus (Potyvirus), Bean common mosaic virus (Potyvirus), Peanut stunt virus (Cucumovirus), Potato virus A (Potyvirus), Potato virus M (Carlavirus), Potato virus S (Carlavirus), Potato virus X (Potexvirus), Potato virus Y (Potyvirus), Prune dwarf virus (Ilarvirus), Primus necrotic ringspot virus (Ilarvirus), Radish mosaic virus (Comovirus), Rice black streaked dwarf virus (Fijivirus), Rice dwarf virus (Reovirus), Rice grassy stunt virus (Tenuivirus), Rice stripe virus (Tenuivirus), Rice tungro spherical virus (Sequivirus), Rice waika virus, Rice tungro spherical virus (Sequivirus), Ryegrass mottle virus, Satsuma dwarf virus (Nepovirus), Soil -borne wheat mosaic virus (Furovirus), Southern bean mosaic virus (Sobemovirus), Soybean mosaic virus (Potyvirus), Soybean stunt virus (Cucumovirus), Cucumber mosaic virus (Cucumovirus), Tobacco mosaic virus (Tobamovirus), Tobacco mosaic virus (Tobamovirus), Tomato mosaic virus (Tobamovirus), Tobacco necrosis virus (Necrovirus), Tobacco rattle virus (Tobravirus), Tobacco ringspot virus (Nepovirus), Tomato aspermy virus (Cucumovirus), Tomato black ring virus (Nepovirus), Tomato mosaic virus (Tobamovirus), Tomato ringspot virus (Nepovirus), Tomato spotted wilt virus (Tospovirus), Turnip mosaic virus (Potyvirus), Watermelon mosaic virus 1 (Potyvirus), Papaya ringspot virus (Potyvirus), Watermelon mosaic virus 2 (Potyvirus), Wheat yellow mosaic virus (Bymovirus), and Zucchini yellow mosaic virus (Potyvirus).
[0017] Embodiment B9: The method of Embodiment B4, wherein the plant pathogen or pest is selected from the group consisting of Botrytis cinerea, Altemaria solani, and Phytophthora infestans.
[0018] Embodiment BIO: The method of any one of Embodiments Al and B1-B9, wherein the miPEP comprises the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, with zero, one, two, three, or four amino acid substitutions, insertions, or deletions, and the plant is an Arabidopsis plant. [0019] Embodiment Bl l: The method of any one of Embodiments Al and B1-B9, wherein the miPEP comprises the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22, with zero, one, two, three, or four amino acid substitutions, insertions, or deletions, and the plant is a strawberry plant.
[0020] Embodiment B12: The method of any one of Embodiments Al and B1-B9, wherein the miPEP comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19, with zero, one, two, three, or four amino acid substitutions, insertions, or deletions, and the plant is a Phaseolus vulgaris plant.
[0021] Embodiment B13: The method of any one of Embodiments Al and B1-B9, wherein the miPEP comprises the amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, with zero, one, two, three, or four amino acid substitutions, insertions, or deletions, and the plant is a tomato plant.
[0022] Embodiment B14: The method of any one of Embodiments Al and Bl -Bl 3, wherein the oligopeptide is provided by expressing a nucleic acid encoding the miPEP.
[0023] Embodiment B 15: The method of Embodiment Bl 4, wherein the nucleic acid is operably linked to a heterologous promoter.
[0024] Embodiment B16: The method of Embodiment B 14 or Bl 5, wherein the heterologous promoter is a constitutive promoter, a tissue specific promoter, or an inducible promoter.
[0025] Embodiment Bl 7: The method of Embodiment Bl 6, wherein the inducible promoter is induced by fungal infection, optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., phenylalanine ammonia lyase, chaicone synthase promoters), a gene that modifies plant cell walls (e.g., hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase promoters), a gene encoding an enzyme that degrade fungal cell walls (e.g., chitinase or glucanase promoters), or a gene encoding a thaumatin-like protein, or or optionally a maize Misl promoter or a flax Fisl promoter.
[0026] Embodiment Bl 8: The method of any one of Embodiments Al and B1-B13, wherein the miPEP is provided by application to the plant or a part thereof.
[0027] Embodiment B 19 : The method of Embodiment B 18, wherein the plant part is a leaf, a bud, a root, a shoot, a floral part, or a seed.
[0028] Embodiment B20: The method of Embodiment B18 or Bl 9, wherein the miPEP is applied as a coating to the seed prior to planting. [0029] Embodiment B21: The method of any one of Embodiments Al and B1-B13, wherein the miPEP is provided by application to the soil in which the plant is planted.
[0030] Embodiment B22: The method of any one of Embodiments Al and B1-B13, wherein the miPEP is provided by addition to water provided to the plant.
[0031] Embodiment B23: The method of any one of Embodiments Al and B1-B13, wherein the oligopeptide is provided following harvest to seeds, fruits, and/or plant parts.
[0032] Embodiment B24: The method of any one of Embodiment B18-B23, wherein the application is by a spreader, a power duster, a boom sprayer, a hand sprayers, a spray dusters, or a granular applicator. [0033] Embodiment B25: The method of any one of Embodiments Al and B1-B24, wherein the miPEP comprises a secretion tag, a tag that promotes entry into a plant cell, or a nuclear localization tag.
[0034] Embodiment B26: The method of Embodiment B25, wherein the tag that promotes entry into a plant cell comprises a cell penetrating peptide linked to the amino acid sequence.
[0035] Embodiment B27: The method of Embodiment B26, wherein the cell penetrating peptide comprises a protein transduction domain, an amphipathic peptide, a synthetic cationic polypeptide, optionally polylysine, polyhistidine, or polyarginine, a dendrimeric polycationic molecule, a peptide vascular endothelial-cadherin cell penetrating peptide, a transportan cell penetrating peptide, a monomer or dimer of HIV- 1 TAT basic domain cell penetrating peptide, a penetratin cell penetrating peptide, a synthetic cationic homoarginine oligopeptide cell penetrating peptide, a gamma zein cell penetrating peptide, a Zea mays knottedl cell penetrating peptide, a Saccharomyces pombe TP10 cell penetrating peptide, a Candida albicans Zebra cell penetrating peptide, an Antennapedia sequence, a TAT sequence , an Antp-3A (Antp mutant) sequence, a Buforin II sequence, a K-FGF sequence, a Ku70 sequence, a prion sequence, a pVEC sequence, a SynBl sequence, a Pep-7 sequence, a HN-1 sequence, a BGSC (Bis- Guanidinium-Spermidine-Cholesterol) sequence, or a BGTC (Bis-Guanidinium-Tren-Cholesterol) sequence.
[0036] Embodiment Cl: An isolated oligopeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NOV, SEQ ID NO: 8, SEQ ID NOV, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NOV, SEQ ID NOV, SEQ ID NO:4, SEQ ID NOV, SEQ ID NO:6, SEQ ID NOV, SEQ ID NO:8, SEQ ID NOV, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, with one, two, three, or four amino acid substitutions, insertions, or deletions, wherein the oligopeptide regulates miR169 or miR396.
[0037] Embodiment C2: An isolated oligopeptide comprising the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID N0:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, or the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, with one, two, three, or four amino acid substitutions, insertions, or deletions, wherein the peptide comprises a proline at the N-terminus and an aspartic acid at the C-terminus, wherein the oligopeptide regulates miRI69 or miR396.
[0038] Embodiment C3: The oligopeptide of Embodiment Cl or C2, wherein the oligopeptide enhances immunity in a plant.
[0039] Embodiment C4: The oligopeptide of Embodiment Cl or C2, wherein the oligopeptide enhances resistance in a plant to a plant pathogen or pest.
[0040] Embodiment C5: The oligopeptide of any one of Embodiments C1-C4, comprising the one, two, three, or four amino acid substitutions, insertions, or deletions.
[0041] Embodiment C6: The oligopeptide of any one of Embodiments C1-C5, wherein the oligopeptide comprises a proline at its N-terminus, an aspartic acid at the C-terminus, or both.
[0042] Embodiment C7: The oligopeptide of any one of Embodiments C1-C6, wherein the oligopeptide comprises one or more D-amino acids.
[0043] Embodiment C8: The oligopeptide of Embodiment C7, wherein the one or more D-amino acids enhance stability of the oligopeptide as compared to an oligopeptide of the same amino acid sequence comprising all L-amino acids.
[0044] Embodiment C9: The oligopeptide of any one of Embodiments C1-C8, wherein the oligopeptide comprises a secretion tag, a tag that promotes entry into a plant cell, or a nuclear localization tag.
[0045] Embodiment CIO: The oligopeptide of Embodiment C9, wherein the tag that promotes entry into a plant cell comprises a cell penetrating peptide linked to the amino acid sequence.
[0046] Embodiment Cl 1: The oligopeptide of Embodiment CIO, wherein the cell penetrating peptide comprises a protein transduction domain, an amphipathic peptide, a synthetic cationic polypeptide, optionally polylysine, polyhistidine, or polyarginine, a dendrimeric polycationic molecule, a peptide vascular endothelial-cadherin cell penetrating peptide, a transportan cell penetrating peptide, a monomer or dimer of HIV- 1 TAT basic domain cell penetrating peptide, a penetratin cell penetrating peptide, a synthetic cationic homoarginine oligopeptide cell penetrating peptide, a gamma zein cell penetrating peptide, a Zea mays knottedl cell penetrating peptide, a Saccharomyces pombe TP10 cell penetrating peptide, a Candida albicans Zebra cell penetrating peptide, an Antennapedia sequence, a TAT sequence , an Antp-3A (Antp mutant) sequence, a Buforin II sequence, a K-FGF sequence, a Ku70 sequence, a prion sequence, a pVEC sequence, a SynBl sequence, a Pep-7 sequence, a HN-1 sequence, a BGSC (Bis- Guanidinium-Spermidine-Cholesterol) sequence, or a BGTC (Bis-Guanidinium-Tren-Cholesterol) sequence.
[0047] Embodiment DI: A composition comprising the oligopeptide of any one of Embodiments Cl- C11 with an agriculturally acceptable formulant that can include water, organic solvents, paraffinic oils, vegetable oils, dispersants, emulsifiers, wetting agents, buffering agents, hydrotrope agents, rheology modifiers, antifoam agents and defoamers, antifreeze agents, biocides, dyes, polymer walls, catalysts, thermosetting materials, cross-polymerizing agents, UV protectants, antioxidants, and chelating agents.
[0048] Embodiment D2: A composition comprising the oligopeptide of any one of Embodiments Cl- C11 with an agriculturally acceptable carrier, diluent, or excipient.
[0049] Embodiment D3: The composition of Embodiment D2, wherein the agriculturally acceptable carrier, diluent, or excipient comprises a buffer and the pH is in the range of from about 3.0 to about 9.0 or about 4.5 to about 8.0.
[0050] Embodiment D4: The composition of Embodiments D1-D3, wherein the oligopeptide is in a concentration range of from about 0. 1 pg/ml to about 100 mg/ml, or from about 5 pg/ml to about 5 mg/ml. [0051] Embodiment D5 : The composition of any one of Embodiments D 1-D4, wherein the composition is in the form of a finely-divided particulate solid, granules, pellets, wettable powders, dust, an aqueous suspension, a dispersion, a gel, or an emulsion.
[0052] Embodiment D6: The composition of any one of Embodiments D1-D5, further comprising one or more other active agents selected from the group consisting of a pesticide, a fertilizer, an insecticide, an attractant, a sterilizing agent, an acaricide, a nematocide, a herbicide, a biostimulant, a biological, and a growth regulator.
[0053] Embodiment E: A plant immunity enhancing peptide identified by: a) selecting an miRNA from a miR169 family or a miR396 family; b) identifying an open reading frame (ORF) from 12 to 303 nucleotides in length contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; d) exogenously adding to the first plant (i) a peptide encoded by the ORF, or (ii) a nucleic acid sequence encoding the peptide; e) comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing immunity enhancing miRNA and the peptide is an immunity enhancing micropeptide.
[0054] Embodiment F 1 : A nucleic acid encoding the oligopeptide of any one of Embodiments Cl-Cl 1. [0055] Embodiment F2: A nucleic acid construct comprising the nucleic acid of Embodiment Fl, operably linked to a promoter.
[0056] Embodiment F3: The nucleic acid construct of Embodiment F2, wherein the promoter is a heterologous promoter.
[0057] Embodiment F4: The nucleic acid construct of Embodiment F2 or F3, wherein the promoter is a constitutive promoter, a tissue specific promoter, a development stage specific promoter, or an inducible promoter.
[0058] Embodiment F5: The nucleic acid construct of claim Embodiment F4, wherein the inducible promoter is induced by fungal infection, optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., phenylalanine ammonia lyase, chaicone synthase promoters), a gene that modifies plant cell walls (e.g., hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase promoters), a gene encoding an enzyme that degrade fungal cell walls (e.g., chitinase or glucanase promoters), or a gene encoding a thaumatin-like protein, or a maize Mis 1 promoter or a flax Fis 1 promoter. [0059] Embodiment G: A cell comprising the nucleic acid of Embodiment Fl or the nucleic acid construct of any one of Embodiments F2-F5.
[0060] Embodiment H: The method of any one of Embodiments A-B27, the oligopeptide of any one of Embodiments Cl-Cl 1, the composition of any one of Embodiments D1-D6, the immunity enhancing peptide of Embodiment E, the nucleic acid of Embodiment F 1, the nucleic acid construct of Embodiments F2-F5, or the cell of Embodiment G, wherein: (a) the miPEP is selected from the group consisting of G/wmiR I 69g. G/wmiR I69m. MP19114, MP19127, MP19478, MP19474, MP19476, and MP19472, wherein the miPEP regulates an immunity enhancing miRNA and the plant is a soybean plant; (b) the miPEP, the oligopeptide, or the immunity enhancing peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 54-59, wherein the miPEP, the oligopeptide, or the immunity enhancing peptide regulates miR169 and the plant is a soybean plant; (c) the miPEP, the oligopeptide, or the immunity enhancing peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 54- 59 with one, two, three, or four amino acid substitutions, insertions, or deletions, wherein the miPEP, the oligopeptide, or the immunity enhancing peptide regulates miR169 and the plant is a soybean plant; (d) the miPEP, the oligopeptide, or the immunity enhancing peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 54-59, wherein the peptide comprises a proline at the N-terminus and/or an aspartic acid at the C-terminus, wherein the miPEP, the oligopeptide, or the immunity enhancing peptide regulates miR169 and the plant is a soybean plant; and/or (e) the miPEP, the oligopeptide, or the immunity enhancing peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 54- 59 with one, two, three, or four amino acid substitutions, insertions, or deletions, wherein the peptide comprises a proline at the N-terminus and/or an aspartic acid at the C-terminus, wherein the miPEP, the oligopeptide, or the immunity enhancing peptide regulates miR169 and the plant is a soybean plant; and (f) optionally in each case of (a)-(e), the plant pathogen or pest is Phakopsora pachyrhizi.
[0061] Embodiment I: A method of increasing immunity in a plant comprising providing to the plant an oligopeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79 and/or providing to the plant an oligopeptide comprising an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions compared to the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79.
[0062] Embodiment J: A method of reducing fungal growth on a plant comprising providing to the plant an oligopeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79 and/or providing to the plant an oligopeptide comprising an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions compared to the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79.
[0063] Embodiment K 1 : A method of inhibiting a plant pathogen or pest on a plant comprising providing to the plant an oligopeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79 and/or providing to the plant an oligopeptide comprising an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions compared to the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79.
[0064] Embodiment K2: The method of Embodiment J or KI, wherein the plant pathogen or pest is selected from fungal pathogens or pests, bacterial pathogens or pests, and viral pathogens or pests.
[0065] Embodiment K3: The method of Embodiment J or KI, wherein the plant pathogen or pest is a fungal pathogen or pest selected from Cercospora spp., Mycosphaerella spp., Glomerella spp., Cladosporium spp., Diplodia maydis, Fusarium oxysporum, Fusarium graminearum, Fusarium monilforme, Fusarium verticillioides, Cochliobolus sativus, Collectotrichum graminicola, Stagonospora nodorum, Stagonospora avenae, Stenocarpella maydis, Sclerotinia minor, Sclerotinia sclerotiorum, Sclerotinia sp., Alternaria spp., Phytophthora spp., Botrytis spp., Pyrenophora tritici-repentis, Phytophthora parasitica, Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichum dematium (Colletotichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v. glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Glomerella glycines, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium deharyanum, Fusarium solani; Canola: Albugo Candida, Altenaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassicicola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter michiganese subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium oxysporum, Verticillium albo-atrum, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulina briosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium botryosum, Leptotrichila medicaginis; Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis fsp. tritici, Puccinia graminis fsp. tritici, Puccinia recondite fsp. tritici, Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola, Pythium aphanidermatum, Plasmopora halstedii, Sclerotinia sclerotiorum, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum pv. carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis; Com: Colletotrichum graminicola, Fusarium verticillioides var. subglutinans, Erwinia stewartii, F. verticillioides, Gibberella zeae (Fusarium graminearum), Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T (Cochliobolus Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganense subsp. nebraskense, Trichoderma viride, Claviceps sorghi, Pseudomonas avenae, Erwinia chrysanthemi pv. zea, Erwinia carotovora, Corn stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium acremonium, Exserohilum turcicum, C. sublineolum, Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium verticillioides, Altemaria altemata, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum, Pythium arrhenomanes, and Pythium graminicola.
[0066] Embodiment K4: The method of Embodiment KI or K2, wherein the plant pathogen or pest is a bacterial pathogen or pest selected from Pseudomonas spp., Pantoua spp., and Erwinia spp.
[0067] Embodiment K5: The method of Embodiment KI or K2, wherein the plant pathogen or pest is a viral pathogen or pest selected from cucumber mosaic, tobacco mosaic, and barley yellow dwarf virus, alfalfa mosaic virus (Alfamovirus), Apple chlorotic leaf spot virus (Trichovirus), Apple scar skin viroid (Viroids), Arabis mosaic virus (Nepovirus), Barley mild mosaic virus (Bymovirus), Barley stripe mosaic virus (Hordeivirus), Barley yellow mosaic virus (Bymovirus), Bean common mosaic virus (Potyvirus), Bean yellow mosaic virus (Potyvirus), Beet necrotic yellow vein virus (Furovirus), Blackeye cowpea mosaic virus (Potyvirus), Bean common mosaic virus (Potyvirus), Broad bean wilt virus (Fabavirus), Butterbur mosaic virus (Carlavirus), Carnation mottle virus (Carmovirus), Carnation vein mottle virus (Potyvirus), Cauliflower mosaic virus (Caulimovirus), Chrysanthemum mild mottle virus (Cucumovirus), Tomato aspermy virus (Cucumovirus), Chrysanthemum stunt viroid (Viroids), Citrus mosaic virus, Citrus tristeza virus (Closterovirus), Clover yellow vein virus (Potyvirus), Cocksfoot mottle virus (Sobemovirus), Cucumber green mottle mosaic virus (Tobamo virus), Cucumber mosaic virus (Cucumovirus), Cycas necrotic stunt virus (Nepovirus), Dasheen mosaic virus (Potyvirus), Grapevine Algerian latent virus (Tombusvirus), Konjac mosaic virus (Potyvirus), Melon necrotic spot virus (Carmovirus), Mulberry ringspot virus (Nepovirus), and Narcissus mosaic virus (Potexvirus). Plant viruses are viruses affecting plants. Additional examples of viruses affecting plants include Odontoglossum ringspot virus (Tobamovirus), Papaya ringspot virus (Potyvirus), Peach latent mosaic viroid, Peanut mottle virus (Potyvirus), Peanut stripe virus (Potyvirus), Bean common mosaic virus (Potyvirus), Peanut stunt virus (Cucumovirus), Potato virus A (Potyvirus), Potato virus M (Carlavirus), Potato virus S (Carlavirus), Potato virus X (Potexvirus), Potato virus Y (Potyvirus), Prune dwarf virus (Ilarvirus), Primus necrotic ringspot virus (Ilarvirus), Radish mosaic virus (Comovirus), Rice black streaked dwarf virus (Fijivirus), Rice dwarf virus (Reovirus), Rice grassy stunt virus (Tenuivirus), Rice stripe virus (Tenuivirus), Rice tungro spherical virus (Sequivirus), Rice waika virus, Rice tungro spherical virus (Sequivirus), Ryegrass mottle virus, Satsuma dwarf virus (Nepovirus), Soil-borne wheat mosaic virus (Furovirus), Southern bean mosaic virus (Sobemovirus), Soybean mosaic virus (Potyvirus), Soybean stunt virus (Cucumovirus), Cucumber mosaic virus (Cucumovirus), Tobacco mosaic virus (Tobamovirus), Tobacco mosaic virus (Tobamovirus), Tomato mosaic virus (Tobamovirus), Tobacco necrosis virus (Necrovirus), Tobacco rattle virus (Tobravirus), Tobacco ringspot virus (Nepovirus), Tomato aspermy virus (Cucumovirus), Tomato black ring virus (Nepovirus), Tomato mosaic virus (Tobamovirus), Tomato ringspot virus (Nepovirus), Tomato spotted wilt virus (Tospovirus), Turnip mosaic virus (Potyvirus), Watermelon mosaic virus 1 (Potyvirus), Papaya ringspot virus (Potyvirus), Watermelon mosaic virus 2 (Potyvirus), Wheat yellow mosaic virus (Bymovirus), and Zucchini yellow mosaic virus (Potyvirus).
[0068] Embodiment K6: The method of Embodiment KI or K2, wherein the plant pathogen or pest is selected from the group consisting of Septoria tritici, Botrytis cinerea, Phytophthora cactorum, and Phakopsora pachyrhizi s.
[0069] Embodiment K7 : The method of any one of Embodiments I-K6, wherein the oligopeptide is provided by expressing a nucleic acid encoding the oligopeptide.
[0070] Embodiment K8: The method of Embodiment K7, wherein the nucleic acid is operably linked to a heterologous promoter.
[0071] Embodiment K9: The method of Embodiment K7 or K8, wherein the heterologous promoter is a constitutive promoter, a tissue specific promoter, or an inducible promoter.
[0072] Embodiment K10: The method of Embodiment K9, wherein the inducible promoter is induced by fungal infection, optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., phenylalanine ammonia lyase, chaicone synthase promoters), a gene that modifies plant cell walls (e.g., hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase promoters), a gene encoding an enzyme that degrade fungal cell walls (e.g., chitinase or glucanase promoters), or a gene encoding a thaumatin-like protein, or optionally a maize Misl promoter or a flax Fisl promoter. [0073] Embodiment K10: The method of any one of Embodiments I-K6, wherein the oligopeptide is provided by application to the plant or a part thereof.
[0074] Embodiment KI 1: The method ofEmbodimentKIO, wherein the plant part is a leaf, a bud, a root, a shoot, a floral part, or a seed.
[0075] Embodiment K 12: The method of Embodiment K 11 or K12, wherein the oligopeptide is applied as a coating to the seed prior to planting.
[0076] Embodiment K13: The method of any one of Embodiments I-K6, wherein the oligopeptide is provided by application to the soil in which the plant is planted.
[0077] Embodiment K14: The method of any one of Embodiments I-K7, wherein the oligopeptide is provided by addition to water provided to the plant.
[0078] Embodiments K15: The method of any one of Embodiments I-K7, wherein the oligopeptide is provided following harvest to seeds, fruits, and/or plant parts.
[0079] Embodiments K16: The method of any one of Embodiments K10-K15, wherein the application is by a spreader, a power duster, a boom sprayer, a hand sprayers, a spray dusters, or a granular applicator. [0080] Embodiments KI 7: The method of any one of Embodiments I-K16, wherein the miPEP comprises a secretion tag, a tag that promotes entry into a plant cell, or a nuclear localization tag.
[0081] Embodiments B28: The method of Embodiment B21, wherein the tag that promotes entry into a plant cell comprises a cell penetrating peptide linked to the amino acid sequence.
[0082] Embodiments B29: The method of Embodiments B28, wherein the cell penetrating peptide comprises a protein transduction domain, an amphipathic peptide, a synthetic cationic polypeptide, optionally polylysine, polyhistidine, or polyarginine, a dendrimeric polycationic molecule, a peptide vascular endothelial-cadherin cell penetrating peptide, a fransportan cell penetrating peptide, a monomer or dimer of HIV- 1 TAT basic domain cell penetrating peptide, a penefratin cell penetrating peptide, a synthetic cationic homoarginine oligopeptide cell penetrating peptide, a gamma zein cell penetrating peptide, a Zea mays knottedl cell penetrating peptide, a Saccharomyces pombe TP10 cell penetrating peptide, a Candida albicans Zebra cell penetrating peptide, an Antennapedia sequence, a TAT sequence , an Antp-3A (Antp mutant) sequence, a Buforin II sequence, a K-FGF sequence, a Ku70 sequence, a prion sequence, a pVEC sequence, a SynBl sequence, a Pep-7 sequence, a HN-1 sequence, a BGSC (Bis- Guanidinium-Spermidine-Cholesterol) sequence, or a BGTC (Bis-Guanidinium-Tren-Cholesterol) sequence.
[0083] Embodiment LI: An isolated oligopeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 54, 58, 59, and 79 or the amino acid sequence set forth in one of SEQ ID NOs: 54, 58, 59, and 79 comprising one, two, three, or four amino acid substitutions, insertions, or deletions. [0084] Embodiment L2: The oligopeptide of Embodiment L, wherein the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.
[0085] Embodiment L3: The oligopeptide of Embodiment LI or L2, comprising the one, two, three, or four amino acid substitutions, insertions, or deletions.
[0086] Embodiment L4: The oligopeptide of any one of Embodiments L1-L3, wherein the oligopeptide comprises a proline at its N-terminus, an aspartic acid at the C-terminus, or both.
[0087] Embodiment L5: The oligopeptide of any one of Embodiments L1-L4, wherein the oligopeptide comprises one or more D-amino acids.
[0088] Embodiment L6: The oligopeptide of Embodiment L5, wherein the one or more D-amino acids enhance stability of the oligopeptide as compared to an oligopeptide of the same amino acid sequence comprising all L-amino acids.
[0089] Embodiment L7: The oligopeptide of any one of Embodiments L1-L6, wherein the oligopeptide comprises a secretion tag, a tag that promotes entry into a plant cell, or a nuclear localization tag.
[0090] Embodiment L8: The oligopeptide of Embodiment L7, wherein the tag that promotes entry into a plant cell comprises a cell penetrating peptide linked to the amino acid sequence.
[0091] Embodiment L9: The oligopeptide of Embodiment L8, wherein the cell penetrating peptide comprises a protein transduction domain, an amphipathic peptide, a synthetic cationic polypeptide, optionally polylysine, polyhistidine, or polyarginine, a dendrimeric polycationic molecule, a peptide vascular endothelial-cadherin cell penetrating peptide, a transportan cell penetrating peptide, a monomer or dimer of HIV- 1 TAT basic domain cell penetrating peptide, a penetratin cell penetrating peptide, a synthetic cationic homoarginine oligopeptide cell penetrating peptide, a gamma zein cell penetrating peptide, a Zea mays knottedl cell penetrating peptide, a Saccharomyces pombe TP10 cell penetrating peptide, a Candida albicans Zebra cell penetrating peptide, an Antennapedia sequence, a TAT sequence , an Antp-3A (Antp mutant) sequence, a Buforin II sequence, a K-FGF sequence, a Ku70 sequence, a prion sequence, a pVEC sequence, a SynBl sequence, a Pep-7 sequence, a HN-1 sequence, a BGSC (Bis- Guanidinium-Spermidine-Cholesterol) sequence, or a BGTC (Bis-Guanidinium-Tren-Cholesterol) sequence.
[0092] Embodiment Ml: A composition comprising the oligopeptide of any one of Embodiments L1-L9 with an agriculturally acceptable formulant that can include water, organic solvents, paraffinic oils, vegetable oils, dispersants, emulsifiers, wetting agents, buffering agents, hydrotrope agents, rheology modifiers, antifoam agents and defoamers, antifreeze agents, biocides, dyes, polymer walls, catalysts, thermosetting materials, cross-polymerizing agents, UV protectants, antioxidants, and chelating agents. [0093] Embodiment M2: A composition comprising the oligopeptide of any one of Embodiments L1-L9 75-83 with an agriculturally acceptable carrier, diluent, or excipient.
[0094] Embodiment M3: The composition of Embodiment M2, wherein the agriculturally acceptable carrier, diluent, or excipient comprises a buffer and the pH is in the range of from about 3.0 to about 9.0, or from about 4.5 to about 8.0.
[0095] Embodiment M4: The composition of any one of Embodiments Ml -M3, wherein the oligopeptide is in a concentration range of from about 0. 1 pg/ml to about 100 mg/ml, or from about 5 pg/ml to about 5 mg/ml.
[0096] Embodiment M5: The composition of any one of Embodiments M1-M4, wherein the composition is in the form of a finely-divided particulate solid, granules, pellets, wettable powders, dust, an aqueous suspension, a dispersion, a gel, or an emulsion.
[0097] Embodiment M6: The composition of any one of Embodiments M1-M5, further comprising one or more other active agents selected from the group consisting of a pesticide, a fertilizer, an insecticide, an attractant, a sterilizing agent, an acaricide, a nematocide, a herbicide, a biostimulant, a biological, and a growth regulator.
[0098] Embodiment N: A nucleic acid encoding the oligopeptide of any one of Embodiments L1-L9.
[0099] Embodiment 01: A nucleic acid construct comprising the nucleic acid of Embodiment N, operably linked to a promoter.
[0100] Embodiment 02: The nucleic acid construct of Embodiment 01, wherein the promoter is a heterologous promoter.
[0101] Embodiment 03: The nucleic acid construct of Embodiment 01 or Embodiment 02, wherein the promoter is a constitutive promoter, a tissue specific promoter, a development stage specific promoter, or an inducible promoter.
[0102] Embodiment 04: The nucleic acid construct of Embodiment 03, wherein the inducible promoter is induced by fungal infection, optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., phenylalanine ammonia lyase, chaicone synthase promoters), a gene that modifies plant cell walls (e.g., hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase promoters), a gene encoding an enzyme that degrade fungal cell walls (e.g., chitinase or glucanase promoters), or a gene encoding a thaumatin-like protein, or a maize Misl promotor or a flax Fisl promoter.
[0103] Embodiment P: A cell comprising the nucleic acid of Embodiment N or the nucleic acid construct of any one of Embodiments O1-O4. BRIEF DESCRIPTION OF THE DRAWINGS
[0104] FIG. 1 shows the experimental screening approach used to determine micropeptides (miPEPs) involved in plant defense. Peptides were applied as a foliar spray on three consecutive days prior to infection, followed by droplet infection with Botrytis cinerea spores incubated in peptide solution (Panel A). At the last day, leaves were harvested and pictures of isolated leaves were acquired (Panel B). Lesion sizes were measured at all infection points (encircled in Panel B), and were compared between control infected and peptide-treated plants.
[0105] FIG. 2 shows the measurements of Botrytis cinerea-inflicted lesions on Arabidopsis thaliana leaves after peptide application of miPEPs targeting members of respective microRNA (miR) families (miR169, miR168, miR396, and miR858). The black arrows indicate miPEPs that significantly decreased lesion size compared to control plants (black dotted line).
[0106] FIG. 3 shows the relative induction of d/pri-miR I69h and d/pri-miR 169c after respective miPEP treatment in A. thaliana.
[0107] FIG. 4 shows the relative induction of 4/pri-miR396b after miPEP treatment in A. benthamiana expressing 35.S'A/pri-miR396b.
[0108] FIG. 5 shows the measurements of Botrytis cinerea-inflicted lesions on strawberry leaves after peptide application of respective miPEPs. The black arrows indicate miPEPs that significantly decreased lesion size compared to control plants (black dotted line). Significance was determined as
* = p < 0.5, ** = p < 0. 1, and *** = p < 0.05, based on a Student’s t-test, with 9 leaves by condition, and 1250 spores by droplet with 100 p.M miPEP.
[0109] FIG. 6 depicts the strawberry fruit infection phenotypes after miPEP application.
[0110] FIG. 7 shows the measurements of Botrytis cinerea-infficted lesions on tomato plants after peptide application of respective miPEPs. The black arrows indicate miPEPs that significantly decreased lesion size compared to control plants (black dotted line). Significance was determined as
* = p < 0.5, ** = p < 0. 1, and *** = p < 0.05, based on a Student’s t-test, with 18 leaves by condition, and 1250 spores by droplet with 100 p.M miPEP.
[oni] FIG. 8 shows the phenotype of lesion development on tomato leaves after peptide application with respective miPEPs. The phenotypes 48 hours after Botrytis cinerea infection are shown.
[0112] FIG. 9 shows the relative fold induction of S/pri-miR169d after miPEP treatments in N. benthamiana expressing 35.8':.S7pri-miR 169d.
[0113] FIGS. 10A-10B show the results of convex hull analysis of tomato plants after Botrytis cinerea spray infection. Tomato plants were sprayed with isolated Botrytis spores and phenotyped every day for five consecutive days. FIG. 10A depicts the convex hull analysis. The convex hull was analyzed with an automated analysis software using the top RGB image. FIG. 10B shows the convex hull measurements of over time. Error bars represent the standard error (n = 8).
[0114] FIGS. 11A-11B show the results of plant fitness analysis of Botrytis czfterea-infected tomato plants after treatment with miPEPs. Tomato plants were sprayed with isolated Botrytis spores, and sprayed with either miPEPs or buffer control after 24 hours. FIG. 11 A shows the phenotype of plants treated with .S7miPEP I69k or buffer control. FIG. 11B shows disease control level in tomato plants treated with either miPEP or buffer control. The quantification of the convex hull in control and miPEP -treated tomato plants was used to calculate a disease control index. The average disease control was reported as a percentage, and three biological replicates (n = 21) forS/miPEP169k-treated and one replicate (n = 7) for S/miPEP169a-treated plants were used to calculate the disease control index. Error bars represent the standard error.
[0115] FIGS. 12A-12B show the disease control after peptide spray application on tomato plants inoculated with Alternaria solani (fungi) or Phytophthora infestans (oomycetes). Improved disease control against fungi and oomycetes was observed after application of S/miPEP169k. The experiments were performed using 4-week-old plants, with five replicates per condition, six inoculated leaves per plant, and a total of 30 plants analyzed per treatment groups (n = 30). FIG. 12A shows the disease control after S/miPEP 169k application on tomato plants inoculated with Alternaria solani. FIG. 12B shows the disease control after S/miPEP169k application on tomato plants inoculated with Phytophthora infestans.
[0116] FIGS. 13A-13B show the comparison of a .S7mi PEP 169k sequence variant. FIG. 13A shows the peptide purity of S7miPEP169k and the S7miPEP169kDlD10 variant. The peptide purity of S7miPEP169k and S7miPEP169kDlD10 was compared after incubation for 40 hours in a tomato wash-off solution. FIG. 13B shows the disease control (%) after application of .S7mi PEP 169k or the S7miPEP169kDlD10 variant at 30 g/ha 2 hours pre-infection of tomato plants with Phytophthora infestans. The graph represents five replicates per condition, six inoculated leaves per plant, and a total of 30 data points (n = 30).
[0117] FIGS. 14A-14B show the analysis of S7miPEP169k and S/miPEP169a sequence variants and other miPEPs encoded in pri-miR169k or pri-miR169a. Different peptides targeting pri-miR169k or pri- miR169a were tested for their biological activity against tomato grey mold (Botrytis cinerea). FIG. 14A shows the analysis of biological activity of S/miPEP169k sequence variants and other miPEPs encoded in pri-miR169k. FIG. 14B shows the analysis of biological activity of S/miPEP169a sequence variants and other miPEPs encoded in pri-miR169a. The disease control is based on convex hull recovery analysis, and 1-2 replicates were performed per condition, with seven plants analyzed per replicate (n = 7).
[0118] FIGS. 15A-15B show bar graph results of disease control by spraying MP19114 or MP19127 peptides (left bars in FIGS. 15A and 15B, respectively) or a reference antifungal peptide (right bars in FIGS. 15A and 15B) onto soybean plants infected with Asian Soybean Rust (ASR). In both FIGS. 15A and 15B, disease control is plotted along the vertical axis in percent of ASR disease control, percentage of disease control is listed within each bar plotted, and each of three replicate trials is plotted along the horizontal axis (“1st rep”, “2nd rep”, and “3rd rep”). FIG. 15A shows the plotted results of percentage of contaminated leaf area for plants sprayed with peptide MP 19114 (medium grey boxes, “MP19114”) compared to plants sprayed with reference peptide MP18279 (dark grey boxes, “Reference”, “*”). Error bars represent standard error, with 18 total measurements (2 leaves on each of 3 plants in each of 3 pots). FIG. 15B shows the plotted results for plants sprayed with peptide MP19127 (medium grey boxes, “MP19127”) compared to plants sprayed with reference peptide MP18279 (dark grey boxes, “Reference”, “*”).
[0119] FIG. 16 shows a schematic of amino acids comprising peptideMP19114. “N-ter” represents the N-terminus of the protein, and “C-ter” represents the C-terminus of the peptide. The two brackets under the schematic indicate the lengths of the peptide’s pair of hydrophobic blocks (“2 Hydrophobic blocks”). The “M” amino acid at the N-terminus end of the peptide represents amino acid methionine. Types of amino acid are categorized by polarity, hydrophobicity, and ionicity, wherein “aa” represents “amino acid”. Cationic amino acids are denoted by positive signs in the schematic, hydrophobic amino acids are denoted by medium grey circles labeled “h” in the schematic, polar amino acids are denoted by dark grey circles labeled “p” in the schematic, nonpolar amino acids are denoted by light grey circles labeled “np” in the schematic, and polar hydrophobic amino acids are denoted by striped circles in the schematic.
[0120] FIG. 17 shows amino acid schematics of two peptide mutation strategies for optimizing synthetic sequences, based on peptide MP 19127. Each row of black circles represents a different example of amino acids edited from peptide MP19127. The first nine amino acids of peptide MP19127 are represented in the topmost row (“MP 19127”), and directly below it is an example of all amino acids potentially causing cleavage changed (“All aa changed”). The locations of MP19127’s amino acids potentially causing cleavage of the peptide are displayed in boxes. On the bottom left panel are examples of sequences generated by mutating one potentially cleaving amino acid to a more acidic counterpart (“One aa changed by acid version”). On the bottom right panel are examples of sequences generated by mutating one potentially cleaving amino acid to a neutral alanine (“Alanine strategy”). Within each amino acid chain, “M” represents methionine, “G” represents glycine, “Q” represents glutamine, “E” represents glutamic acid, “N” represents asparagine, “D” represents aspartic acid, and “Ala” represents alanine. Various other amino acids are labeled by position in the amino acid sequence (“3”, “6”, “7”, and “8”).
DETAILED DESCRIPTION
[0121] Provided herein are methods of identifying and synthetizing immunity enhancing peptides, methods of increasing plant immunity, and methods of increasing resistance of a plant to a pathogen or pest. Also provided herein are immunity enhancing peptides, and nucleic acids encoding said peptides.
[0122] The methods and polypeptides of the disclosure are based, at least in part, on Applicant’s discovery of short micropeptides encoded by open reading frames contained in microRNA genes can be used to regulate plant immunity and confer resistance to common plant fungal pathogens. In particular, Applicants found that truncated versions of the micropeptides (having around 10 amino acids in length) had the same activity as the full peptide encoded in the microRNA gene, and were sufficient to activate expression of their target miRNA upon external application. Moreover, Applicants identified and synthetized micropeptides targeting members of different miRNA families. Applicant surprisingly discovered that micropeptides targeting the miR169 and miR396 can confer broad protection against fungal pathogens across various plant species. Application of micropeptides targeting miR169 or miR396 led to modulation of Botrytis infection, with some micropeptides producing increased resistance to infection with this fungal pathogen in four different plant species (Arabidopsis thaliana, Phaseolus vulgaris, Fragaria vesca, Solarium lycopersicum). Micropeptides targeting the same miRNA families also led to increased resistance to other common fungal pathogens, Pseudomonas and Alternaria, indicating a role for these miRNAs in immunity and resistance to fungal pathogens in plants.
[0123] As used herein, an “amino acid” or “amino acid residue” refers to any naturally occurring amino acid, any non-naturally occurring amino acid, any modified amino acid, including derivatized amino acids, or any amino acid mimetic known in the art. In some embodiments, the amino acids are D-amino acids. In some embodiments, the amino acids are L-amino acids. The amino acid may be referred by both their common three letter abbreviation and single letter abbreviation.
[0124] As used herein, “microRNA”, “non-coding microRNA” and “miRNA” are equivalent and may be used interchangeably to refer to small molecules of RNA of about 21 nucleotides, which regulate certain genes via post-transcriptional mechanisms, for example by means of the RISC complex. The primary transcript of a microRNA or “pri-miRNA” corresponds to the RNA molecule obtained directly 1 from transcription of the miRNA gene. Generally, this primary transcript undergoes one or more post- transcriptional modifications, involving for example a particular structure of the RNA or cleavage of certain portions of the RNA by splicing phenomena, and which lead to the precursor form of the microRNA or “pre-miRNA”, then to the mature form of the microRNA or “miRNA”. In some embodiments, the pri-miRNA contains an open reading frame in addition to the miRNA sequence.
[0125] As used herein, “micropeptides” and “miPEPs” (microRNA encoded PEPtides) are equivalent and may be used interchangeably to refer to a peptide that is encoded by an open reading frame present in the primary transcript of a microRNA, and which is capable of modulating the accumulation of said microRNA. In some embodiments, the miPEPs are 4 to 50 or 6-30 amino acids in length.
[0126] As used herein, “open reading frame” or “ORF” are equivalent and may be used interchangeably to refer to a nucleotide sequence in a DNA or RNA molecule that may potentially encode a peptide or a protein. The ORF may be identified by having a plant recognized start codon (the start codon generally encoding a methionine), followed by a series of codons (each codon encoding an amino acid), and ends with a stop codon (the stop codon not being translated). In some embodiments, the ORF is present in the primary transcript of a miRNA. In some embodiments, the ORF is 12 to 303 nucleotides in length. In some embodiments, the ORF encodes a peptide of 3 to 100 amino acids in length. As used herein, the term “peptide” refers to any peptide structure comprising or consisting of two or more amino acids, including chemical modifications and derivatives of amino acids. In some embodiments, the peptide is a short peptide (e.g., 4 to 50 amino acids in length). In some embodiments, the ORF is located in a primary transcription of an miRNA.
[0127] As used herein, the term “purified” molecule refers to biological or synthetic molecules that are removed from their natural environment and are isolated or separated and are free from other components with which they are naturally associated.
[0128] The term "isolated" in reference to a molecule including a nucleic acid, construct, vector, etc., may refer to a molecule that is not found in nature and/or is present in a context in which it is not found in nature. The term "isolated" may also refer to a molecule that has undergone at least one step towards being isolated or concentrated or enriched from a more complex solution or source. The term "isolated," however, is in no way intended to limit the molecule to a particular location or state. For example, an isolated nucleic acid molecule includes the nucleic acid molecule introduced into the genome of a cell in a position where it is not found in nature or when it is resident in progeny of cells into which the nucleic acid molecule has been introduced into its genome in a position where it is not found in nature.
[0129] The term “sequence identity” refers to the degree of similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, and otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
[0130] As used herein, “immunity enhancing peptide”” refer to an amino acid sequence capable of inducing an increased immune response in living cells and/or organisms that are exposed to said amino acid sequence. In some embodiments, the immunity enhancing peptide increases immunity against one or more pathogens or pests. In some embodiments, the immunity enhancing peptide is a plant immunity enhancing peptide.
I. Methods of Identifying and Synthetizing Immunity Enhancing Peptides
[0131] Some aspects of the present disclosure provide for methods of identifying and synthetizing an immunity enhancing micropeptide (miPEP) or a nucleic acid encoding the miPEP. Without being limited by theory, miPEPs are thought to be transcriptional regulators, in particular transcriptional activators that can operate at the transcription level to modulate the accumulation of a pri-miR or downstream miRNA. [0132] In some embodiments, the method for identifying and synthesizing (i) an immunity enhancing micropeptide (miPEP) encoded by a nucleotide sequence contained in the sequence of the primary transcript of a microRNA (miRNA), or (ii) a nucleic acid sequence encoding the miPEP that does not comprise the mature miRNA, comprises: a) selecting an miRNA from a miR169 family or a miR396 family; b) identifying an open reading frame (ORF) from 12 to 303 nucleotides in length contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; exogenously adding to the first plant (i) a peptide encoded by the ORF or (ii) a nucleic acid sequence encoding the peptide; comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing miRNA and the peptide is the miPEP; and synthesizing the miPEP or the nucleic acid sequence encoding the miPEP. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37. microRNAs
[0133] In some embodiments, the method comprises selecting a miRNA. In some embodiments, the miRNA is a plant miRNA. In some embodiments, the miRNA is a miRNA from a miR169 or miR396. MicroRNA families can consist of different number of members, with different plant species having a different number of members of these families. Each microRNA family defined by the mature microRNA produced from miRNA genes in these families, which has a high degree of homology amongst members of a particular family. The mature miRNA sequence dictates the subset of genes regulated by a particular family. For example, the miR169 family is known to target HAPLESS2 (HAP2) genes, while the miR396 family targets growth regulating factors (GRFs) genes in plant. The secondary mRNA structure of each member of a particular family can vary.
[0134] In some embodiments, the miRNA (such as miR169 or miR396) controls genes involved in plant immunity or resistance to plant pathogens or pests. In some embodiments, the miR169 or miR396 genes contain an ORF encoding a micropeptide. In some embodiments, the miRNA is a miR169 or miR396 from a dicot or monocot plant. In some embodiments, the miR169 or miR396 from are from an Arabidopsis spp., Phaseolus spp., Fragaria spp., or Solarium spp.. In some embodiments, the miR169 or miR396 from are from Arabidopsis thaliana, Phaseolus vulgaris, Fragaria vesca, or Solarium lycopersicum. In some embodiments, the miR169 or miR396 genes contain an ORF encoding a micropeptide.
Identification of ORFs
[0135] In some embodiments, the method comprises identifying an ORF contained in the primary transcript sequence of the miRNA. In some embodiments, the ORF has a size of 12 to 303 nucleotides. In some embodiments, the ORF has a size of 12, 15, 18, 21, 24, 27 , 30, 33, 36, 39, 42, 45, 47, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300 or 303 nucleotides in length. In some embodiments, the ORF has a size of 15 to 153 nucleotides. In some embodiments, the ORF has a size of 21 to 93 nucleotides. In some embodiments, the ORF encodes a peptide of 3 to 100 amino acids in length. In some embodiments, the ORF encodes a peptide of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 20 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 amino acids in length. In some embodiments, the ORF encodes a miPEP of 4 to 50 amino acids in length. In some embodiments, the ORF encodes a miPEP of 6 to 30 amino acids in length. In some embodiments, the ORF is contained in a pri-miR169 or pri-miR396. In some embodiments, the ORF encodes a miPEP that regulates a miR169 or a miR396. In some embodiments, the ORF encodes a miPEP having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
[0136] ORFs contained in miRNA genes may be identified by any method known to those in the art, including, without limitation, bioinformatics, RNA sequencing, DNA sequencing, or mass spectrometry- based approaches. For example, protein extract from cells may be analyzed through mass spectrometry to identify novel short peptides and then identify nucleic acid sequences corresponding to a sequence encoding the peptide. Methods that detect translation of peptides encoded by ORFs contained in noncoding RNAs, such as ribosome profiling, may also be used to identify ORFs.
[0137] In some embodiments, identifying an ORF contained in the primary transcript sequence of the miRNA comprises analysis of genomic databases and/or genomic sequences. In some embodiments, the genomic database is analyzed to identify genes or transcript sequences comprising a start codon, at least three codons coding an amino acid, and a stop codon. The gene or transcript may also be analyzed to further identify sequences that regulate translation. The analysis may further comprise comparing the identified ORF to a known ORF or amino acid sequence encoded therein.
[0138] In some embodiments, identifying an ORF contained in the primary transcript sequence of the miRNA comprises searching for ORFs on a genomic database. In some embodiments, the genomic database is a plant genomic database. In some embodiments, identifying an ORF contained in the primary transcript sequence of the miRNA comprises searching for ORFs on a pri-miR database.
Plant Cells
[0139] In some embodiments, the method comprises providing a first plant cell and second plant cell that both express the miRNA. In some embodiments, the first and second plant cells are from the same type of plant. In some embodiments, the first and second plant cell express a miR169 or miR396. In some embodiments, the first and second plant cell express a miPEP having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
[0140] In some embodiment, the first and second plant cell are from a dicot or monocot plant. In some embodiments, the first and second plant are from a crop plant. In some embodiments, the first and second cell are from a row crop plant, a fruit-producing plant, a free, a vine, a vegetable, or an ornamental plants (e.g. ornamental flowers, trees, shrubs, groundcovers, and turf grasses). In some embodiments, the first and second plant cell are from an Arabidopsis spp., Phaseolus spp., Fragaria spp., or Solanum spp. In some embodiments, the first and second plant cell are from Arabidopsis thaliana, Phaseolus vulgaris, Fragaria vesca, or Solanum lycopersicum. Other Exemplary types of types of plants include, without limitation, Medicago sativa, Prunus dulcis, Malus domestica, Prunus spp., Asparagus officinalis, Arabidopsis spp., Musa spp., Hordeum vulgare, Phaseolus spp., Vaccinium spp., Theobroma cacao, Brassica spp.,, Dianthus caryophyllus, Daucus carota sativus, Manihot esculentum, Prunus avium, Cicer arietinum, Cichorium intybus, Capsicum spp., Chrysanthemum spp., Cocos nucifera, Coffea spp., Gossypium hirsutum L., Vigna spp., Vicia faba, Cucumis sativus, Ribes spp., Phoenix dactylifera, Solanum spp., Eucalyptus spp., Linum usitatissumum L), Pelargonium spp., Vitus spp., Psidium guajava, Humulus lupulus, Cannabis sativa and Cannabis spp., Iris spp), Lactuca sativa, Citrus spp., Zea mays L, Mangifera indie), Garcinia mangostan), melon Cucumis mel), (Setaria spp., Echinochloa spp., Eleusine spp., Panicum spp., Pennisetum spp), Avena sativ), Ellis quineensis, Olea europaea, Allium spp., Carica papaya, Prunus persic), Pyrus spp , Pisa sativum, Arachis hypogaea, Paeonia spp, Petunia spp., Ananas comosus, Musa spp., Prunus domestica, (Euphorbia pulcherrima, Populus spp), , Cucurbita spp., Oryza sativa L, Rosa spp., Hevea brasiliensis, Secale cereale, Carthamus tinctorius L, Sesame indium, Sorghum bicolor, Glycine max L., Fragaria spp., Beta vulgari), Saccharum spp., Helianthus annuus, Ipomoea batatas, Camellia sinensis, Nicotiana tabacum L., Lycopersicon esculentum, Tulipa spp., Juglans spp. L., Citrulus lanatus, Triticum aestivum, and Discorea spp. In some embodiments, the plant is a wild plant variety. In some embodiments, the plant is a domesticated plant variety. In some embodiments, the plant is a hybrid plant variety. In some embodiments, the plant is a genetically modified plant and/or a gene- edited plant.
Delivery of Peptides or Nucleic Acids to Plants
[0141] In some embodiments, the method comprises exogenously adding to the first plant (i) a peptide encoded by the ORF or (ii) a nucleic acid sequence encoding the peptide.
[0142] In some embodiments, the method comprises exogenously adding to the first plant a peptide encoded by the ORF. The peptide may be added by any method known in the art. For instance, the peptide may be added by foliar spray, foliar drench, drip irrigation, coating, mixing, pouring, dusting, atomizing, soil irrigation, fumigation, soil injection, seepage irrigation, sprinklers or manual irrigation, or a combination thereof. In some embodiments, exogenously adding to the first plant a peptide encoded by the ORF comprises applying the peptide by foliar spray. In some embodiments, exogenously adding to the first plant a peptide encoded by the ORF comprises adding a cell that expresses the peptide encoded by the ORF. In some embodiments, the peptide encoded by the ORF is a miPEP. In some embodiments, the miPEP that regulates a miR169 or a miR396. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
[0143] In some embodiments, the miPEP is provided as a composition. In some embodiments, the composition is in the form of a finely-divided particulate solid, granules, pellets, wettable powders, dust, an aqueous suspension, a dispersion, a gel, or an emulsion. In some embodiments, the composition comprises a miPEP at a concentration range of from about 0. 1 pg/ml to about 100 mg/ml, or from about 5 pg/ml to about 5 mg/ml. In some embodiments, the composition comprises a miPEP at a concentration of about 0.1 pg/ml, 0.5 pg/ml, 1 pg/ml, 5 pg/ml, 10 pg/ml, 20 pg/ml, 30 pg/ml, 40 pg/ml, 50 pg/ml, 60 pg/ml, 70 pg/ml, 80 pg/ml, 90 pg/ml, 1 mg/ml, 5 mg/ml, 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, or 100 mg/ml. In some embodiments, the miPEP regulates a miR169 or a miR396. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
[0144] In some embodiments, the miPEP is provided by application to a plant or part thereof. In some embodiments, the plant part is a leaf, a bud, a root, a shoot, a floral part, or a seed. In some embodiments, the miPEP is provided to a plant or part thereof after harvest. In some embodiments, the miPEP is provided to a plant or part thereof prior to harvest. In some embodiments, the miPEP is provided to a plant by application to the soil in which the plant is planted. In some embodiments, the miPEP is provided to a plant by addition of water provided to the plant. In some embodiments, the miPEP is applied to a plant by a spreader, a power duster, a boom sprayer, a hand sprayers, a spray dusters, or a granular applicator. In some embodiments, the miPEP regulates a miR169 or a miR396. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
[0145] In some embodiments, the method comprises exogenously adding to the first plant a nucleic acid sequence encoding the peptide encoded by the ORF. In some embodiments, nucleic acids encoding the peptide are added to a plant by transformation. In some embodiment, transformation comprises Agrobacterium-mediated transformation, micro-projectile-mediated transformation, sonication, electroporation, or liposome- or spheroplast-mediated vector delivery. In some embodiments, the nucleic acids encoding the peptide are added to a plant by genome editing. Gene editing may be performed by any method known in the art, including, but not limited to using zinc fmger-nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), oligonucleotide-directed mutagenesis (ODM), a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, or by gene writing (see, e.g., PCT Patent Application Publication W02020/047124).In some embodiments, nucleic acids encoding the peptide are added to a plant by crossing a first plant comprising the nucleic acids encoding the peptide to a second plant.
Plant Immunity
[0146] In some embodiments, the method comprises comparing immunity in the first plant cell and the second plant cell after exogenously adding to the first plant a peptide encoded by the ORF, or a nucleic acid sequence encoding the peptide, wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing miRNA and the peptide is the miPEP. In some embodiments, the miPEP regulates a miR169 or a miR396. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
[0147] Any suitable method of determining plant immunity known in the art may be used. For example, plant immunity may be measure as a reduction in pest number on the plant when compared to an untreated plant, or as a reduction in physical damage to the plant when compared to an untreated plant. Physical damage includes feeding damage and boring damage, and may manifest in a variety of plant phenotypes, including, but not limited to, chewed or ragged leaves, missing leaves, tunnels in leaves, holes in stems, leaf distortion, leaf discoloration, leaf spotting, wilting, stunted growth, girdled or dead stems, yellowing, breakage damage, or root damage. Convex hull analysis may be used to examine the physical damage to an infected plant. In some embodiments, plant immunity is determined by convex hull analysis.
[0148] In some embodiments, plant immunity is enhanced when an increase in disease control is detected. The term "disease control," as used herein, means killing, reducing in numbers, and/or reducing growth, feeding or normal physiological development of any or all life stages of a plant pathogen or pest, and/or reduction of the effects of a plant pest infection and/or infestation. In some embodiments, an increase of disease control of 10% to 90% as compared to an untreated infected plant indicates enhanced immunity. In some embodiments, disease control In some embodiments, an increase of disease control of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% as compared to an untreated infected plant indicates enhanced immunity. In some embodiments, a 2-to 20-fold increase in disease control of as compared to an untreated infected plant indicates enhanced immunity. In some embodiments, a 2-folf, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, or 20-fold increase in disease control as compared to an untreated infected plant indicates enhanced immunity.
Synthesis
[0149] In some embodiments, the method comprises synthetizing a miPEP, or a nucleic acid encoding the miPEP. In some embodiments, the miPEP regulates a miR169 or a miR396. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
[0150] In some embodiments, the method comprises synthetizing a miPEP, or a nucleic acid encoding the miPEP by chemical synthesis. Various approaches been developed for chemical synthesis of peptides, such as for example, by solid-phase methods. Solid phase peptide synthesis is a method for chemically synthesizing peptides on a solid support. In solid phase peptide synthesis, amino acids or peptides are typically attached to a solid support via the C-terminus. The new amino acid is added to the bound amino acid or peptide by a coupling reaction. Translation-based approaches for peptide synthesis have also been developed, whereby a peptide is produced from an encoding transcript by in vitro translation. Synthetic nucleic acids (DNA, RNA or their analogues) may be prepared using column-based synthesizers, or produced from an existing nucleic acid by PCR or in vitro transcription. Other methods of nucleic synthesis are described for example in U.S. Pat. No. 6,586,211 Bl, in PCT/EP2004/013131, in WO 00/13017 A2, in S. Rayner et al., PCR Methods and Applications 8 (7), 741-747, 1998, in WO 90/00626 Al, in EP 385 410 A2, in WO 94/12632 Al, in WO 95/17413 Al, in EP 316 018 A2, in EP 022 242 A2, in L. E. Sindelar and J. M. Jaklevic, Nucl. Acids Res. 23 (6), 982-987, 1995, in D. A. Lashkari, Proc. Nat. Acad. Sci. USA 92 (17), 7912-7915, 1995, and in WO 99/14318 Al.
[0151] In some embodiments, the method comprises synthetizing a miPEP, or a nucleic acid encoding the miPEP by biological synthesis. Any suitable method of biological synthesis of peptides or nucleic acids may be used. Biological synthesis may comprise expressing a peptide in a cell and purifying the peptide from the cell. Nucleic acids may introduced into a cell, such as a bacteria, and allowed to be replicated before isolating them.
[0152] In some embodiments, synthetizing a miPEP comprises expressing a fusion polypeptide comprising a polypeptide that forms inclusion bodies in a cell operably linked to two or more miPEPs. In some embodiments, synthetizing a miPEP comprises expressing a fusion polypeptide comprising an carrier polypeptide operably linked to two or more miPEPs by a peptide bond. In some embodiments, synthetizing a miPEP comprises expressing a fusion polypeptide comprising an carrier polypeptide operably linked to two or more miPEPs by a peptide bond, and releasing the two or more miPEPs from the carrier polypeptide. In some embodiments, synthetizing a miPEP comprises expressing a fusion polypeptide comprising an carrier polypeptide operably linked to two or more miPEPs by a peptide bond, and releasing the two or more miPEPs from the carrier polypeptide. In some embodiments, the two or more miPEPs in the fusion polypeptide are released from the carrier polypeptide and/or from each other by sequence-specific chemical cleavage of the peptide bond. In some embodiments, the peptide bond is an Asp-Pro bond and the sequence-specific cleavage is performed using acetic acid. In some embodiments, releasing the two or more miPEPs from the carrier polypeptide is performed in the absence of a chaotropic agent. In some embodiments, releasing the two or more miPEPs from the carrier polypeptide is performed without a column-based purification step. In some embodiments, the miPEP is 4-50 amino acids long. In some embodiments, the miPEP comprises an N-terminal proline and a C-terminal aspartic acid. In some embodiments, the miPEP regulates a miR169 or miR396. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
II. Methods of Increasing Plant Immunity or of Increasing Resistance to a Pathogen
[0153] Also provided herein are methods of increasing plant immunity, and methods of increasing resistance to a fungal pathogen.
Peptide Application [0154] In some embodiments, the method comprises providing to the plant a miPEP that regulates an immunity enhancing miR169 or an immunity enhancing miR396. The miPEP may be provided to a plant by any suitable method known in the art. In some embodiments, the miPEP is provided to a plant by foliar spray, foliar drench, drip irrigation, coating, mixing, pouring, dusting, atomizing, soil irrigation, fiimigation, soil injection, seepage irrigation, sprinklers or manual irrigation. In some embodiments, the miPEP is applied to a plant by a spreader, a power duster, a boom sprayer, a hand sprayers, a spray dusters, or a granular applicator. In some embodiments, the miPEP is provided to a plant by foliar spray. In some embodiments, the miPEP is provided to the plant at a concentration of 2 g/ha to 200 g/ha. In some embodiments, the miPEP is provided to the plant at a concentration of 2 g/ha, 5 g/ha, 10 g/ha , 15 g/ha, 20 g/ha, 25 g/ha, 30 g/ha, 35 g/ha, 40 g/ha, 45 g/ha, 50 g/ha, 55 g/ha, 60 g/ha, 65 g/ha, 70 g/ha, 75 g/ha, 80 g/ha, 85 g/ha, 90 g/ha, 95 g/ha, 100 g/ha, 110 g/ha, 120 g/ha, 130 g/ha, 140 g/ha, 150 g/ha, 160 g/ha, 170 g/ha, 180 g/ha, 190 g/ha, or 200 g/ha. In some embodiments, the miPEP is provided to the plant by foliar spray at a concentration of 100 g/ha. In some embodiments, the miPEP is provided to one or more of a leaf, a bud, a root, a shoot, a floral part, or a seed. In some embodiments, the miPEP is provided to a plant or part thereof after harvest. In some embodiments, the miPEP is provided to a plant or part thereof prior to harvest. In some embodiments, the miPEP is provided to the plant prior, during, or after an infection with a pathogen or pest. In some embodiments, the miPEP is provided to a plant by application to the soil in which the plant is planted. In some embodiments, the miPEP is provided to a plant by addition of water provided to the plant. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
[0155] In some embodiments, the method comprises preparing a composition comprising the miPEP. In some embodiments, the composition is an agriculturally acceptable composition. In some embodiments, the composition comprises a miPEP and an agriculturally acceptable formulant. In some embodiments, the agriculturally acceptable formulant comprises one or more of a water, organic solvents, paraffinic oils, vegetable oils, dispersants, emuiers, wetting agents, buffering agents, hydrotrope agents, rheology modifiers, antifoam agents and defoamers, antifreeze agents, biocides, dyes, polymer walls, catalysts, thermosetting materials, cross-polymerizing agents, UV protectants, antioxidants, and chelating agents. In some embodiments, the composition comprises a miPEP at a concentration range of from about 0. 1 pg/ml to about 100 mg/ml, or from about 5 pg/ml to about 5 mg/ml. In some embodiments, the composition fiirther comprises one or more other active agents selected from the group consisting of a pesticide, a fertilizer, an insecticide, an attractant, a sterilizing agent, an acaricide, a nematocide, a herbicide, a biostimulant, a biological, and a growth regulator. In some embodiments, the composition is formulated as a liquid, a gel, an emulsion, a suspension, an encapsulation, a solid, a powder, an aerosol, a paste, a coating, a spray, a soil drench, a microcapsule, an emulsifiable concentrate, or as granules. In some embodiments, the composition is in the form of a finely-divided particulate solid, granules, pellets, wettable powders, dust, an aqueous suspension, a dispersion, a gel, or an emulsion. In some embodiments, the agriculturally acceptable composition is formulated as a seed treatment, a foliar spray, a foliar drench, a Ready-To-Use (RTU) formulation, a produce coating, a suspension concentrate, a tank-mix, an aerosol, a root dip, a soil treatment, a dipping formulation, an irrigation formulation, or a sprinkler formulation. In some embodiments, the agriculturally acceptable composition comprising a miPEP is a foliar spray composition. In some embodiments, the composition comprises a miPEP that regulates a miR169 or a miR396. In some embodiments, the composition comprises a miPEP having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
[0156] In some embodiments, the method comprises preparing composition comprising a miPEP and an agriculturally acceptable carrier, diluent or excipient. In some embodiments, the composition comprises a miPEP and an agriculturally acceptable carrier, diluent or excipient. In some embodiments, the agriculturally acceptable carrier, diluent, or excipient comprises a buffer and the pH is in the range of from about 3.0 to about 9.0 or about 4.5 to about 8.0. In some embodiments, the agriculturally acceptable carrier comprises a solid carrier, a liquid carrier, a gel carrier, a suspension, or an emulsion. In some embodiments, the agriculturally acceptable carrier comprises an adjuvant, an inert component, a dispersant, a surfactant, a humectant, an emulsifier, a thickener, a wetting agent, a fertilizer, a mineral, a solvent, a tackifier, a binder, or a stabilizer. In some embodiments, the agriculturally acceptable composition comprising a miPEP and a surfactant or humectant.
[0157] In some embodiments, the method comprises providing a composition comprising an miPEP to a plant or part thereof. In some embodiments, the plant part is a leaf, a bud, a root, a shoot, a floral part, or a seed. In some embodiments, the composition is provided to a plant or part thereof after harvest. In some embodiments, the miPEP is provided to a plant or part thereof prior to harvest. In some embodiments, the miPEP is provided to the plant prior, during, or after an infection with a pathogen or pest. In some embodiments, the miPEP is provided to a plant by application to the soil in which the plant is planted. In some embodiments, the miPEP is provided to a plant by addition of water provided to the plant. In some embodiments, the composition comprises a miPEP that regulates a miR169 or a miR396. In some embodiments, the composition comprises a miPEP having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
Plant Immunity
[0158] In some embodiments, the method comprises increasing plant immunity. In some embodiments, increasing plant immunity comprises inducing an increase in disease control. In some embodiments, increasing plant immunity comprises inducing an increase in of 10% to 90% as compared to an untreated infected plant indicates enhanced immunity. In some embodiments, disease control. In some embodiments, increasing plant immunity comprises inducing an increase in at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% as compared to an untreated infected plant indicates enhanced immunity. In some embodiments, increasing plant immunity comprises inducing an increase in 2-to 20-fold increase in disease control as compared to an untreated infected plant indicates enhanced immunity. In some embodiments, increasing plant immunity comprises inducing an increase in a 2-folf, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, or 20-fold increase in disease control as compared to an untreated infected plant indicates enhanced immunity.
Plant Pathogens and Pests
[0159] Also provided herein are methods of increasing resistance to a plant pathogen or pest in a plant. In some embodiments, the plant pathogen or pest is a fungus, bacteria, virus, or an eukaryote.
[0160] In some embodiments, the plant pathogen or pest is a fungus. Examples of plant pathogenic fungi include, without limitation, Cercospora spp., Mycosphaerellasp ., Glomerella spp., Cladosporium spp., Fusarium oxysporum, Fusarium graminearum, Fusarium monilforme, Cochliobolus sativus, Collectotrichum graminicola, Stagonospora nodorum, Stagonospora avenae, Stenocarpella maydis, Sclerotinia minor, Sclerotinia sclerotiorum, Sclerotinia sp., Alternaria spp., Phytophthora spp., Botrytis spp., Pyrenophora tritici-repentis, and Phytophthora parasitica. Other plant fungal pathogens or pests for which resistance may be provided include, Collelolrichum graminicola, Diplodia maydis, Fusarium graminearum, and Fusarium verticillioides. Specific pathogens for major crops include: Soybeans: Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichum dematium (Colletotichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v. glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Glomerella glycines, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Fusarium solani; Canola: Albugo Candida, Altenaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassicicola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter michiganese subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium oxysporum, Verticillium albo-atrum, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulina briosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium botryosum, Leptotrichila medicaginis; Whca/: Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v. syringae, Alternaria altemata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis fsp. tritici, Puccinia graminis fsp. tritici, Puccinia recondite fsp. tritici, Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae, P seudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola, Pythium aphanidermatum, Sunflower: Plasmopora halstedii, Sclerotinia sclerotiorum, Septoria helianthi, Cephalosporium maydis, Cephalosporium acremonium, Sorghum: Exserohilum turcicum, C. sublineolum, Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium verticillioides, Alternaria altemata, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas civenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum, Pythium arrhenomanes, Pythium graminicola, etc.
[0161] In some embodiments, the plant pathogen or pest is a bacteria. Examples of plant pathogenic bacteria include those belonging to Pseudomonas spp., Pantoua spp., and Erwinia spp. Additional plant pathogenic bacteria are described in Robert W. Jackson, Plant Pathogenic Bacteria: Genomics and Molecular Biology, published by Horizon Scientific Press, 2009, ISBN 1904455379, 9781904455370; Samuel S. Gnanamanickam, Plant-Associated Bacteria, published by Springer, 2007, ISBN 1402045379, 9781402045370; Martin Dworkin et al., The Prokaryotes: a handbook on the biology of bacteria, Published by Springer, 2006, ISBN 0387254927, 9780387254920; George N. Agrios, Plant pathology, published by Academic Press, 2005, ISBN 0120445654, 9780120445653; and David W. Parry, Plant pathology in agriculture, published by CUP Archive, 1990, ISBN 0521368901, 9780521368902.
[0162] In some embodiments, the plant pathogen or pest is a virus. Examples of viruses that may cause infections in plants include, without limitation, cucumber mosaic, tobacco mosaic, and barley yellow dwarf virus, alfalfa mosaic virus (Alfamovirus), Apple chlorotic leaf spot virus (Tricho virus), Apple scar skin viroid (Viroids), Arabis mosaic virus (Nepovirus), Barley mild mosaic virus (Bymovirus), Barley stripe mosaic virus (Hordeivirus), Barley yellow mosaic virus (Bymovirus), Bean common mosaic virus (Potyvirus), Bean yellow mosaic virus (Potyvirus), Beet necrotic yellow vein virus (Furovirus), Blackeye cowpea mosaic virus (Potyvirus), Bean common mosaic virus (Potyvirus), Broad bean wilt virus (Fabavirus), Butterbur mosaic virus (Carlavirus), Carnation mottle virus (Carmovirus), Carnation vein mottle virus (Potyvirus), Cauliflower mosaic virus (Caulimovirus), Chrysanthemum mild mottle virus (Cucumovirus), Tomato aspermy virus (Cucumovirus), Chrysanthemum stunt viroid (Viroids), Citrus mosaic virus, Citrus tristeza virus (Closterovirus), Clover yellow vein virus (Potyvirus), Cocksfoot mottle virus (Sobemovirus), Cucumber green mottle mosaic virus (Tobamo virus), Cucumber mosaic virus (Cucumovirus), Cycas necrotic stunt virus (Nepovirus), Dasheen mosaic virus (Potyvirus), Grapevine Algerian latent virus (Tombusvirus), Konjac mosaic virus (Potyvirus), Melon necrotic spot virus (Carmovirus), Mulberry ringspot virus (Nepovirus), Narcissus mosaic virus (Potexvirus), Plant viruses are viruses affecting plants. Examples of viruses that may cause Odontoglossum ringspot virus (Tobamovirus), Papaya ringspot virus (Potyvirus), Peach latent mosaic viroid, Peanut mottle virus (Potyvirus), Peanut stripe virus (Potyvirus), Bean common mosaic virus (Potyvirus), Peanut stunt virus (Cucumovirus), Potato virus A (Potyvirus), Potato virus M (Carlavirus), Potato virus S (Carlavirus), Potato virus X (Potexvirus), Potato virus Y (Potyvirus), Prune dwarf virus (liarvirus), Primus necrotic ringspot virus (Ilarvirus), Radish mosaic virus (Comovirus), Rice black streaked dwarf virus (Fijivirus), Rice dwarf virus (Reovirus), Rice grassy stunt virus (Tenuivirus), Rice stripe virus (Tenuivirus), Rice tungro spherical virus (Sequivirus), Rice waika virus, Rice tungro spherical virus (Sequivirus), Ryegrass mottle virus, Satsuma dwarf virus (Nepovirus), Soil -borne wheat mosaic virus (Furovirus), Southern bean mosaic virus (Sobemovirus), Soybean mosaic virus (Potyvirus), Soybean stunt virus (Cucumovirus), Cucumber mosaic virus (Cucumovirus), Tobacco mosaic virus (Tobamovirus), Tobacco mosaic virus (Tobamovirus), Tomato mosaic virus (Tobamovirus), Tobacco necrosis virus (Necrovirus), Tobacco rattle virus (Tobravirus), Tobacco ringspot virus (Nepovirus), Tomato aspermy virus (Cucumovirus), Tomato black ring virus (Nepovirus), Tomato mosaic virus (Tobamovirus), Tomato ringspot virus (Nepovirus), Tomato spotted wilt virus (Tospovirus), Turnip mosaic virus (Potyvirus), Watermelon mosaic virus 1 (Potyvirus), Papaya ringspot virus (Potyvirus), Watermelon mosaic virus 2 (Potyvirus), Wheat yellow mosaic virus (Bymovirus), Zucchini yellow mosaic virus (Potyvirus). More plant viruses have been described in F. C. Bawden, Plant Viruses and Virus Diseases, Publisher Biotech Books, 2002, ISBN 8176220647, 9788176220644.
[0163] In some embodiments, the plant pathogen or pest is an eukaryote. In some embodiments, the eukaryote is an insect or a nematode. Agricultural insect pests can be classified into: chewing insects, sucking insects, and soil insects. Common chewing insects are, for example, beet armyworm (Spodoptera exigua), diamondback moth ( lulella xylostella), com earworm (He I loth is zea. a.k.a. bollworm and tomato fruitworm), blister beetles (Epicauta and others), carrot weevils (Listronotus oregonensis, Hyperodes texana), cabbage looper (Trichopulsia m), grasshopper (several species), flea beetles (e.g., tobacco fleabeetle (Epitrix hirtipennis), eggplant fleabeetle (E. fuscula), potato fleabeetle (E. cucumeri) and other species), fall armyworm (Spodoptera frugiperda), Lesser cornstalk borer (Elasmopalpus lignosellus), Texas leafcutting ant (Atta texana), citrus leafminer (Phyllocnistis citrella), leafminers (Liiriomyza spp.), yellowstriped armyworm (Spodoptera ornithogalli). Common sucking insects are, for example, stink bugs (e.g. Nezara viridula and other species), sharpshooters (Homalodisca spp. and Oncopmetopia spp.), whiteflies (e.g. sliverleaf whitefly, greenhouse whitefly, sweetpotato whitefly (Bemisia tabaci)), greenhouse whitefly (Trialeuroides vaporariorum), psyllid (e.g. Asian citrus psyllid), squash bug (Anasa tristis), leaffooted bugs (Leptoglossus spp.), leafhoppers (e.g., bean leafhopper, Empoasca solana, aster leafhopper, Macrosteles fascifrons, western potato leafhopper, Empoasca abrupta, grape leafhopper, variegated leafhopper, beet leafhopper, Circulifer tenellus), aphids (Aphidoidea, e.g. green peach aphid, turnip aphid, melon aphid, potato aphid, rosy apple aphid, spirea aphid,). Common rasping insects include, but are not limited to, thrips (e.g. citrus thrips, western flower thrips (Frankliniella occidentalis), onion thrips (Thrips tabaci), melon thrips, chili thrips). Common soil insects are, for example, granulate cutworm (Feltia subterranea), mole crickets (e.g. northern mole cricket, Neocurtilla hexadactyla, southern moire cricket Scapteriscus acletus), com rootworm (e.g. Diabrotica undecimpunctata howardi), pillbugs and sowbugs (several species), sweetpotato weevil Cylas formicarius elegantulus), white grubs Pyllophaga spp.), wireworms (several species). Exemplary nematodes that act as plant pathogens or pest can be classified into parasitic nematodes such as root-knot nematodes, including Heterodera spp., Meloidogyne spp., and Globodera spp.; cyst nematodes, which include Heterodera spp. Meloidogyne spp. (cotton cyst nematode) and Globodera spp.; and lesion neamtodes, which include Pratylenchus spp.
[0164] In some embodiments, the pathogen is a pathogen of Arabidopsis thaliana, Phaseolus vulgaris, Fragaria vesca, or Solanum lycopersicum. In some embodiments, the plant pathogen or pest is a fungi. In some embodiments, the fungi is Botrytis spp., Phytophthora spp. or Alternaria spp. In some embodiments, the fungi is Botrytis cinerea, Phytophthora infestans or Alternaria solani.
Peptides
[0165] In some embodiments, the method comprises providing to a plant a miPEP. In some embodiments, the miPEP is selected from the group consisting of A/miPEP169c, A/miPEP169h, AtPEP396b, PvmiPEP169e, PvmiPEP169kI, PvmiPEP169pI, FvmiPEP169hI, FvmiPEP1691I, FvmiPEP369a, S7miPEP169a, S7miPEP169d, S7miPEP169k, S7miPEP396c, MP19043, MP19042, S7miPEP169kDlD10, MP19051, MP19048, and MP19049. In some embodiments, the miPEP regulates an immunity enhancing miRNA. In some embodiments, the immunity enhancing miRNA is a miR169 or a miR396.
[0166] In some embodiments, the method comprises providing to the a plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NOs: 1-13. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ IDNO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the miPEP further comprises an N-terminal proline and a C-terminal aspartate. In some embodiments, the miPEP regulates miR169 or miR396.
[0167] In some embodiments, the method comprises providing to a plant a miPEP comprising a proline at the N-terminus and an aspartate at the C-terminus. In some embodiments, the method comprises providing to a plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C- terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169 or miR396.
[0168] In some embodiments, the method comprises providing to miPEP to an Arabidopsis thaliana plant.
[0169] In some embodiment, the method comprises providing .4 /mi PEP 169c to an Arabidopsis thaliana plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 1 to an Arabidopsis thaliana plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 1. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to an Arabidopsis thaliana plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 14. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 14. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
[0170] In some embodiment, the method comprises providing 4/miPEP I 69h to an Arabidopsis thaliana plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 2 to an Arabidopsis thaliana plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 2. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to an Arabidopsis thaliana plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 15. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 15. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
[0171] In some embodiment, the method comprises providing 4/miPEP396b to an Arabidopsis thaliana plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 3 to an Arabidopsis thaliana plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 3. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to an Arabidopsis thaliana plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 16. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 16. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 16. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR396.
[0172] In some embodiments, the method comprises providing to miPEP to a Phaseolus vulgaris plant. [0173] In some embodiment, the method comprises providing PvmiPEP169e to a Phaseolus vulgaris plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 4 to a Phaseolus vulgaris plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 4. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 4. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to an Phaseolus vulgaris plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 17. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 17. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
[0174] In some embodiment, the method comprises providing PvmiPEP 169kl to a Phaseolus vulgaris plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 5 to a Phaseolus vulgaris plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 5. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 5. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to an Phaseolus vulgaris plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 18. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 18. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO18. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miRI69.
[0175] In some embodiment, the method comprises providing PvmiPEP 169pl to a Phaseolus vulgaris plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 6 to a Phaseolus vulgaris plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 6. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 6. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to a Phaseolus vulgaris plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 19. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 19. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 19. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miRI69.
[0176] In some embodiments, the method comprises providing to miPEP to a Fragaria vesca plant. [0177] In some embodiment, the method comprises providing FvmiPEP 169hl to a Fragaria vesca plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 7 to a Fragaria vesca plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 7. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C-terminus. In some embodiments, the method comprises providing to a Fragaria vesca plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 20. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 20. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 20. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
[0178] In some embodiment, the method comprises providing FvmiPEP 16911 to a Fragaria vesca plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 8 to a Fragaria vesca plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 8. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C-terminus. In some embodiments, the method comprises providing to a Fragaria vesca plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 21. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 21. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 21. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
[0179] In some embodiment, the method comprises providing FvmiPEP369a to a Fragaria vesca plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 9 to a Fragaria vesca plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 9. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C-terminus. In some embodiments, the method comprises providing to a Fragaria vesca plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 22. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 22. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 22. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR396.
[0180] In some embodiments, the method comprises providing to miPEP to a Solarium lycopersicum plant.
[0181] In some embodiment, the method comprises providing S7miPEP169a to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 10 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 10. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 10. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
[0182] In some embodiment, the method comprises providing S7miPEP169d to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 11 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 11. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 11. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth SEQ ID NO: 28. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 28. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 28. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
[0183] In some embodiment, the method comprises providing MP 19043 to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 26 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 26. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 26. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 24. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 24. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 24. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
[0184] In some embodiment, the method comprises providing MP 19042 to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 1 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 1. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 27. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 25. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 25. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 25. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
[0185] In some embodiment, the method comprises providing S7miPEP169k to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 12 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 12. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 12. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
[0186] In some embodiment, the method comprises providing S/miPEP169kDlD10 to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 33 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 33. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 33. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the miPEP regulates miR169.
[0187] In some embodiment, the method comprises providing MP 19051 to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 34 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 34. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 34. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 30. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 30. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 30. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
[0188] In some embodiment, the method comprises providing MP 19048 to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 35 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 35. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 35. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth SEQ ID NO: 31. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 31. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 31. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
[0189] In some embodiment, the method comprises providing MP 19049 to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 36 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 36. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 36. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to a Solarium lycopersicum plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 32. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 32. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 32. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR169.
[0190] In some embodiment, the method comprises providing S7miPEP396c to a Solarium lycopersicum plant. In some embodiment, the method comprises providing to miPEP having an amino acid sequence as set forth in SEQ ID NO: 13 to a Solarium lycopersicum plant. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 13. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13. In some embodiments, the miPEP further comprises a proline at the N-terminus and the aspartate at the C- terminus. In some embodiments, the method comprises providing to a Solanum lycopersicum plant a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 37. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to SEQ ID NO: 37. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 37. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartate at the C-terminus result from fusion polypeptide expression of the peptide. In some embodiments, the miPEP regulates miR396.
III. Peptides
[0191] Also provided herein are immunity enhancing peptides. In some embodiments, the immunity enhancing peptides are antimicrobial or antifungal oligopeptides. In some embodiments, the immunity enhancing peptides are miPEPs. In some embodiments, the miPEP regulates a miR169 or miR396.
[0192] In some embodiments, the miPEP comprises an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ IDNO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 1. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 4. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 5. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 6. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 10. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 11. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 12. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the miPEP fiirther comprises an N-terminal proline and a C-terminal aspartate. In some embodiments, the miPEP regulates miRI69 or miR396.
[0193] In some embodiments, the miPEP comprises a proline at the N-terminus and an aspartic acid at the C-terminus. In some embodiments, the miPEP comprises an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 36, or SEQ ID NO: 37. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 14. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 15. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 16. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 17. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 18. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 19. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 20. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 21. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 22. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 23. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 24. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 25. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 26. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 28. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 29. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 30. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 31. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 32. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 33. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 34. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 35. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 36. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 37. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, proline at the N-terminus and the aspartic acid at the C-terminus result from fusion polypeptide expression of the miPEP.In some embodiments, the miPEP regulates miR169 or miR396.
[0194] In some embodiments, the miPEP comprises a protease-resistant amino acid sequence. In some embodiments, the miPEP comprise one or more D-amino acids. In some embodiments, the one or more D-amino acids enhance the stability of the miPEP in a cell. In some embodiments, the one or more D- amino acids enhance stability of the oligopeptide as compared to an oligopeptide of the same amino acid sequence comprising all L-amino acids. In some embodiments, the miPEP comprising one or more D- amino acids regulates a miR169 or a miR396. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37, with one or more D-amino acids.
[0195] In some embodiments, the immunity enhancing peptide is identified by a) selecting an miRNA from a miR169 family or a miR396 family; b) identifying an ORF contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; d) exogenously adding to the first plant (i) a peptide encoded by the ORF, or (ii) a nucleic acid sequence encoding the peptide; e) comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing immunity enhancing miRNA and the peptide is an immunity enhancing miPEP. In some embodiments, the ORF is 12 to 303 nucleotides in length. In some embodiments, the plant is an Arabidopsis spp., Phaseolus spp., Fragaria spp., or Solarium spp. In some embodiments, the plant is Arabidopsis thaliana, Phaseolus vulgaris, Fragaria vesca, or Solarium lycopersicum.
IV. Nucleic acids
[0196] Also provided are nucleic acids encoding immunity enhancing peptides. In some embodiments, the immunity enhancing peptide is a miPEP that regulates a plant miR169 or miR396. In some embodiments, the plant miR169 or miR396 are from Arabidopsis thaliana, Phaseolus vulgaris, Fragaria vesca, or Solanum lycopersicum. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
[0197] The nucleic acids of the disclosure may be prepared using any technique known in the art. Without limitation, these may include cloning, DNA isolation, amplification and purification, enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques, such as gel electrophoresis and chromatography. A number of standard techniques are described in Ausubel et al. (1992) Current Protocols in Molecular Biology, Green/Wiley, New York, N. Y.; Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose (1981) Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK; Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York; and Ausubel et al. (1992) Current Protocols in Molecular Biology, Greene/Wiley, New York, N.Y. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein.
[0198] Also provided herein are vectors comprising nucleic acids encoding any of the fusion polypeptides, carrier polypeptides or oligopeptides disclosed herein.
[0199] A “vector” is a nucleic acid that is capable of transporting another nucleic acid. Vectors may be, for example, plasmids, viruses, cosmids or phage. An “expression vector” is a vector that is capable of directing expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment. Examples of vectors are those that can autonomously replicate and express structural gene products present in the DNA segments to which they are operatively linked. Vectors, therefore, can contain the replicons and selectable markers described earlier. Vectors include, but are not necessarily limited to, expression vectors.
[0200] In some embodiments, the vector is a bacterial vector. In some embodiments, the vector is a yeast vector. Examples of expression vectors that can be used in prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pET plasmids (Novagen, Madison, Wis., USA) or pBR322 (ATCC 37017). The pBR322 vector contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. To construct an expression vector using pBR322, an appropriate promoter and a DNA sequence encoding one or more of the polypeptides of the invention are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM-1 (Promega Biotec, Madison, Wis., USA). Other commercially available vectors include those that are specifically designed for the expression of proteins; these would include pMAL-p2 and pMAL-c2 vectors that are used for the expression of proteins fused to maltose binding protein (New England Biolabs, Beverly, Mass., USA).
[0201] In bacterial systems a number of expression vectors may be advantageously selected depending upon the use intended for the variant squalene synthase enzyme expressed, and whether it is desired to isolate the enzyme and in what state of purity. For example, when large quantities are to be produced, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
[0202] In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review, see Current Protocols in Molecular Biology, Vol. 2, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13 (1988); Bitter et al., Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp. 516-544 (1987); Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3 (1986); Bitter, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684 1987); and The Molecular Biology of the Yeast Saccharomyces, Eds. Strathem et al., Cold Spring Harbor Press, Vols. I and II (1982). A constitutive yeast promoter such as ADH1 or LEU2 or an inducible promoter such as GAL4 may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol. 11, A Practical Approach, Ed. D M Glover, IRL Press, Wash., D.C. (1986)). Alternatively, vectors may be used which promote integration of foreign DNA sequences into the yeast or bacterial chromosome.
[0203] Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include the bacteriophage T7 promoter (Studier and Moffatt, J. Mol. Biol. 189: 113 (1986)), [3-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; Goeddel et al., Nature 281:544 (1979)), tryptophan (tap) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057 (1980); EP- A-36776), and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; p. 412 (1982)). A particularly useful prokaryotic host cell expression system employs a phage PL promoter and a cl857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection (ATCC), which incorporate derivatives of the PL promoter, include plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).
[0204] In some embodiments, the nucleic acid encoding the immunity enhancing peptide is operably linked to a heterologous promoter. In some embodiments, the heterologous promoter is a is a constitutive promoter, a tissue specific promoter, or an inducible promoter. In some embodiments, the inducible promoter is induced by fungal infection, optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., phenylalanine ammonia lyase, chaicone synthase promoters), a gene that modifies plant cell walls (e.g., hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase promoters), a gene encoding an enzyme that degrade fungal cell walls (e.g., chitinase or glucanase promoters), a gene encoding a thaumatin-like protein, or a maize or flax promoters, designated as Mis 1 and Fisl, respectively, are also induced by fungal infections in plants and can be used (US Patent Appl. Pub. No. 20020115849).
[0205] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a polypeptide that forms inclusion bodies in a cell operably linked to two or more miPEPs. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an carrier polypeptide operably linked to two or more miPEPs. In some embodiments, the operable linkage comprises a peptide bond susceptible to sequence-specific chemical cleavage. In some embodiments, the operable linkage is an Asp-Pro bond and the sequence-specific cleavage is acetic acid cleavage. In some embodiments, the miPEP is 4-50 amino acids long. In some embodiments, the miPEP comprises an N-terminal proline and a C-terminal aspartic acid. In some embodiments, the miPEP regulates a miR169 or miR396. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
[0206] In some embodiments, the nucleic acids encode an miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ IDNO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the miPEP further comprises an N-terminal proline and a C-terminal aspartate. In some embodiments, the miPEP regulates miR169 or miR396.
[0207] In some embodiments, the nucleic acid encodes a miPEP comprising a proline at the N-terminus and an aspartic acid at the C-terminus. In some embodiments, the nucleic acid encodes an miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartic acid at the C-terminus result from fiision polypeptide expression of the miPEP. In some embodiments, the miPEP regulates a miRI69 or miR396. [0208] In some embodiments, the nucleic acid encodes a miPEP identified by: a) selecting an miRNA from a miRI69 family or a miR396 family; b) identifying an ORF contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; d) exogenously adding to the first plant (i) a peptide encoded by the ORF, or (ii) a nucleic acid sequence encoding the peptide; e) comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing immunity enhancing miRNA and the peptide is an immunity enhancing miPEP. In some embodiments, the ORF is from 12 to 303 nucleotides in length.
[0209] In some embodiments, the nucleic acid encodes an miPEP further comprising a tag. In some embodiments, the tag is a secretion tag, a tag that promotes entry into a plant cell, or a nuclear localization tag.
V. Cells
[0210] Also provided are cells comprising immunity enhancing peptides or nucleic acids encoding immunity enhancing peptides. In some embodiment, the cell comprises an immunity enhancing peptide or a nucleic acid encoding the immunity enhancing peptide. In some embodiments, the immunity enhancing peptide is a miPEP that regulates a miRI69 or miR396. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37. In some embodiments, the cell is a host cell.
[0211] In some embodiments, the cell is a plant cell. In some embodiments, the cell is a cell of the plant. In some embodiments, the plant cell is a dicot or monocot plant cell. In some embodiments, the plant cell is a cell from an Arabidopsis spp., Phaseolus spp., Fragaria spp., or Solarium spp. plant. In some embodiments, the cell is a transgenic cell. In some embodiments, the cell expresses a miRI69 or miR396. In some embodiments, the cell has increased immunity to a fiingal pathogen.
[0212] In some embodiments, the cell is a bacterial cell. In some embodiments, the bacteria is an E. colt strain. In some embodiments, the cells is a cell that is capable of forming inclusion bodies. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a fiingal cell. In some embodiments, the cell is an algal cell. In some embodiments, the cell is an animal cell. In some embodiments, the animal cell is a mammalian cell or an insect cell.
[0213] Microorganism host cells usefill in the present invention may include, but are not limited to, bacteria, such as the enteric bacteria {Escherichia and Salmonella for example) as well as Bacillus, Acinetobacter, Streptomyces, Methylobacter, Rhodococcus and Pseudomonas,' Cyanobacteria, such as Rhodobacter and Synechocystis,' yeasts, such as Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia, Yarrowia, and Torulopsis,' and filamentous fungi such as Aspergillus and Arthrobotrys, and algae for example.
[0214] In some embodiments, the host cell comprises at least one copy of a nucleic acid sequence encoding an immunity enhancing miPEP. The at least one copy of the nucleic acid sequence encoding an immunity enhancing miPEP can be present in the chromosome of a prokaryotic (bacterial) cell or in one chromosome of a eukaryotic cell. Alternatively, the at least one copy of the nucleic acid sequence encoding an immunity enhancing miPEP can be present in a vector or plasmid that is present in the cell. The host cell, as described above, can be a prokaryotic or eukaryotic cell. If it is a prokaryotic cell, it can be a bacterial cell. If it is a eukaryotic cell, it can be a yeast cell, a plant cell, or an animal cell. Suitable host cells are described herein.
[0215] In some embodiments, the cell comprises a miPEP or a nucleic acid encoding an miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ IDNO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the miPEP fiirther comprises an N-terminal proline and a C-terminal aspartate. In some embodiments, the miPEP regulates miRI69 or miR396.
[0216] In some embodiments, the cell comprises a miPEP or a nucleic acid encoding a miPEP comprising a proline at the N-terminus and an aspartic acid at the C-terminus. In some embodiments, the cell comprises an miPEP or a nucleic acid encoding a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the miPEP regulates miRI69 or miR396.
[0217] In some embodiments, the cell comprises an miPEP, or a nucleic acid encoding an miPEP, identified by: a) selecting an miRNA from a miRI69 family or a miR396 family; b) identifying an ORF contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; d) exogenously adding to the first plant (i) a peptide encoded by the ORF, or (ii) a nucleic acid sequence encoding the peptide; e) comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing immunity enhancing miRNA and the peptide is an immunity enhancing miPEP. In some embodiments, the ORF is from 12 to 303 nucleotides in length.
VI. Kits
[0218] Some aspects of the present disclosure provide for kits comprising an immunity enhancing peptide and a package insert comprising instructions for the application of the immunity enhancing peptide. In some embodiments, the immunity enhancing peptide is a miPEP.
[0219] In some embodiments, the kit comprises a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NOV, SEQ ID NO:8, SEQ ID NOV, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, and instructions for the application of the miPEP. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NOV, SEQ ID NO:8, SEQ ID NOV, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 1, SEQ ID NOV, SEQ ID NOV, SEQ ID NO:4, SEQ ID NOV, SEQ ID NOV, SEQ ID NOV, SEQ IDNO:8, SEQ ID NOV, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the miPEP further comprises an N-terminal proline and a C-terminal aspartate. In some embodiments, the miPEP regulates miR169 or miR396.
[0220] In some embodiments, the kit comprises a miPEP comprising a proline at the N-terminus and an aspartic acid at the C-terminus. In some embodiments, the kit comprises a miPEP comprising an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NOVO, SEQ ID NO:21, SEQ ID NOV2, SEQ ID NO:23, SEQ ID NOV4, SEQ ID NOV5, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NOV8, SEQ ID NOV9, SEQ ID NOVO, SEQ ID NO:31, SEQ ID NOV2, SEQ ID NOV3, SEQ ID NOV4, SEQ ID NO:35, SEQ ID NOV6, or SEQ ID NO:37, and instructions for the application of the miPEP. In some embodiments, the miPEP comprises an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions as compared to any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NOVO, SEQ ID NO:21, SEQ ID NOV2, SEQ ID NOV3, SEQ ID NOV4, SEQ NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the miPEP comprises amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the miPEP is 4-50 or 5-30 amino acids in length. In some embodiments, the proline at the N-terminus and the aspartic acid at the C- terminus result from fiision polypeptide expression of the miPEP. In some embodiments, the miPEP regulates miRI69 or miR396.
[0221] In some embodiments, the kit comprises instructions and a miPEP identified by: a) selecting an miRNA from a miRI69 family or a miR396 family; b) identifying an ORF contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; d) exogenously adding to the first plant (i) a peptide encoded by the ORF, or (ii) a nucleic acid sequence encoding the peptide; e) comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing immunity enhancing miRNA and the peptide is an immunity enhancing miPEP. In some embodiments, the ORF is from 12 to 303 nucleotides in length.
[0222] In some embodiments, the kit comprises a composition comprising a miPEP and, and a package insert comprising instructions for application of the composition. In some embodiments, the composition comprises a miPEP and an agriculturally acceptable formulant. In some embodiments, the agriculturally acceptable formulant comprises one or more of a water, organic solvents, paraffinic oils, vegetable oils, dispersants, emulsifiers, wetting agents, buffering agents, hydrotrope agents, rheology modifiers, antifoam agents and defoamers, antifreeze agents, biocides, dyes, polymer walls, catalysts, thermosetting materials, cross-polymerizing agents, UV protectants, antioxidants, and chelating agents. In some embodiments, the composition is in the form of a finely-divided particulate solid, granules, pellets, wettable powders, dust, an aqueous suspension, a dispersion, a gel, or an emulsion. In some embodiments, the composition further comprises one or more other active agents selected from the group consisting of a pesticide, a fertilizer, an insecticide, an attractant, a sterilizing agent, an acaricide, a nematocide, a herbicide, a biostimulant, a biological, and a growth regulator. In some embodiments, the kit comprises a composition comprising a miPEP that regulates a miRI69 or a miR396. In some embodiments, the kit comprises a composition comprising a miPEP comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
[0223] In some embodiments, the kit comprises a composition comprising a miPEP and a agriculturally acceptable carrier, diluent or excipient. In some embodiments, the kit comprises a composition comprising a miPEP and a package insert comprising instructions for applying the miPEP to a plant or part thereof. In some embodiments, the plant part is a leaf, a bud, a root, a shoot, a floral part, or a seed. In some embodiments, the package insert comprises instructions for providing the composition to a plant by application to the soil in which the plant is planted. In some embodiments, the package insert comprises instructions for providing the composition by addition of water provided to the plant. In some embodiments, the package insert comprises instructions for applying the composition by a spreader, a power duster, a boom sprayer, a hand sprayers, a spray dusters, or a granular applicator. In some embodiments, the miPEP regulates a miRI69 or a miR396. In some embodiments, the miPEP comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
[0224] Also provided herein are kits comprising cells comprising a miPEP, or a nucleic acid encoding the miPEP, and a package insert comprising instructions for application of the cells. In some embodiments, the cells comprise a miPEP that regulates a miRI69 or a miR396. In some embodiments, the cells comprises a miPEP having s an amino acid sequence as set forth in any one of SEQ ID NOs: 1-37.
EXAMPLES
Example 1: miPEP candidate screening on Arabidopsis thaliana to determine microRNA families involved in defense against Botrytis cinerea
[0225] Micropeptides (miPEPs) are characterized by short peptide sequences (7-44 amino acids in length) defined by one or several short open reading frames (ORFs) located in the pri-miR sequence of a specific miR family member. In this example, shortened micropeptides (miPEP) targeting various microRNAs (miRs) were designed and tested to identify miR families involved in the defense against grey mold in Arabidopsis thaliana.
Results
[0226] Several short peptides sequences of about 10 amino acids were designed based on the pri-miR of respective miR members, and tested for their effect on defense against Botrytis cinerea (grey mold) in Arabidopsis thaliana.
[0227] To determine miR families involved in plant defense, the miRI68, miRI69, miR396 and miR848 families were chosen for screening of miR families involved in the defense against Botrytis cinerea (grey mold) in Arabidopsis thaliana. Open reading frames (ORFs) in pri-miRs from these families were analyzed to design 10 amino acid-long miPEP sequences to screen. A total of seven miPEPs for four different miR families (miR169, miR168, miR386 and miR858) were designed and chemically synthesized.
[0228] To analyze the effect of the miPEPs on the development of Botrytis cinerea infection, a peptide application and infection protocol was developed. The protocol consisted of three consecutive days of peptide spray application on Arabidopsis leaves, followed by a droplet infection with Botrytis spores, and a post infection peptide treatment (FIG. 1).
[0229] As Botrytis cinerea infection leads to brown lesions on leaf surfaces, the lesions were measured to determine the spread of infection. Of the seven miPEPs tested, three miPEPs (targeting .4 /miR 169c. A tai PEP 168a, A tali R396b) led to a more resistant phenotype compared to control treated plants (FIG. 2), as determined based on lesions that were significantly smaller compared to control. Application of miPEPs targeting the family AtaiiR858 led to a susceptible phenotype. AtaiiPEP396a also increased the lesion sizes compared to control plants. AtaiiPEP168a did not show any difference to control treatment, suggesting that general peptide application does not interfere with Botrytis infection (FIG. 2). At least two miR families, miR169 and miR396, were identified as positively controlling Botrytis growth on Arabidopsis leaves.
Example 2: Induction of specific miRNA transcript by miPEPs
[0230] In this example, the effect of miPEP application on expression of target pri-miRs in Arabidopsis thaliana was evaluated.
Results
[0231] As described in Lauressergues, D., etal. (2015) Nature. 520(7545): 90-3 (“Lauressergues, etal ”), external application of miPEPs leads to the increased transcript production of their target pri-miR. To investigate whether shortened miPEPs lead to a similar pri-miR induction as full length miPEPs, RNA was extracted from Arabidopsis leaves sprayed with peptides and control-treated plants and pri-MIR expression was analyzed. Treatment with AtaiiPEP169h and AtmiPEP 169c led to detection of a 2 to 5 -fold increase in pri-miR (FIG. 3)
[0232] AtaiiR396b was not detectable in Arabidopsis leaves. To test the effect of this miPEP on its pri- miR expression, the pri-miR sequence was amplified from cDNA, sequenced and cloned into a plant transformation vector for transient expression of in Nicotiana benthamiana leaves. Nicotiana benthamiana leaves were transformed with the pri-AtaiiR396b sequence construct and then sprayed with AtmiPEP396b. RNA was extracted and pri-miR transcript expression was analyzed. A 2-fold induction of pri-miR transcript after peptide application compared to control plants (FIG. 4). [0233] These experiments showed that external application of d/miPEP I 69h. d/miPEP I69c. d/miPEP396B induce the transcription of the respective pri-miR targets and function on the molecular level as previously described in Lauressergues, et al.
Example 3: miPEP screening on Phaseolus vulgaris (bean) to analyze the effect of family members of PvmiR169 and PvmiR396 involved in defense against Botrytis cinerea.
[0234] This example describes the screening of miPEPs targeting the miR169 and miR396 families in bean plant defense against Botrytis infection.
Results
[0235] To investigate whether the miPEPs have a role in the defense against Botrytis cinerea in other host plants, Phaseolus vulgaris (bean) pri-miR sequences were analyzed and miPEPs for 16 PvmiR169 family members and one PvmiR396 family member were designed and tested. Peptide application and infection were performed as described in Example 1. Of 17 miPEPs tested, three (Pvmi PEP 169c. PvmiPEP 169kl, PvmiPEP 169pl) significantly reduced Botry //.s-induccd lesions size on bean leaves after peptide application.
Example 4: miPEP screening on Fragaria vesca (strawberry) to analyze the effect of family members of FvmiR169 and FvmiR396 involved in defense against Botrytis cinerea.
[0236] This example describes the screening of miPEPs targeting the miR169 and miR396 families in strawberry plant defense against Botrytis infection.
Results
[0237] Fragaria vesca (strawberry) pri-miR sequences were analyzed, and miPEPs for twelve members of theFvmiR169 family and six members of the FvmiR396 family were designed and tested. Peptide application and infection were then performed as described in Example 1. Of 18 miPEPs tested, four (FvmiPEP169hI, FvmiPEP1691I, FvmiPEP396a, FvmiPEP396f) significantly reduced Botrytis- induced lesions size on strawberry leaves after peptide application (FIG. 5).
[0238] These four miPEPs were consequently tested for their effect to control Botrytis growth on mature Strawberry fruits. Strawberry fruits were sprayed with the miPEPs prior to Botrytis infection. Botrytis spores were deposited as droplet on the fruit, and the peptides were applied on the infection sites for three days after infection. Control strawberry fruits were heavily infected with Botrytis, whereas strawberries treated with FvmiPEP169hI, FvmiPEP 16911, FvmiPEP396a showed significantly less Botrytis growth on the fruit body (FIG. 6, bolded). Control fruits and fruits treated with FvmiPEP396- 1 showed strong Botrytis cinerea infection (FIG. 6, black arrows) and tissue collapse.
Example 5: miPEP screening on Solanum lycopersicum (tomato) to analyze the effect of family members of SlvmiR169 and SlmiR396 involved in defense against Botrytis cinerea.
[0239] This example describes the screening of miPEPs targeting the miR169 and miR396 families in tomato plant defense against Botrytis infection.
Results
[0240] To investigate the broad host control of miPEPs against Botrytis cinerea, we analyzed Solanum lycopersicum (tomato) pri-miR sequences were analyzed and miPEPs for 18 S/miR169 family members and 4 S/miR396 family members were designed and tested. Peptide application and infection were performed as described in Example 1. Of 22 miPEPs tested, application of four peptides (S/miPEP169a, S/miPEP169k, S/miPEP169d, and S/miPEP396c) led to reduced Botrytis- induced lesions size on tomato leaves after peptide application (FIG. 7 and FIG. 8). These miPEPs significantly decreased lesion compared to control (water-treated) plants (FIG. 8, highlighted in black). S/miPEP 169-8 increased lesions after application (FIG. 8, highlighted in grey).
Example 6: Induction of SLmiR169a transcript by the SlmiPEP169a miPEP in tomato.
[0241] In this example, the ability of the S/miPEP169a miPEP to induce S/miR169a expression was evaluated.
Results
[0242] To test the effect in the \miPEP169a miPEP on its pri-miR expression, the pri-miR S/miR169a sequence was synthesized, sequenced and cloned into a plant transformation vector for transient expression in Nicotiana benthamiana leaves. Nicotiana benthamiana leaves were transformed with the pri-S/miR169a sequence construct, and the plants were then sprayed with S/miPEP169a. RNA was then extracted and pri-miR transcript expression was analyzed. A 2-3 fold induction of pri-miR transcript was observed after peptide application as compared to control plants (FIG. 9). Thus, external application of S/miPEP169a activated the expression of its respective pri-miR target. Example 7: Foliar spray application of SlmiPEP169a and SlmiPEP169k controls Botrytis growth on tomato leaves.
[0243] This example describes the evaluation of peptide application at filed conditions on defense against Botrytis cinerea in tomato plants.
Results
[0244] Previously, Botrytis spores were applied as droplets in high concentrations to assess quantitatively the lesion sizes, with non-formulated miPEPs sprayed preventively or applied curatively as droplets on the site of infection. To improve the peptide application protocol and be closer to field applications, Botrytis spores were applied at low concentrations and sprayed on tomato plants in order to mimic the infection occurring in field conditions. The effect of pre- and post-spray application of miPEPs was then evaluated using S/miPEP169a and S/miPEP169k miPEPs. Additionally, to improve the coverage of peptides, formulated buffers with spreader and humectant ingredients were used to enhance the peptide effect on leaves.
[0245] Overall plant fitness was evaluated after peptide application. Side and top RGB images of tomato plants were used to calculate the convex hull, which serves as readout to evaluate the overall infection on plants (FIG. 10A). Non-infected and control plants showed an increase in convex hull over the measured time points (dai, days after infection), while infected plants showed a decrease in convex hull due to overall leaf infection (FIG. 10B). The convex hull was significantly reduced in Botrytis- infected plants, the as compared to the control plants (FIG. 10B).
[0246] To further analyze the effects of S7miPEP169a and S/miPEP169k application, tomato plants were spray-infected with Botrytis spores and their leaves sprayed with miPEP 18 hours after infection, covering the leaf area. The plants were inspected daily and images were taken to analyze the convex hull. Disease control was calculated as disease control % = (Treated- Blanker)* I OO/fBIankNi-Blankiuf). where Blanks and Blanker indicate non-infected and infected and untreated plants, respectively. On the final day (4 days after infection), leaves were harvested and leaf symptoms were visually scored and ranked in a scale from non-infected to highly infected. Single post-infection application of S7miPEP169k and S7miPEP169a increased plant fitness after Botrytis infection (FIG. 11 A). Postinfection treatment with S7miPEP169k showed a significant increase in disease control, with up to 43% disease control observed (FIGS. 11 A) with S/miPEP169k-treated plants showing more healthy plant phenotypes than control plants at three days after infection. Interestingly, no improvement in disease control was detected when using a higher miPEP concentration (FIG. 1 IB). Application of S/miPEP169k at 100 grams per hectare (g/ha) showed 43% disease control compared to control infected plants (FIG. 1 IB). Both peptides showed a disease control of around 30% at 200 grams per hectares (g/ha), and application of S/miPEP169k at 200 g/ha did not lead to increased disease control (FIG. 11B). Thus, application of miPEP at 100 g/ha 24 hours post-infection was determined as the best tested condition to improve disease control.
Example 8: Foliar spray application of SlmiPEP169k increases disease control in tomato plants against Alternaria solani and Phytophthora inf estans.
[0247] This example describes the evaluation of a miPEP targeting a miR169 family member in defense against Alternaria solani (fungi) and Phytophthora infestans (oomycetes) infection in tomato plants.
Results
[0248] To further investigate the effect of S7miPEP169k on defense against different pathogens in tomato plants, experiments were conducted using tomato plants infected with Alternaria solani and Phytophthora infestans. Peptides were applied pre- and post-infection to evaluate defense against Alternaria solani, and 2 hours pre-infection to evaluate defense against Phytophthora infestans. Disease symptoms were visually inspected, and disease control was analyzed by calculating the infected leaf area in non-treated plants, control peptide plants and plants treated with the respective miPEPs. All peptides were diluted in formulated buffer and control plants were sprayed on the same time points with unrelated peptides and inoculated with the respective pathogens. In both pathosystems, application of S/miPEP 169k led to enhanced disease control compared to control plants (FIG. 12), with 24% and 39% disease control for Alternaria and Phytophthora infections, respectively. Tomato plants sprayed with SlmiPEP169k (40 g/ha or 4 g/ha) 24 hours pre- and 2 hours postinoculation with Alternaria solani showed improved disease control (24%) as compared to control plants (FIG. 12A). Tomato plants sprayed with S7miPEP169k (3 g/ha) 2 hours pre-inoculation with Phytophthora infestans showed improved disease control (39%) as compared to control plants. Higher concentrations of S/miPEP 169k did not further improve disease control (FIG. 12B). Thus, foliar spray application of S7miPEP169k increased tomato plant immunity against not only Botrytis, but also Alternaria and Phytophthora.
Example 9: Identification of more stable and biologically active variants of SlmiPEP169k
[0249] In this example, a variant miPEP with improved stability is developed and evaluated.
Results [0250] The stability of the S7miPEP169k miPEP in tomato leaf wash-off solution, which represents the microorganisms present on the leaf surface, was tested. S7miPEP169k was found to be degraded rapidly after storage for 40 hours in this solution (FIG. 13A).
[0251] To test whether miPEP stability could be improved and to overcome the biological degradation of S7miPEP169k, a variant peptide with the D-form of the first and the last amino acid in the S/miPEP 169k sequence replaced by (S/miPEP 169kD 1 D 10) was chemically synthetized and evaluated. As D-amino acids are not naturally derived amino acids, they cannot be metabolized and should be less susceptible to degradation by microorganisms. Indeed, the S7miPEP169kDlD10 variant showed a 20% improved stability as compared to the S7miPEP169k. when stored for the same time in leaf wash-off solution when compared to the original peptide (FIG. 13 A). To further analyze the impact of D-amino acids on the biological activity of S7miPEP169k, tomato plants infected with Phytophthora infestans were sprayed with either S7miPEP169k or S7miPEP169kDlD10 at 30 g/ha 2 hours pre-inoculation. Similar disease control (18-27%) was observed for both peptides (FIG. 13B), and the S/miPEP169kDlD10 variant was still biologically active, showing 27% disease control. The S7miPEP169kDlD10 maintains its full biological activity while appearing to be less prone to degradation.
Example 10: Identification of miPEPs targeting miR169 family members with increased biological activity or stability.
[0252] This example describes the identification of additional miPEPs targeting miR169i and miR169d that regulate defense against Botrytis cinerea in tomato plants.
Results
[0253] To identify new peptides with higher biological activity or improved stability, several miPEPs targeting priMIR169k (FIG. 14A) or priMIR169a (FIG. 14B) were designed. These peptides were tested for their biological activity against Botrytis cinerea (grey mold) in tomato plants (FIGS. 1 TAUB).
[0254] In addition to the S7miPEP169k (MP 18537), five different peptides from three different ORFs on pri-miR169i were designed and tested. Three of these peptides (MP19051, MP19048 and MP19049) showed similar activity to S7miPEP169k (MP18537), with 30-40% disease control (FIG. 14A). MP 19052, a truncated form of the active peptide MP 19051, did not show activity, while MP 19050 was less active, with 25% disease control (FIG. 14A). [0255] In addition to the S7miPEP169a (MP18356) peptide evaluated in the previous examples, three different peptides on two other ORFs found on miR169a were designed and tested. MP 19043 showed similar activity to S/miPEP169a (MP18356), with 30% disease control, whereas MP19042 and MP 19045 did not show activity (FIG. 14B). The data shows that different peptides can target the same pri-miR with similar biological activity, with activity of the miPEP being dependent on the specific peptide sequence.
Example 11: Identification of miPEPs boosting soybean immunity against Asian Soybean Rust
(ASR) caused by Phakopsora pachyrhifL
[0256] This example describes the identification of additional miPEPs targeting miR169g and miR169m that regulate defense against Phakopsora pachyrhizi (commonly known as Asian Soybean Rust, or ASR) in Glycine max (soybean) plants.
Results
[0257] The miR169 family in Glycine max (referred to herein as gma-MIR169 and G/wmiR 169, with family members indicated by a letter suffixed thereon), is known to be involved in plant defense pathways. To identify new peptides with higher biological activity or improved stability, 44 miPEPs collectively targeting 22 members of the G/wmiR 169 family (including both miR169g and miR169m) were designed, with two peptides designed per G/wmiR 169 family member.
[0258] The 44 miPEP peptides were chemically synthetized and screened on ASR-infected soybean plants. Peptides solutions were foliar sprayed at 24 hours pre-infection and at 24 hours post-infection, both at 200 g/ha. Peptides that displayed more than 30% disease control compared to blank controls (comprising reference antifungal peptide MP18279; SEQ ID NO: 60) were tested a second time. Peptides that displayed more than 30% disease control compared to blank controls in the second replicate were tested a third time. These tests validated two miPEP peptides 3 times each with > 30% disease control: MP19114 and MP19127 (FIGS. 15A-15B and SEQ ID NOs: 54-55, respectively), each of which were found to be as efficient as the MP 18279 reference in controlling ASR disease progression.
[0259] The impacts of timing and dosing of various peptides were studied in different in planta experiments testing two doses: 200 and 20 g/ha, with 2 applications each: 24h pre- and post-infection. Results from 2 replicates are shown in Table 1 below. Peptides were either chemically synthesized (labeled “synthetic” in Table 1) or produced in vivo in bacteria (labeled “bioproduced” in Table 1). The differences in disease control levels between 200 and 20 g/ha from the results in Table 1 are shown in Table 2.
Table 1: Disease control results of in planta experiments
Table 2: Difference in disease control levels between 200 and 20 g/ha
[0260] No significant dose effects were observed in Replicate 1 (Tables 1-2). In Replicate 2, decreasing the dose 10-fold (from 200 g/ha to 20 g/ha) did not seem to tremendously impact the level of disease control, with the maximum observed difference being only 25 percentage points for MP 19474 (synthetic).
[0261] Peptide stability was tested in leaf wash-off of tomato (soybean leaf wash off was not available). After 24h, MP19114 and MP19127 were confirmed to be stable. However, LC/MS measurements n MP19127 revealed cleavage of Q4-G5. MP19114 cleavage was not studied.
Example 12: Antifungal properties of MP19114
[0262] This example describes the identification of direct antifungal properties of peptide MP 19114.
Results
[0263] Disease control following peptide application can be due to a boost in plant immunity or by antifungal properties of the peptide (e.g., wherein the peptide itself has a negative impact on, e.g., fungal spore development). In this project, peptides were designed as miPEPs to boost plant immunity only. However, it was possible that some peptides displayed antifungal properties due to their structures and/or physiochemical properties. In vitro tests were designed in order to efficiently check for potential antifungal activities against Septoria tritici, Phytophthora cactorum, and Botrytis cinerea. In vitro tests could not be run on ASR, however, because it is an obligate parasite that cannot survive outside a plant. Results of these in vitro tests, shown in Table 3, demonstrated that MP 19114 exhibited antifungal activities against each of the three tested pathogens, ft is possible that the observed antifungal activity of MP19114, a 13-amino-acid-long miPEP with a hydrophobicity index of 34.05 and an isoelectric point (PI) of 12.5, is due to the presence of two hydrophobic blocks within its peptide structure (FIG. 16). MP19114 was assigned an antifungal score of 0.91. Antifungal score was measured via a antimicrobial peptide prediction model that was trained on public and private data with an algorithm based on Natural Language Processing and deep learning methods.
Table 3: In vitro tests of MP19114 and MP19127 peptides against Septoria tritici, Phytophthora cactorum, and Botrytis cinerea
* No observed antifungal activity; ** low observed antifungal activity; *** medium observed antifunga activity; **** high observed antifungal activity.
Example 13: Bioproduction feasibility ofMP19114 and MP19127
[0264] This example describes experiments testing the feasibility of large-scale bioproduction of MP19114 and of MP19127.
Results
[0265] In order to assess bioproduction feasibility of MP19114 and MP19127, a coding nucleic acid including one copy, 3 copies, or 6 copies of the nucleotide sequence encoding the peptide sequence ofMP19114 or MP19127 (Table 4) were integrated into the E. coli genome. For multi-copy insertions, the coding nucleic acid encoded a fusion polypeptide comprising a polypeptide designed to form inclusion bodies in a cell operably linked to 3 or 6 copies of MP 19114 or 3 or 6 copies of MP 19127. The copies of the nucleic acid sequences encoding the peptide were linked by nucleic acids encoding a cleavable linker peptide, such that individual copies of MP19114 or MP19127 could be cleaved from the inclusion bodies.
Table 4: Nucleotide Sequences of Disease Control Peptides
[0266] Bioproduction was assayed by checking the expression of peptides and fusion proteins embedded in inclusion bodies. Then the efficiency of cleavage of the peptides from fusion proteins was assessed and peptide quantification was performed (Table 5). MP 19114 displayed good yield in a 2L fermenter and did not display multiple chromatogram peaks. However, although MP19127 demonstrated good expression by gel test, it displayed many peaks by chromatogram, indicating potentially poor bioproducibility.
Table 5: Bioproduction Results
Example 14: In planta activity of MP19114 and MP19127 variants with P and DP additions
[0267] This example describes experiments testing the in planta activities of variants of MP 19114 and MP 19127 having additions of P or DP amino acids.
Results
[0268] Concatemeric expression strategies such as those described in Example 13 involve adding, for example, up to 6 copies of a peptide sequence into a bacterial genome in order to increase yield. However, when cleaving out the individual peptides, P and/or D amino acids remain appended to one or both ends of the peptide. Thus, the MP19114 and MP19127 peptides expressed as concatemers necessarily had the amino acids DP present between each copy. Acidic cleavage of the cleavable linker then cut 2 peptide copies between D and P and released copies of the peptide flanked by D and P (e.g., with P on the N-terminus and/or D on the C-terminus). Thus, the sequence of the released peptides was not the original MP 19114 or MP 19127 sequence, but had 2 additional amino acids: D and P. The relationship between each of these peptides and their names and SEQ ID NOs herein are summarized in Table 6.
Table 6: Original peptide sequences vs. P- and PD-added variants
[0269] Because sequences of the cleaved concatemer peptides with P and/or D additions (SEQ ID NOs: 56-59) were not the same as the originally tested sequences (SEQ ID NOs: 54-55), it was crucial to assess in planta activities of SEQ ID NOs: 56-59 to check if sequence changes disturbed peptide activity. These results are displayed in Table 1. As shown in Table 1, when sprayed 2 times (24h pre- and post-infection) on soybean plants at 2 x 200 g/ha, MP19114 showed in planta efficiency (>30% disease control compared to a blank control in at least one replicate), as did the bioproduced P-appended version thereof (MP19472; SEQ ID NO: 59), but the bioproduced PD version thereof (MP 19476; SEQ ID NO: 58) did not. Further, when sprayed 2 times (24h pre- and post-infection) on soybean plants at 2 x 200 g/ha, MP 19127 showed in planta efficiency, as did the P- and PD-appended versions thereof (MP 19474, SEQ ID NO: 57; and MP 19478, SEQ ID NO: 56, respectively). Further, MP 19114 was found to be bioproducible, while MP 19127 was not (Table 5). However, the multiple chromatogram peaks seen for MP 19127 (Table 5) could potentially be overcome through optimization. Because it retained activity with the DP addition, MP 19127 was chosen as the most promising candidate for field tests and optimization, while MP19114 was chosen as the second-most- promising candidate.
Example 15: Design of additional sequence variants ofMP19127
[0270] This example describes additional synthetic variants of MP 19127 designed based on potential acidic deamination of MP 19127.
Results
[0271] The multiple peaks observed in the chromatogram data for MP19127 (Table 5) may have been due to deamination from glutamine to glutamic acid and/or from asparagine to aspartic acid in the acidic environment. Based on this theory, additional sequence variants of MP19127 were designed by either changing amino acids to acidic versions or by replacing amino acids with a neutral amino acid (alanine) (FIG. 17). Sequences of these additional sequence variants are listed as SEQ ID NOs: 66-69 in Table 7, along with the corresponding strategy from FIG. 17 used to generate each sequence variant.
Table 7: Additional sequence variants and sources
SEQUENCES BY SEQ ID NO (SID)
MIRNA SEQUENCES BY SEQ ID NO (SID)
[0272] SEQ ID NO: 61 is a 2021-nt-long nucleic acid sequence that corresponds to an extended version of the hairpin sequence gma-MIR169g referenced in miRBase 22. 1 under ID MI0017837, extended based on genomic positions from assembly Wm82.v4.4PTR (soybase.org) glyma.Wm82.gnm4. Gm 17 (4593256-4595276)-.
[0273] SEQ ID NO: 62 is a 2021-nt-long nucleic acid sequence that corresponds to an extended version of the hairpin sequence gma-MIR169m referenced in miRBase 22. 1 under ID MI0018665, extended based on genomic positions from assembly Wm82.v4.4PTR (soybase.org) glyma.Wm82.gnm4. Gm 13 (20517719-20519739)+.

Claims

1. A method for identifying and synthesizing (i) an immunity enhancing micropeptide (miPEP) encoded by a nucleotide sequence contained in the sequence of the primary transcript of a microRNA (miRNA), or (ii) a nucleic acid sequence encoding the miPEP that does not comprise the mature miRNA, comprising: a) selecting an miRNA from a miR169 family or a miR396 family; b) identifying an open reading frame (ORF) from 12 to 303 nucleotides in length contained in the
5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; d) exogenously adding to the first plant (i) a peptide encoded by the ORF or (ii) a nucleic acid sequence encoding the peptide; e) comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing immunity enhancing miRNA and the peptide is the miPEP; and f) synthesizing the miPEP or the nucleic acid sequence encoding the miPEP.
2. A method of increasing immunity in a plant comprising providing to the plant a miPEP that regulates an immunity enhancing miR169 or an immunity enhancing miR396.
3. The method of claim 2, where the miPEP is selected from the group consisting of d /mi PEP 169c. 4/miPEP I69h. AtPEP396b, PvmiPEP169e, PvmiPEP169kI, PvmiPEP169pI, FvmiPEP169hI, FvmiPEP1691I, FvmiPEP369a, S7miPEP169a, S7miPEP169d, S7miPEP169k, S7miPEP396c, MP19043, MP19042, S/miPEP169kDlD10, MP19051, MP19048, and MP19049, wherein the miPEP regulates an immunity enhancing miRNA.
4. A method of increasing immunity in a plant comprising providing to the plant a miPEP comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, or the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37 with one, two, three, or four amino acid substitutions, insertions, or deletions, wherein the miPEP regulates miRI69 or miR396.
5. A method of increasing resistance to a plant pathogen or pest in a plant comprising providing to the plant a miPEP comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID
NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, or the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37 with one, two, three, or four amino acid substitutions, insertions, or deletions, wherein the miPEP regulates miRI69 or miR396.
6. The method of claim 5, wherein the plant pathogen or pest is selected from fungal pathogens or pests, bacterial pathogens or pests, and viral pathogens or pests.
7. The method of claim 5, wherein the plant pathogen or pest is a fiingal pathogen or pest selected from Cercospora spp., Mycosphaerella spp., Glomerella spp., Cladosporium spp., Diplodia maydis, Fusarium oxysporum, Fusarium graminearum, Fusarium monilforme, Fusarium verticillioides, Cochliobolus sativus, Collectotrichum graminicola, Stagonospora nodorum, Stagonospora avenae, Stenocarpella maydis, Sclerotinia minor, Sclerotinia sclerotiorum, Sclerotinia sp., Alternaria spp., Phytophthora spp., Botrytis spp., Pyrenophora tritici-repentis, Phytophthora parasitica, Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichum dematium (Colletotichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v. glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Glomerella glycines, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium deharyanum, Fusarium solani; Canola: Albugo Candida, Altenaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassicicola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter michiganese subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium oxysporum, Verticillium albo-atrum, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulina briosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium botryosum, Leptotrichila medicaginis; Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis fsp. tritici, Puccinia graminis fsp. tritici, Puccinia recondite fsp. tritici, Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola, Pythium aphanidermatum, Plasmopora halstedii, Sclerotinia sclerotiorum, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum pv. carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis; Com: Colletotrichum graminicola, Fusarium verticillioides var. subglutinans, Erwinia stewartii, F. verticillioides, Gibberella zeae (Fusarium graminearum), Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T (Cochliobolus heterostrophus),
8. The method of claim 5, wherein the plant pathogen or pest is a bacterial pathogen or pest selected from Pseudomonas spp., Pantoua spp., spp.
9. The method of claim 5, wherein the plant pathogen or pest is a viral pathogen or pest selected from cucumber mosaic, tobacco mosaic, and barley yellow dwarf virus, alfalfa mosaic virus (Alfamovirus), Apple chlorotic leaf spot virus (Trichovirus), Apple scar skin viroid (Viroids), Arabis mosaic virus (Nepovirus), Barley mild mosaic virus (Bymovirus), Barley stripe mosaic virus (Hordeivirus), Barley yellow mosaic virus (Bymovirus), Bean common mosaic virus (Potyvirus), Bean yellow mosaic virus (Potyvirus), Beet necrotic yellow vein virus (Furovirus), Blackeye cowpea mosaic virus (Potyvirus), Bean common mosaic virus (Potyvirus), Broad bean wilt virus (Fabavirus), Butterbur mosaic virus (Carlavirus), Carnation mottle virus (Carmovirus), Carnation vein mottle virus (Potyvirus), Cauliflower mosaic virus (Caulimovirus), Chrysanthemum mild mottle virus (Cucumovirus), Tomato aspermy virus (Cucumovirus), Chrysanthemum stunt viroid (Viroids), Citrus mosaic virus, Citrus tristeza virus (Closterovirus), Clover yellow vein virus (Potyvirus), Cocksfoot mottle virus (Sobemovirus), Cucumber green mottle mosaic virus (Tobamo virus), Cucumber mosaic virus (Cucumovirus), Cycas necrotic stunt virus (Nepovirus), Dasheen mosaic virus (Potyvirus), Grapevine Algerian latent virus (Tombusvirus), Konjac mosaic virus (Potyvirus), Melon necrotic spot virus (Carmovirus), Mulberry ringspot virus (Nepovirus), and Narcissus mosaic virus (Potexvirus). Plant viruses are viruses affecting plants. Additional examples of viruses affecting plants include Odontoglossum ringspot virus (Tobamovirus), Papaya ringspot virus (Potyvirus), Peach latent mosaic viroid, Peanut mottle virus (Potyvirus), Peanut stripe virus (Potyvirus), Bean common mosaic virus (Potyvirus), Peanut stunt virus (Cucumovirus), Potato virus A (Potyvirus), Potato virus M (Carlavirus), Potato virus S (Carlavirus), Potato virus X (Potexvirus), Potato virus Y (Potyvirus), Prune dwarf virus (Ilarvirus), Prunus necrotic ringspot virus (Ilarvirus), Radish mosaic virus (Comovirus), Rice black streaked dwarf virus (Fijivirus), Rice dwarf virus (Reovirus), Rice grassy stunt virus (Tenuivirus), Rice stripe virus (Tenuivirus), Rice tungro spherical virus (Sequivirus), Rice waika virus, Rice tungro spherical virus (Sequivirus), Ryegrass mottle virus, Satsuma dwarf virus (Nepovirus), Soil-borne wheat mosaic virus (Furovirus), Southern bean mosaic virus (Sobemovirus), Soybean mosaic virus (Potyvirus), Soybean stunt virus (Cucumovirus), Cucumber mosaic virus (Cucumovirus), Tobacco mosaic virus (Tobamovirus), Tobacco mosaic virus (Tobamovirus), Tomato mosaic virus (Tobamovirus), Tobacco necrosis virus (Necrovirus), Tobacco rattle virus (Tobravirus), Tobacco ringspot virus (Nepovirus), Tomato aspermy virus (Cucumovirus), Tomato black ring virus (Nepovirus), Tomato mosaic virus (Tobamovirus), Tomato ringspot virus (Nepovirus), Tomato spotted wilt virus (Tospovirus), Turnip mosaic virus (Potyvirus), Watermelon mosaic virus 1 (Potyvirus), Papaya ringspot virus (Potyvirus), Watermelon mosaic virus 2 (Potyvirus), Wheat yellow mosaic virus (Bymovirus), and Zucchini yellow mosaic virus (Potyvirus).
10. The method of claim 5, wherein the plant pathogen or pest is selected from the group consisting of Botrytis cinerea, Alternaria solani, and Phytophthora infestans.
11. The method of any one of claims 1-10, wherein the miPEP comprises the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID
NO: 16, with zero, one, two, three, or four amino acid substitutions, insertions, or deletions, and the plant is an Arabidopsis plant.
12. The method of any one of claims 1-10, wherein the miPEP comprises the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22, with zero, one, two, three, or four amino acid substitutions, insertions, or deletions, and the plant is a strawberry plant.
13. The method of any one of claims 1-10, wherein the miPEP comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID
NO: 19, with zero, one, two, three, or four amino acid substitutions, insertions, or deletions, and the plant is a Phaseolus vulgaris plant.
14. The method of any one of claims 1-10, wherein the miPEP comprises the amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, with zero, one, two, three, or four amino acid substitutions, insertions, or deletions, and the plant is a tomato plant.
15. The method of any one of claims 1-14, wherein the oligopeptide is provided by expressing a nucleic acid encoding the miPEP.
16. Tire method of claim 15, wherein the nucleic acid is operably linked to a heterologous promoter.
17. The method of claim 15 or claim 16, wherein the heterologous promoter is a constitutive promoter, a tissue specific promoter, or an inducible promoter.
18. The method of claim 17, wherein the inducible promoter is induced by fungal infection, optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., phenylalanine ammonia lyase, chaicone synthase promoters), a gene that modifies plant cell walls (e.g., hydroxyproline -rich glycoprotein, glycinc-rich protein, and peroxidase promoters), a gene encoding an enzyme that degrade fungal cell walls (e.g., chitinase or glucanase promoters), or a gene encoding a thaumatin-like protein, or or optionally a maize Mis 1 promoter or a flax Fis 1 promoter.
19. The method of any one of claims 1-14, wherein the miPEP is provided by application to the plant or a part thereof.
20. The method of claim 19, wherein the plant part is a leaf, a bud, a root, a shoot, a floral part, or a seed.
21. The method of claim 19 or 20, wherein the miPEP is applied as a coating to the seed prior to planting.
84
RECTIFIED SHEET (RULE 91) ISA/EP
22. The method of any one of claims 1-14, wherein the miPEP is provided by application to the soil in which the plant is planted.
23. The method of any one of claims 1-14, wherein the miPEP is provided by addition to water provided to the plant.
24. The method of any one of claims 1-14, wherein the oligopeptide is provided following harvest to seeds, fruits, and/or plant parts.
25. The method of any one of claims 19-24, wherein the application is by a spreader, a power duster, a boom sprayer, a hand sprayers, a spray dusters, or a granular applicator.
26. The method of any one of claims 1-25, wherein the miPEP comprises a secretion tag, a tag that promotes entry into a plant cell, or a nuclear localization tag.
27. The method of claim 26, wherein the tag that promotes entry into a plant cell comprises a cell penetrating peptide linked to the amino acid sequence.
28. The method of claim 27, wherein the cell penetrating peptide comprises a protein transduction domain, an amphipathic peptide, a synthetic cationic polypeptide, optionally polylysine, polyhistidine, or polyarginine, a dendrimeric polycationic molecule, a peptide vascular endothelial-cadherin cell penetrating peptide, a transportan cell penetrating peptide, a monomer or dimer of HIV-1 TAT basic domain cell penetrating peptide, a penetratin cell penetrating peptide, a synthetic cationic homoarginine oligopeptide cell penetrating peptide, a gamma zein cell penetrating peptide, a Zea mays knotted 1 cell penetrating peptide, a Saccharomyces pombe TP 10 cell penetrating peptide, a Candida albicans Zebra cell penetrating peptide, an Antennapedia sequence, a TAT sequence , an Antp-3A (Antp mutant) sequence, a Buforin II sequence, a K-FGF sequence, a Ku70 sequence, a prion sequence, a pVEC sequence, a SynBl sequence, a Pep-7 sequence, a HN-1 sequence, a BGSC (Bis-Guanidinium- Spermidine-Cholesterol) sequence, or a BGTC (Bis-Guanidinium-Tren-Cholesterol) sequence.
29. An isolated oligopeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 or the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID
85
RECTIFIED SHEET (RULE 91) ISA/EP NO: 12, or SEQ ID NO: 13, with one, two, three, or four amino acid substitutions, insertions, or deletions, wherein the oligopeptide regulates miR169 or miR396.
30. An isolated oligopeptide comprising the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID
NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID
NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, or the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID
NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID
NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID
NO:37, with one, two, three, or four amino acid substitutions, insertions, or deletions, wherein the peptide comprises a proline at the N-terminus and an aspartic acid at the C-terminus, wherein the oligopeptide regulates miR169 or miR396.
31. The oligopeptide of claim 29 or claim 30, wherein the oligopeptide enhances immunity in a plant.
32. Tire oligopeptide of claim 29 or claim 30, wherein the oligopeptide enhances resistance in a plant to a plant pathogen or pest.
33. The oligopeptide of any one of claims 29-32, comprising the one, two, three, or four amino acid substitutions, insertions, or deletions.
34. The oligopeptide of any one of claims 29-33, wherein the oligopeptide comprises a proline at its N- terminus, an aspartic acid at the C-terminus, or both.
35. The oligopeptide of any one of claims 29-34, wherein the oligopeptide comprises one or more D- amino acids.
36. The oligopeptide of claim 35, wherein the one or more D-amino acids enhance stability of the oligopeptide as compared to an oligopeptide of the same amino acid sequence comprising all L-amino acids.
37. Tire oligopeptide of any one of claims 29-36, wherein the oligopeptide comprises a secretion tag, a tag that promotes entry into a plant cell, or a nuclear localization tag.
86
RECTIFIED SHEET (RULE 91) ISA/EP
38. The oligopeptide of claim 37, wherein the tag that promotes entry into a plant cell comprises a cell penetrating peptide linked to the amino acid sequence.
39. The oligopeptide of claim 38, wherein the cell penetrating peptide comprises a protein transduction domain, an amphipathic peptide, a synthetic cationic polypeptide, optionally polylysine, polyhistidine, or polyargmine, a dendrimenc polycationic molecule, a peptide vascular endothelial-cadherin cell penetrating peptide, a transportan cell penetrating peptide, a monomer or dimer of HIV-1 TAT basic domain cell penetrating peptide, a penetratin cell penetrating peptide, a synthetic cationic homoarginine oligopeptide cell penetrating peptide, a gamma zein cell penetrating peptide, a Zea mays knotted 1 cell penetrating peptide, a Saccharomyces pombe TP 10 cell penetrating peptide, a Candida albicans Zebra cell penetrating peptide, an Antennapedia sequence, a TAT sequence , an Antp-3A (Antp mutant) sequence, a Buforin 11 sequence, a K-FGF sequence, a Ku70 sequence, a prion sequence, a pVEC sequence, a SynBl sequence, a Pep-7 sequence, a HN-1 sequence, a BGSC (Bis-Guanidinium- Spermidine-Cholesterol) sequence, or a BGTC (Bis-Guanidinium-Tren-Cholesterol) sequence.
40. A composition comprising the oligopeptide of any one of claims 29-39 with an agriculturally acceptable formulant that can include water, organic solvents, paraffinic oils, vegetable oils, dispersants, emulsifiers, wetting agents, buffering agents, hydrotrope agents, rheology modifiers, antifoam agents and defoamers, antifreeze agents, biocides, dyes, polymer walls, catalysts, thermosetting materials, cross-polymerizing agents, UV protectants, antioxidants, and chelating agents.
41. A composition comprising the oligopeptide of any one of claims 29-39 with an agriculturally acceptable carrier, diluent, or excipient.
42. The composition of claim 41, wherein the agriculturally acceptable carrier, diluent, or excipient comprises a buffer and the pH is in the range of from about 3.0 to about 9.0 or about 4.5 to about 8.0.
43. The composition of any one of claims 40-42, wherein the oligopeptide is in a concentration range of from about 0.1 pg/ml to about 100 mg/ml, or from about 5 pg/ml to about 5 mg/ml.
44. The composition of any one of claims 40-43, wherein the composition is in the form of a finely- divided particulate solid, granules, pellets, wettable powders, dust, an aqueous suspension, a dispersion, a gel, or an emulsion.
87
RECTIFIED SHEET (RULE 91) ISA/EP
45. The composition of any one of claims 40-44, further comprising one or more other active agents selected from the group consisting of a pesticide, a fertilizer, an insecticide, an attractant, a sterilizing agent, an acaricide, a nematocide, a herbicide, a biostimulant, a biological, and a growth regulator.
46. A plant immunity enhancing peptide identified by: a) selecting an miRNA from a miR169 family or a miR396 family; b) identifying an open reading frame (ORF) from 12 to 303 nucleotides in length contained in the 5' or 3' portion of the primary transcript sequence of the miRNA; c) providing a first plant cell and second plant cell that both express the miRNA and are the same type of plant; d) exogenously adding to the first plant (i) a peptide encoded by the ORF, or (ii) a nucleic acid sequence encoding the peptide; e) comparing immunity in the first plant cell and the second plant cell after step d), wherein a change of the immunity of the first plant compared to the immunity of the second plant indicates that the miRNA is an immunity enhancing immunity enhancing miRNA and the peptide is an immunity enhancing micropeptide.
47. A nucleic acid encoding the oligopeptide of any one of claims 29-39.
48. A nucleic acid construct comprising the nucleic acid of claim 46, operably linked to a promoter.
49. The nucleic acid construct of claim 48, wherein the promoter is a heterologous promoter.
50. Tire nucleic acid construct of claim 48 or claim 49, wherein the promoter is a constitutive promoter, a tissue specific promoter, a development stage specific promoter, or an inducible promoter.
51. The nucleic acid construct of claim 50, wherein the inducible promoter is induced by fungal infection, optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., phenylalanine ammonia lyase, chaicone synthase promoters), a gene that modifies plant cell walls (e.g., hydroxyproline -rich glycoprotein, glycine -rich protein, and peroxidase promoters), a gene encoding an enzyme that degrade fungal cell walls (e.g., chitinase or glucanase promoters), or a gene encoding a thaumatin-like protein, or a maize Mis 1 promoter or a flax Fis 1 promoter.
52. A cell comprising the nucleic acid of claim 47 or the nucleic acid construct of any one of claims 48- 51.
88
RECTIFIED SHEET (RULE 91) ISA/EP
53. The method of any one of claims 1-27, the oligopeptide of any one of claims 28-38, the composition of any one of claims 39-44, the immunity enhancing peptide of claim 45, the nucleic acid of claim 46, the nucleic acid construct of any one of claims 47-50, or the cell of claim 51, wherein:
(a) the miPEP is selected from the group consisting of GwmiRl 69g, QmniR 169m. MP19114, MP19127, MP19478, MP19474, MP19476, and MP19472, wherein the miPEP regulates an immunity enhancing miRNA and the plant is a soybean plant;
(b) the miPEP, the oligopeptide, or the immunity enhancing peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 54-59 and 66-79, wherein the miPEP, the oligopeptide, or the immunity enhancing peptide regulates miR169 and the plant is a soybean plant;
(c) the miPEP, the oligopeptide, or the immunity enhancing peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 54-59 and 66-79 with one, two, three, or four amino acid substitutions, insertions, or deletions, wherein the miPEP, the oligopeptide, or the immunity enhancing peptide regulates miR169 and the plant is a soybean plant;
(d) the miPEP, the oligopeptide, or the immunity enhancing peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 54-59 and 66-73, wherein the peptide comprises a proline at the N-terminus and/or an aspartic acid at the C-terminus, wherein the miPEP, the oligopeptide, or the immunity enhancing peptide regulates miR169 and the plant is a soybean plant; and/or
(e) the miPEP, the oligopeptide, or the immunity enhancing peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 54-59 and 66-79 with one, two, three, or four amino acid substitutions, insertions, or deletions, wherein the peptide comprises a proline at the N-terminus and/or an aspartic acid at the C-terminus, wherein the miPEP, the oligopeptide, or the immunity enhancing peptide regulates miRl 69 and the plant is a soybean plant; and
(f) optionally in each case of (a)-(e), the plant pathogen or pest is Phakopsora pachyrhizi.
54. A method of increasing immunity in a plant comprising providing to the plant an oligopeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79 and/or providing to the plant an oligopeptide comprising an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions compared to the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79.
55. A method of reducing fungal growth on a plant comprising providing to the plant an oligopeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79 and/or providing to the plant an oligopeptide comprising an amino acid sequence with one, two, three, or four
89
RECTIFIED SHEET (RULE 91) ISA/EP amino acid substitutions, insertions, or deletions compared to the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79.
56. A method of inhibiting a plant pathogen or pest on a plant comprising providing to the plant an oligopeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79 and/or providing to the plant an oligopeptide comprising an amino acid sequence with one, two, three, or four amino acid substitutions, insertions, or deletions compared to the amino acid sequence set forth in any one of SEQ ID NOs: 54, 58, 59, and 79.
57. The method of claim 55 or 56, wherein the plant pathogen or pest is selected from fungal pathogens or pests, bacterial pathogens or pests, and viral pathogens or pests.
58. The method of claim 55 or 56, wherein the plant pathogen or pest is a fungal pathogen or pest selected from Cercospora spp., Mycosphaerella spp., Glomerella spp., Cladosporium spp., Diplodia maydis. Fusarium oxysporum, Fusarium graminearum, Fusarium monilforme, Fusarium verticillioides, Cochliobolus sativus, Collectotrichum graminicola, Stagonospora nodorum, Stagonospora avenae,
St enocar pella maydis, Sclerotinia minor, Sclerotinia sclerotiorum, Sclerotinia sp., Alternaria spp., Phytophthora spp., Botrytis spp., Pyrenophora tritici-repentis, Phytophthora parasitica, Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichum dematium (Colletotichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v. glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Glomerella glycines, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Fusarium solani; Canola: Albugo Candida, Altenaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassicicola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter michiganese subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium oxysporum, Vertlcillium albo-atrum, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulina briosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium botryosum,
90
RECTIFIED SHEET (RULE 91) ISA/EP Leptotrichila medicaginis; Pseudomonas syringae p.v. atrofaciens, Urocyst is agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v. syringae, Alternaria altemata, Cladosporium herbarum, Fusarium grammearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici, Puccinia graminis f.sp. tritici, Puccinia recondite f.sp. tritici, Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, Claviceps purpurea, Tilletia tritici, Tilleda laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola, Pythium aphanidermatum, Plasmopora halstedii, Sclerotinia sclerotiorum, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum pv. carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis; Com: Colletotrichum graminicola, Fusarium verticillioides var. subglutinans, Erwinia stewartii, F. verticillioides, Gibberella zeae (Fusarium graminearum), Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T (Cochliobolus heterostrophus), Helminthosporium carbonum I, II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III, Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganense subsp. nebraskense, Trichoderma viride, Claviceps sorghi, Pseudomonas avenae, Erwinia chrysanthemi pv. zea, Erwinia carotovora, Corn stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium acremonium, Exserohilum turcicum, C. sublineolum, Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium verticillioides, Alternaria altemata, Bipolaris sorghicola, Helminthosporium sorghi cola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Claviceps sorghi, Rhizoctonia solani, Acremonium
91
RECTIFIED SHEET (RULE 91) ISA/EP strictum, Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum, Pythium arrhenomanes, and Py thrum graminicola.
59. The method of claim 56 or 57, wherein the plant pathogen or pest is a bacterial pathogen or pest selected from Pseudomonas spp., Pantoua spp., and Erwinia spp.
60. The method of claim 56 or 57, wherein the plant pathogen or pest is a viral pathogen or pest selected from cucumber mosaic, tobacco mosaic, and barley yellow dwarf virus, alfalfa mosaic vims (Alfamovirus), Apple chlorotic leaf spot vims (Trichovims), Apple scar skin viroid (Viroids), Arabis mosaic vims (Nepovims), Barley mild mosaic vims (Bymovirus), Barley stripe mosaic vims (Hordeivims), Barley yellow mosaic vims (Bymovims), Bean common mosaic vims (Potyvims), Bean yellow mosaic vims (Potyvims), Beet necrotic yellow vein vims (Furovims), Blackeye cowpea mosaic vims (Potyvims). Bean common mosaic vims (Potyvims), Broad bean wilt vims (Fabavims), Butterbur mosaic vims (Carlavirus), Carnation mottle vims (Carmovims), Carnation vein mottle vims (Potyvims). Cauliflower mosaic vims (Caulimovims), Chrysanthemum mild mottle vims (Cucumovims), Tomato aspermy vims (Cucumovims), Chrysanthemum stunt viroid (Viroids), Citms mosaic vims, Citms tristeza vims (Closterovims), Clover yellow vein vims (Potyvims), Cocksfoot mottle vims (Sobemovims), Cucumber green mottle mosaic vims (Tobamovims), Cucumber mosaic vims (Cucumovims), Cycas necrotic stunt vims (Nepovims), Dasheen mosaic vims (Potyvims), Grapevine Algerian latent vims (Tombusvims), Konjac mosaic vims (Potyvims), Melon necrotic spot vims (Carmovims), Mulberry ringspot vims (Nepovims), and Narcissus mosaic vims (Potexvims). Plant vimses are vimses affecting plants. Additional examples of vimses affecting plants include Odontoglossum ringspot vims (Tobamovims), Papaya ringspot vims (Potyvims), Peach latent mosaic viroid, Peanut mottle vims (Potyvims), Peanut stripe vims (Potyvims), Bean common mosaic vims (Potyvims), Peanut stunt vims (Cucumovims), Potato vims A (Potyvims), Potato vims M (Carlavims), Potato vims S (Carlavims), Potato vims X (Potexvims), Potato vims Y (Potyvims), Pmne dwarf vims (Ilarvims), Prunus necrotic ringspot vims (Ilarvims), Radish mosaic vims (Comovims), Rice black streaked dwarf vims (Fijivims), Rice dwarf vims (Reovims), Rice grassy stunt vims (Tenuivims), Rice stripe vims (Tenuivims), Rice tungro spherical vims (Sequivims), Rice waika vims. Rice tungro spherical vims (Sequivims), Ryegrass mottle vims, Satsuma dwarf vims (Nepovims), Soil-bome wheat mosaic vims (Furovims), Southern bean mosaic vims (Sobemovims), Soybean mosaic vims (Potyvims), Soybean stunt vims (Cucumovims), Cucumber mosaic vims (Cucumovims), Tobacco mosaic vims (Tobamovims), Tobacco mosaic vims (Tobamovims), Tomato mosaic vims
92
RECTIFIED SHEET (RULE 91) ISA/EP (Tobamovirus), Tobacco necrosis virus (Necrovirus), Tobacco rattle virus (Tobravirus), Tobacco ringspot virus (Nepovirus), Tomato aspermy virus (Cucumovirus), Tomato black ring virus (Nepovirus), Tomato mosaic virus (Tobamovirus), Tomato ringspot virus (Nepovirus), Tomato spotted wilt virus (Tospovirus), Turnip mosaic virus (Potyvirus), Watermelon mosaic virus 1 (Potyvirus), Papaya ringspot virus (Potyvirus). Watermelon mosaic virus 2 (Potyvirus), Wheat yellow mosaic virus (Bymovirus), and Zucchini yellow mosaic virus (Potyvirus).
61. The method of claim 56 or 57, wherein the plant pathogen or pest is selected from the group consisting of Septoria tritici, Botrytis cinerea, Phytophthora cactorum, and Phakopsora pachyrhizi s.
62. The method of any one of claims 54-61, wherein the oligopeptide is provided by expressing a nucleic acid encoding the oligopeptide.
63. The method of claim 62, wherein the nucleic acid is operably linked to a heterologous promoter.
64. The method of claim 62 or claim 63, wherein the heterologous promoter is a constitutive promoter, a tissue specific promoter, or an inducible promoter.
65. The method of claim 64, wherein the inducible promoter is induced by fungal infection, optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., phenylalanine ammonia lyase, chaicone synthase promoters), a gene that modifies plant cell walls (e.g., hydroxyproline -rich glycoprotein, glycine-rich protein, and peroxidase promoters), a gene encoding an enzyme that degrade fungal cell walls (e.g., chitinase or glucanase promoters), or a gene encoding a thaumatin-like protein, or optionally a maize Mis 1 promoter or a flax Fis 1 promoter.
66. The method of any one of claims 54-61, wherein the oligopeptide is provided by application to the plant or a part thereof.
67. The method of claim 66, wherein the plant part is a leaf, a bud, a root, a shoot, a floral part, or a seed.
68. Tire method of claim 66 or 67, wherein the oligopeptide is applied as a coating to tire seed prior to planting.
69. The method of any one of claims 54-61, wherein the oligopeptide is provided by application to the soil in which the plant is planted.
93
RECTIFIED SHEET (RULE 91) ISA/EP
70. The method of any one of claims 54-62, wherein the oligopeptide is provided by addition to water provided to the plant.
71. The method of any one of claims 54-62, wherein the oligopeptide is provided following harvest to seeds, fruits, and/or plant parts.
72. The method of any one of claims 66-71, wherein the application is by a spreader, a power duster, a boom sprayer, a hand sprayers, a spray dusters, or a granular applicator.
73. The method of any one of claims 54-72, wherein the miPEP comprises a secretion tag, a tag that promotes entry into a plant cell, or a nuclear localization tag.
74. The method of claim 22, wherein the tag that promotes entry into a plant cell comprises a cell penetrating peptide linked to the amino acid sequence.
75. The method of claim 74, wherein the cell penetrating peptide comprises a protein transduction domain, an amphipathic peptide, a synthetic cationic polypeptide, optionally polylysine, polyhistidine, or polyarginine, a dendrimeric polycationic molecule, a peptide vascular endothelial-cadherin cell penetrating peptide, a transportan cell penetrating peptide, a monomer or dimer of HIV-1 TAT basic domain cell penetrating peptide, a penetratin cell penetrating peptide, a synthetic cationic homoarginine oligopeptide cell penetrating peptide, a gamma zein cell penetrating peptide, a Zea mays knotted 1 cell penetrating peptide, a Saccharomyces pombe TP 10 cell penetrating peptide, a Candida albicans Zebra cell penetrating peptide, an Antennapedia sequence, a TAT sequence , an Antp-3A (Antp mutant) sequence, a Buforin II sequence, a K-FGF sequence, a Ku70 sequence, a prion sequence, a pVEC sequence, a SynBl sequence, a Pep-7 sequence, a HN-1 sequence, a BGSC (Bis-Guanidinium- Spermidine-Cholesterol) sequence, or a BGTC (Bis-Guanidinium-Tren-Cholesterol) sequence.
76. An isolated oligopeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 54, 58, 59, and 79 or the amino acid sequence set forth in one of SEQ ID NOs: 54, 58, 59, and 79 comprising one, two, three, or four amino acid substitutions, insertions, or deletions.
77. The oligopeptide of claim 76, wherein the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.
78. The oligopeptide of claim 76 or claim 77, comprising the one, two, three, or four amino acid substitutions, insertions, or deletions.
94
RECTIFIED SHEET (RULE 91) ISA/EP
79. The oligopeptide of any one of claims 76-78, wherein the oligopeptide comprises a proline at its N- terminus, an aspartic acid at the C-terminus, or both.
80. The oligopeptide of any one of claims 76-79, wherein the oligopeptide comprises one or more D- amino acids.
81. The oligopeptide of claim 80, wherein the one or more D-amino acids enhance stability of the oligopeptide as compared to an oligopeptide of the same amino acid sequence comprising all L-amino acids.
82. The oligopeptide of any one of claims 76-81, wherein the oligopeptide comprises a secretion tag, a tag that promotes entry into a plant cell, or a nuclear localization tag.
83. The oligopeptide of claim 82, wherein the tag that promotes entry into a plant cell comprises a cell penetrating peptide linked to the amino acid sequence.
84. The oligopeptide of claim 83, wherein the cell penetrating peptide comprises a protein transduction domain, an amphipathic peptide, a synthetic cationic polypeptide, optionally polylysine, polyhistidine, or polyarginine, a dendrimeric polycationic molecule, a peptide vascular endothelial-cadherin cell penetrating peptide, a transportan cell penetrating peptide, a monomer or dimer of HIV-1 TAT basic domain cell penetrating peptide, a penetratin cell penetrating peptide, a synthetic cationic homoarginine oligopeptide cell penetrating peptide, a gamma zein cell penetrating peptide, a Zea mays knotted 1 cell penetrating peptide, a Saccharomyces pombe TP 10 cell penetrating peptide, a Candida albicans Zebra cell penetrating peptide, an Antcnnapcdia sequence, a TAT sequence , an Antp-3A (Antp mutant) sequence, a Buforin II sequence, a K-FGF sequence, a Ku70 sequence, a prion sequence, a pVEC sequence, a SynBl sequence, a Pep-7 sequence, a HN-1 sequence, a BGSC (Bis-Guanidinium- Spcrmidinc-Cholcstcrol) sequence, or a BGTC (Bis-Guanidinium-Trcn-Cholcstcrol) sequence.
85. A composition comprising the oligopeptide of any one of claims 76-84 with an agriculturally acceptable formulant that can include water, organic solvents, paraffinic oils, vegetable oils, dispersants, emulsifiers, wetting agents, buffering agents, hydrotrope agents, rheology modifiers, antifoam agents and defoamers, antifreeze agents, biocides, dyes, polymer walls, catalysts, thermosetting materials, cross-polymerizing agents, UV protectants, antioxidants, and chelating agents.
86. A composition comprising the oligopeptide of any one of claims 76-84 with an agriculturally acceptable carrier, diluent, or excipient.
95
RECTIFIED SHEET (RULE 91) ISA/EP
87. The composition of claim 86, wherein the agriculturally acceptable carrier, diluent, or excipient comprises a buffer and the pH is in the range of from about 3.0 to about 9.0, or from about 4.5 to about 8.0.
88. The composition of any one of claims 85-87, wherein the oligopeptide is in a concentration range of from about 0.1 pg/ml to about 1 0 mg/ml, or from about 5 pg/ml to about 5 mg/ml.
89. The composition of any one of claims 85-88, wherein the composition is in the form of a finely- divided particulate solid, granules, pellets, wettable powders, dust, an aqueous suspension, a dispersion, a gel, or an emulsion.
90. The composition of any one of claims 85-89, further comprising one or more other active agents selected from the group consisting of a pesticide, a fertilizer, an insecticide, an attractant, a sterilizing agent, an acaricide, a nematocide, a herbicide, a biostimulant, a biological, and a growth regulator.
91. A nucleic acid encoding the oligopeptide of any one of claims 76-84.
92. A nucleic acid construct comprising the nucleic acid of claim 91, operably linked to a promoter.
93. The nucleic acid construct of claim 92, wherein the promoter is a heterologous promoter.
94. The nucleic acid construct of claim 92 or claim 93, wherein the promoter is a constitutive promoter, a tissue specific promoter, a development stage specific promoter, or an inducible promoter.
95. The nucleic acid construct of claim 94, wherein the inducible promoter is induced by fungal infection, optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., phenylalanine ammonia lyase, chaicone synthase promoters), a gene that modifies plant cell walls (e.g., hydroxyproline -rich glycoprotein, glycine -rich protein, and peroxidase promoters), a gene encoding an enzyme that degrade fungal cell walls (e.g., chitinase or glucanase promoters), or a gene encoding a thaumatin-like protein, or a maize Misl promotor or a flax Fisl promoter.
96. A cell comprising the nucleic acid of claim 91 or the nucleic acid construct of any one of claims 92- 95.
96
RECTIFIED SHEET (RULE 91) ISA/EP
EP24707495.8A 2023-02-23 2024-02-23 Micropeptides for improving plant immunity and their application Pending EP4669656A1 (en)

Applications Claiming Priority (4)

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US202363486652P 2023-02-23 2023-02-23
US202363486653P 2023-02-23 2023-02-23
US202363486654P 2023-02-23 2023-02-23
PCT/EP2024/054668 WO2024175780A1 (en) 2023-02-23 2024-02-23 Micropeptides to improve plant immunity and application thereof

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