CN118812680B

CN118812680B - Application of endogenous plant peptide in regulation and control of wheat scab and stem basal rot resistance

Info

Publication number: CN118812680B
Application number: CN202410584023.5A
Authority: CN
Inventors: 李刚; 何漪; 董一帆; 张正光; 杨东雷; 夏小波; 王渝华
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2024-05-11
Filing date: 2024-05-11
Publication date: 2026-04-17
Anticipated expiration: 2044-05-11
Also published as: CN118812680A

Abstract

The invention discloses application of three endogenous plant peptides in preventing and treating wheat scab and stem basal rot. Three endogenous Pep precursors (TaPROPEP) can be cut to generate endogenous plant peptides TaPep, the endogenous plant peptides can be used as immune elicitors to cause plant immune response, the infection degree of wheat scab and stem basal rot is reduced, and the resistance of wheat to the scab and the stem basal rot is obviously improved in a greenhouse and in a field.

Description

Application of endogenous plant peptide in regulation and control of wheat scab and stem basal rot resistance

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of three endogenous plant peptides in wheat scab and stem base rot resistance.

Background

Crops are continually subjected to various biotic stresses during growth and cause significant yield losses (Savary,S.,Willocquet,L.,Pethybridge,S.J.,Esker,P.,McRoberts,N.,and Nelson,A.(2019).The global burden of pathogens and pests on major food crops.Nat.Ecol.&Evol.3(3),430-439). fungal pathogens that can cause many devastating diseases, over 2000 million dollars per year, accounting for 10% -20% (www.ars.usda.gov) of the global crop losses. Wheat is one of the most important staple food crops that are widely planted worldwide, particularly susceptible to infection by pathogens. Wheat scab (Fusarium head blight, FHB) is mainly caused by fusarium graminearum (Fusarium graminearum, f.graminearum) complex, one of ten crop diseases worldwide, and is prevalent in wheat planting areas. The disease not only can lead to yield reduction, but also can generate mycotoxin mainly comprising Deoxynivalenol (DON) and endanger human and animal health. The wide use of germicides such as tebuconazole can prevent the food toxin from becoming popular and reduce the yield loss, but cause environmental and health problems (Mawcha,K.T.,Zhang,N.,Wang,Y.,and Yang,W.(2022).Advances in wheat breeding for resistance to Fusarium head blight.Czech J.Genet.Plant.58(4),167-188).

Over the past three decades, significant progress has been made in plant scab resistance studies using genetic approaches and the like. Hundreds of resistance QTLs have been identified at present, but only a few resistance QTLs such as Fhb1 show a stabilizing effect. Thus, there is a need to find and utilize more efficient methods for controlling wheat scab. However, unlike other diseases of wheat, immune-related proteins such as those directly involved in wheat resistance to powdery mildew and rust have not been reported. For example, fhb1 and Fhb7 encode histidine-rich calbindin and glutathione S-transferase, respectively, but are both involved in wheat scab resistance and alleviating toxin contamination (Su,Z.,Bernardo,A.,Tian,B.,Chen,H.,Wang,S.,Ma,H.,Cai,S.,Liu,D.,Zhang,D.,Li,T.,et al.(2019).A deletion mutation in TaHRC confers Fhb1 resistance to Fusarium head blight in wheat.Nat.Genet.51(7),1099-1105). by unknown mechanisms, although the use of plant' S innate defense mechanisms is a potentially effective strategy to increase crop disease resistance, our understanding of how to use such mechanisms to increase wheat scab resistance remains lacking.

In plant innate immune responses, conserved pathogen-associated molecular patterns (PAMPs) produced by pathogenic microorganisms can be recognized by plant cell surface anchor Pattern Recognition Receptors (PRRs), triggering PTI immunity. Many PAMPs act as immune inducers, triggering defensive signals and conferring disease resistance to crops, such as induction of PTI with the bacterial flagellin conserved 22 amino acid peptide flg22, which can enhance resistance of arabidopsis to bacterial diseases. In addition to microbial elicitors, plants secrete and release endogenous immune factors or polypeptides into the extracellular space during pathogen interaction, which are recognized by plasma membrane localized PRRs, activate the PTI response, promoting plant survival (Hou,S.,Liu,D.,and He,P.(2021).Phytocytokines function as immunological modulators of plant immunity.Stress Biol.1(1),8).

In plants, only a few endogenous peptides were identified as plant cytokines involved in plant immune regulation, such as systemin-like peptides of the Solanaceae family, with anti-herbivory defenses. Notably, pep1 (plant elicitor peptide), a plant endogenous peptide consisting of 23 amino acids, was the first discovered arabidopsis thaliana immunoendogenous polypeptide that triggered a labeled PTI response through a cell surface anchored Pep receptor (PEPR), promoting resistance to various stresses (Huffaker,A.,Pearce,G.,and Ryan,C.A.(2006).An endogenous peptide signal in Arabidopsis activates components of the innate immune response.P.Natl.Acad.Sci.103(26),10098-10103).

The Pep endogenous peptide is cleaved by cysteine protease from its precursor protein PROPEP, and ultimately functions as the mature body at the N-terminus. PROPEP proteins are found in many angiosperms such as maize, rice and potato. However, the sequences of these precursor proteins are not conserved, and the resulting Peps mature forms are generally not recognized between species, so the activity of the Peps exciton is species specific. Arabidopsis Pep1 is reported to have functions (Yamaguchi,Y.,Huffaker,A.,Bryan,A.C.,Tax,F.E.,and Ryan,andC.A.(2010).PEPR2 is a second receptor for the Pep1 and Pep2 peptides and contributes to defense responses in Arabidopsis.Plant Cell,22(2),508-522). of enhancing various aspects of plant pathogen resistance, pests, injury stress and the like, corn homologous proteins ZmPep1 and ZmPep3 respectively regulate the resistance (Huffaker,A.,Pearce,G.,Veyrat,N.,Erb,M.,Turlings,Ted C.,Sartor,R.,Shen,Z.,Briggs,S.P.,Vaughan,M.M.,Alborn,H.T.,et al.(2013).Plant elicitor peptides are conserved signals regulating direct and indirect antiherbivore defense.P.Natl.Acad.of Sci.,110(14),5707-5712). of corn to pathogens and pests, rice homologous OsPep3 can enhance the resistance (Shen,W.,Zhang,X.,Liu,J.,Tao,K.,Li,C.,Xiao,S.,Zhang,W.,Li,J.F.(2022).Plant elicitor peptide signalling confers rice resistance to piercing-sucking insect herbivores and pathogens.Plant Biotechnol.J.20(5),991-1005). of crops to pests, the resistance (Shen,W.,Zhang,X.,Liu,J.,Tao,K.,Li,C.,Xiao,S.,Zhang,W.,Li,J.F.(2022).Plant elicitor peptide signalling confers rice resistance to piercing-sucking insect herbivores and pathogens.Plant Biotechnol.J.20(5),991-1005). of crops to diseases and pests can enhance the disease resistance of crops by utilizing endogenous Peps, and the method is an economic and effective way, and although most Peps realizes the protection and defense of plants through immune response, the application and development of the Peps immune inducer still have great challenges at present.

Disclosure of Invention

Based on the studies of the present inventors, it was found for the first time that overexpression of endogenous Pep precursors (TaPROPEP) can reduce the infection degree of wheat scab and stalk rot, and these four endogenous Pep precursors can be cleaved by cysteine proteases to form their respective TaPeps mature body plant peptide excitons (three mature body sequences SEQ ID NO:3 and SEQ ID NO:9 are identical, for example), thereby regulating wheat resistance to scab and stalk rot. The TaPROPEP gene is over-expressed in wheat and has obvious resistance to wheat scab and stem basal rot. Thus, the present invention has been completed.

The invention firstly provides four endogenous Pep precursors (TaPROPEP), and the amino acid sequences of the precursors are shown as SEQ ID No.2, 5, 8 and 11.

Further, the invention provides an expression element, a recombinant vector and a host cell containing the gene.

Preferably, the gene is overexpressed in the plant by transgenic means.

The invention also provides application of the gene in creating disease-resistant transgenic plants, wherein the gene is overexpressed in the transgenic plants by a transgenic method.

Preferably, the plant is a monocotyledonous plant, preferably the plant is wheat. More preferably, the disease resistance is fusarium-induced wheat scab or stalk based rot.

The invention relates to application of four endogenous Pep precursors (TaPROPEP) in disease-resistant process, wherein the over-expression of TaPROPEP gene does not influence the growth and yield of wheat plants, but the over-expression transgenic plant of TaPROPEP gene has obvious resistance to wheat scab and stem basal rot and can inhibit the expansion of gibberella in host cells. In the process of infection of the gibberella, taPROPEP can be cut by wheat cysteine proteinase to form three TaPep mature plant peptides which are used as excitons, so that the resistance to the wheat scab and the stem basal rot is obviously improved. The transgenic plants of the present invention are not affected by normal growth and set.

Further, the invention provides a method for enhancing the resistance of plants to wheat scab and stem basal rot, which is obtained by over-expressing the genes in transgenic plants by a transgenic method, or directly treating plants by using three synthetic TaPep mature plant peptides, for example, the endogenous Pep precursor TaPROPEP is prepared by a genetic engineering method, or is synthesized by a TaPeps mature chemical method.

Furthermore, the present invention also provides an agent for enhancing disease resistance of plants, which is characterized by comprising the TaPep mature polypeptide. The agent can be used for wheat scab or stem basal rot caused by fusarium of wheat crops.

The research of the inventor discovers that three types of wheat which are exogenously sprayed with TaPep mature bodies or over-express Pep precursor genes (TaPROPEP 7 and TaPROPEP 10) show resistance to fusarium graminearum, and can be popularized and applied practically. The invention is beneficial to the cultivation of wheat disease-resistant varieties and provides basis for later screening of high-resistance wheat varieties. For example, the present invention can provide TaPROPEP over-expressed plants, and in addition, the present invention can further provide three TaPep mature plant peptides synthesized as elicitors to treat plants, thereby increasing the resistance of plants to scab and stem basal rot.

Drawings

FIG. 1 is a graph showing that exogenous application TaPep induces wheat scab resistance under greenhouse conditions. Panel A shows pretreatment of TaPep prior to infection of wheat ears by Fusarium graminearum. Panel B is a representative picture of Fielder ears 17 days after infection with Fusarium graminearum pre-treated with 20 μ M TaPep under greenhouse conditions. Panel C shows quantification of the severity of the disease after 10-20 days of infection with F.graminearum as mentioned in panel B.

FIG. 2 is a graph showing that exogenous application TaPep induces wheat scab resistance under field conditions. Panel A is a representative image of wheat ears 15 days after infection with F.graminearum after pretreatment of the Fielder variety under field conditions. Panel B is a quantification of the severity of the disease after infection with Fusarium graminearum mentioned in panel A.

FIG. 3 is a graph showing that exogenous application TaPeps induces resistance of wheat to stalk rot. Panel A shows the onset of wheat stems 14 days after Fusarium graminearum inoculation with 20. Mu.M TaPep-e, taPep-g pretreatment. And B, quantitatively measuring the pathogenic stem length of the wheat stem-based rot.

FIG. 4 shows that overexpression TaPep of the precursor in wheat enhances its resistance to wheat scab. Panel A shows the relative expression levels of TaPROPEP and TaPROPEP10 in over-expressed wheat lines. Panel B is a representative image of wheat ears 15 days after infection of F.graminearum with WT (Fielder), taPROPEP-OE and TaPROPEP-OE under greenhouse conditions. Panel C is a quantitative analysis of the indices of the onset of TaPROPEP-OE and TaPROPEP-OE.

FIG. 5 is a representative image (top) and lesion area (bottom) of WT, taPROPEP7-OE and TaPROPEP-OE leaf infections and Fusarium graminearum at day 5.

FIG. 6 shows that over-expression TaPep of the precursor in wheat can enhance its resistance to wheat stem basal rot. Panel A shows that three plants, WT (Fielder), taPROPEP-OE and TaPROPEP-OE, were inoculated with F.graminearum conidia for 14 days. Panel B shows quantitative determination of the pathogenic stem length of wheat stem-based rot WT and TaPROPEP-OE seedlings.

Detailed Description

The invention will now be illustrated by means of specific examples for a better understanding of the invention, without however being limited thereto.

Example 1 exogenous application TaPep induces wheat resistance to wheat scab

Considering that several peps in arabidopsis and maize regulate immune and biological responses as plant cytokines, we speculate that wheat homologues may also function as disease resistance inducers.

Formation of 4 wheat endogenous Pep precursors (TaPROPEP-10) 3 mature bodies TaPep-e (SEQ ID NO:3, 9), taPep-f (SEQ ID NO: 6), taPep-g (SEQ ID NO: 12), precursor TaPROPEP (SEQ ID NO: 2), taPROPEP9 (SEQ ID NO: 8) TaPep-e, precursor TaPROPEP (SEQ ID NO: 5) TaPep-f, precursor TaPROPEP (SEQ ID NO: 11) TaPep-g.

To verify this hypothesis, we artificially synthesized mature TaPep-e, taPep-f, taPep-g (Sangon Biotech, china) (Table 1) and applied them exogenously to wheat ears (Fielder) and then observed the symptoms of wheat scab onset (FIG. 1A). Under the condition of a greenhouse, the severity of wheat scab of TaPep-e, taPep-f and TaPep-g after pretreatment is obviously lower than that of a control group, and the number of the developed spikes of TaPep-e, taPep-f and TaPep-g after treatment is obviously lower than that of the control group (B and C in fig. 1) through observation and disease index statistics, so that the resistance of wheat to wheat scab is improved by externally applying TaPep-e, taPep-f and TaPep-g.

Furthermore, three groups of exogenous Tapeps were applied to wheat ears (field) under field conditions, and TaPep-e, taPep-f, taPep-g were found to similarly reduce the number of spiked ears that developed under natural conditions, indicating that TaPep-e, taPep-f, taPep-g treatments enhanced resistance to wheat scab (FIG. 2A, B).

TABLE 1

Wherein, the peptide treatment method in the above experiment comprises the following steps:

Dissolving peptide powder in distilled water to working concentration of 0.5-50 μm. For phenotypic analysis, flowers of three central spikelets on one spike were treated with 20. Mu. L TaPep-e, taPep-f or TaPep-g one day before F.graminearum treatment.

The wheat scab phenotype in the above experiment was evaluated as follows:

Fusarium graminearum (Fg 1312 isolate) of Jiangsu China is used as an inoculum to carry out infection of the Fusarium graminearum. The wheat head in the early stage of wheat flower is the optimal period for wheat scab infection. Flowers of an inflorescence center spikelet were infested with 10 μl of Fg1312 conidium suspension (about 1×10 ⁵ spores/mL), covered with plastic bags, and kept moist for 2 days (greenhouse) or 3 days (field), thus ensuring that fusarium graminearum could be infested normally. Wheat scab spread between ears (type II resistance) was assessed by counting symptomatic spikes 7, 9, 11, 13, 15, 17 and 21 days post inoculation.

Example 2 exogenous application TaPep induces wheat resistance to wheat stem basal rot

To explore the role of TaPeps in wheat stem rot caused by fusarium graminearum, wheat stem rot (Fusarium crown rot, FCR) detection was performed after pretreatment of TaPeps wheat seedlings. Pretreatment TaPep-e, g resulted in significantly less length of brown stems than control, taPep-e, taPep-g significantly reduced plaque size of basal stem brown necrosis (a, B in fig. 3), indicating that group III TaPeps also confers wheat resistance to wheat basal stem rot.

Wherein the peptide is prepared by pretreating wheat seedlings with TaPep-e, taPep-g one day before infection of fusarium graminearum spores.

The evaluation method of the wheat stem basal rot symptoms is as follows:

Wheat seeds were sterilized with 70% ethanol for 2 minutes, then rinsed 3-4 times with sterile water and germinated on three-layer soaked filter paper in petri dishes. When the seedling length reached 0.5-1cm, it was immersed in spore suspension of Fg1312 (containing 1X 10 ⁶ spores/mL) for 1-2 minutes. 10 seedlings inoculated with Fusarium graminearum were wrapped in a wet tissue and grown in plastic containers at 22℃and 60% humidity for 16 hours in the growth chamber. Resistance assessment was performed by measuring the length of the dark brown lesions at the basal stem.

Example 3 overexpression TaPep of the precursor in wheat enhances its resistance to wheat scab

For wheat overexpression constructs, the full-length CDS fragment TaPROPEP7 (SEQ ID NO: 1), taPROPEP (SEQ ID NO: 10) was inserted into pUbiGW vector with the Ubiquitin promoter via BamHI site using In-Fusion cloning technology (Clontech, cat. No. 638910). All constructs were transformed into agrobacterium EHA105 strain. Wheat transformation (Goetz H.,Cornelia M.,and Jochen K.(2021).Agrobacterium-Mediated Transformation of Wheat Using Immature Embryos.Rom.Agric.Res.38,99-107(2021)). genotyping of transgenic plants was performed AS described previously using EnviologixQuickStix Kit (Envirologix, catalog No. AS 013). It was further confirmed by qRT-PCR whether T1 and T2 generation lines were overexpressed or homozygous mutation identified using gene specific primer sequencing (a in fig. 4).

Subsequently, wheat transgenic material overexpressing TaPROPEP (TaPROPEP-OE) (SEQ ID NO: 1) and TaPROPEP (TaPROPEP 10-OE) (SEQ ID NO: 10) precursors was identified for scab resistance. After TaPROPEP-OE and TaPROPEP-OE onset under greenhouse conditions, the spikelet onset was significantly less severe than that of wild-type field (B, C in FIG. 4). We also tested disease symptoms caused by fusarium graminearum in the leaves of the over-expressed material. Notably, taPROPEP-OE and TaPROPEP-OE exhibited a significant reduction in lesion area in leaves compared to wild-type (FIG. 5).

The processing method of the in-vitro blade comprises the following steps:

Wild type (Fielder) and transgenic wheat plants were grown in growth chambers at 22 ℃ with photoperiod 16h light and 8h darkness. The 2 week old plant was collected from the secondary leaves and transferred to square dishes containing 1% water agar. mu.L of the conidium suspension (about 5X 10 ⁴ conidium/mL) was cultured in the center of the wheat leaf. Symptoms of infection were recorded 5-6 days after infection using ImageJ (https:// www.computerbild.de/download/ImageJ-422527. Html) to assess necrotic lesions.

Example 4 overexpression TaPep of the precursor in wheat enhances its resistance to wheat stem rot

To understand the role of endogenous TaPep in responding to wheat stem basal rot, we also tested disease symptoms caused by fusarium graminearum in overexpressing material stems. Notably, taPROPEP-OE and TaPROPEP-OE exhibited a significant reduction in the plaque area in the basal stem region compared to the wild-type (FIG. 6A, B).

Claims

1. Application of an endogenous Pep precursor TaPROPEP or its encoding gene in improving plant disease resistance;

The plant in question is wheat; the disease resistance refers to wheat scab or stem rot caused by Fusarium wilt.

The amino acid sequence of the endogenous Pep precursor TaPROPEP is shown in SEQ ID No. 2 or 11.

2. The application of an endogenous Pep precursor TaPROPEP or its encoding gene, an expression element containing the encoding gene, and a recombinant vector containing the encoding gene in the preparation of transgenic plants with enhanced disease resistance;

3. A method for preparing transgenic plants with enhanced disease resistance, characterized by comprising the steps of overexpressing a gene encoding the endogenous Pep precursor TaPROPEP in transgenic plants via transgenic methods, and screening to obtain transgenic plants with enhanced resistance to wheat scab.

4. The application of an endogenous TaPep mature polypeptide in enhancing plant disease resistance, wherein the plant is wheat; the disease resistance refers to wheat scab or stem rot caused by Fusarium wilt;

The amino acid sequence of the endogenous TaPep mature polypeptide is shown in SEQ ID No. 3, 6, 9 or 12.

5. A method for enhancing plant disease resistance, characterized in that the plant is directly treated with endogenous TaPep mature polypeptide to increase its disease resistance;