Sciences

Salicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease-resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB-A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauli¯ower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c-SAS plants) or to the chloroplast (p-SAS plants). In p-SAS plants, the amount of free and conjugated SA was increased more than 20-fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P-SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwar®sm could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a signi®cant threefold increase in conjugated SA, probably re¯ecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR-1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.

The endogenous plant hormones salicylic acid (SA) and jasmonic acid (JA), whose levels increase on pathogen infection, activate separate sets of genes encoding antimicrobial proteins in Arabidopsis thaliana. The pathogen-inducible genes PR-1, PR-2, and PR-5 require SA signaling for activation, whereas the plant defensin gene PDF1.2, along with a PR-3 and PR-4 gene, are induced by pathogens via an SA-independent and JA-dependent pathway. An Arabidopsis mutant, coi1, that is affected in the JA-response pathway shows enhanced susceptibility to infection by the fungal pathogens Alternaria brassicicola and Botrytis cinerea but not to Peronospora parasitica, and vice versa for two Arabidopsis genotypes (npr1 and NahG) with a defect in their SA response. Resistance to P. parasitica was boosted by external application of the SA-mimicking compound 2,6-dichloroisonicotinic acid [Delaney, T., et al. (1994) Science 266, 1247-1250] but not by methyl jasmonate (MeJA), whereas treatment with MeJA but not 2,6-dichloroisonicotinic acid elevated resistance to Alternaria brassicicola. The protective effect of MeJA against A. brassicicola was the result of an endogenous defense response activated in planta and not a direct effect of MeJA on the pathogen, as no protection to A. brassicicola was observed in the coi1 mutant treated with MeJA. These data point to the existence of at least two separate hormone-dependent defense pathways in Arabidopsis that contribute to resistance against distinct microbial pathogens.

Induced resistance was studied in the model pathosystem Arabidopsis -Phytophthora brassicae (formerly P. porri ) in comparison with the agronomically important late blight disease of potato caused by Phytophthora infestans . For the quantification of disease progress, both Phytophthora species were transformed with the vector p34GFN carrying the selectable marker gene neomycine phosphotransferase ( nptII ) and the reporter gene green fluorescent protein ( gfp ). Eighty five per cent of the transformants of P. brassicae and P. infestans constitutively expressed GFP at high levels at all developmental stages both in vitro and in planta . Transformants with high GFP expression and normal in vitro growth and virulence were selected to quantify pathogen growth by measuring the in planta emitted GFP fluorescence. This nondestructive monitoring of the infection process was applied to analyse the efficacy of two chemical inducers of disease resistance, a functional SA-analogue, benzothiadiazole (BTH), and βaminobutyric acid (BABA) which is involved in priming mechanisms of unknown nature. BABA pre-treatment (300 µ M ) via soil drench applied 24 h before inoculation completely protected the susceptible Arabidopsis accession Landsberg erecta (Ler) from infection with P. brassicae . A similar treatment with BTH (330 µ M ) did not induce resistance. Spraying the susceptible potato cultivar Bintje with BABA (1 m M ) 2 days before inoculation resulted in a phenocopy of the incompatible interaction shown by the resistant potato cultivar Matilda while BTH (1.5 m M ) did not protect Bintje from severe infection. Thus, in both pathosystems, the mechanisms of induced resistance appeared to be similar, suggesting that the Arabidopsis -P. brassicae pathosystem is a promising model for the molecular analysis of induced resistance mechanisms of potato against the late blight disease.

New insights into the phenomenon of systemic acquired resistance have been gained in recent years, by the use of techniques in molecular genetics and biology that have replaced the largely descriptive approach of earlier work. The isolation of mutants in the signal transduction pathway from induction to expression of resistance as well as the use of transgenic plants over-expressing or suppressing the expression of putative candidate genes involved in systemic acquired resistance and its signalling have identified several steps in the establishment of plant resistance. In this review the latest developments implicating salicylic acid as a signal molecule in systemic resistance are discussed and contrasted with new signalling pathways which, seemingly, are based on alternative mechanisms.

The influence of site on resistance of grapevine (cv. Chasselas) to Plasmopara viticola was evaluated. Grapevine leaves from three vineyards in the region of Lake Neuchâtel (Switzerland) were tested for their susceptibility to P. viticola in the lab in five successive years (2004–2008), and the expression levels of four selected defence-related genes (Glucanase, Lipoxygenase 9, 9-cis epoxycarotenoid dioxygenase, Stilbene synthase) were studied in 1 year. In all 5 years of examination, differences between sites were substantial. In four out of 5 years, plants from site Hauvernier were much less susceptible to P. viticola than plants from site Auvernier. In another year, differences were less pronounced but still significant for one leaf age. Susceptibility of plants from a third site (Concise) varied from year to year. Differences in the genetic background were excluded by microsatellite analysis. Differences in susceptibility were mirrored in the constitutive expression pattern of four defence-related genes, with samples from the Hauterive site clearly separated from samples of the other two sites in redundancy analysis. Furthermore, it was evaluated whether site-specific resistance can be modulated by agronomic practices such as the application of organic amendments. In two commercial vineyards (cv. Pinot noir), soils had either not (control) or yearly (compost) been amended with a compost for the last 9 years. Leaves from plants grown in any of the two treatments did not differ in their susceptibility to P. viticola in both years of examination. Additionally, under controlled conditions, none of 19 different composts amended to the substrate of grapevine seedlings or cuttings affected their susceptibility to P. viticola, but 8 out of 19 composts reduced severity in the control bioassay Arabidopsis thaliana—Hyaloperonospora arabidopsidis, indicating that a modulation of site-specific susceptibility of grapevine plants by organic amendments is at the very least, difficult.

The mechanisms controlling plant resistance to necrotrophic fungal pathogens are poorly understood. We previously reported on Ep5C, a gene shown to be induced by the H 2 O 2 generated during a plant-pathogen interaction. To identify novel plant components operating in pathogen-induced signaling cascades, we initiated a large-scale screen using Arabidopsis thaliana plants carrying the b-glucuronidase reporter gene under control of the H 2 O 2 -responsive Ep5C promoter. Here, we report the identification and characterization of a mutant, ocp3 (for overexpressor of cationic peroxidase 3), in which the reporter construct is constitutively expressed. Healthy ocp3 plants show increased accumulation of H 2 O 2 and express constitutively the Glutathione S-transferase1 and Plant Defensine1.2 marker genes, but not the salicylic acid (SA)-dependent pathogenesisrelated PR-1 gene. Strikingly, the ocp3 mutant shows enhanced resistance to the necrotrophic pathogens Botrytis cinerea and Plectosphaerella cucumerina. Conversely, resistance to virulent forms of the biotrophic oomycete Hyaloperonospora parasitica and the bacterial pathogen Pseudomonas syringae pv tomato DC3000 remains unaffected in ocp3 plants when compared with wild-type plants. Consistently with this, ocp3 plants are not affected in SA perception and express normal levels of PR genes after pathogen attack. To analyze signal transduction pathways where ocp3 operates, epistasis analyses between ocp3 and pad4, nahG, npr1, ein2, jin1, or coi1 were performed. These studies revealed that the resistance signaling to necrotrophic infection in ocp3 is fully dependent on appropriate perception of jasmonic acid through COI1 and does not require SA or ethylene perception through NPR1 or EIN2, respectively. The OCP3 gene encodes a homeodomain transcription factor that is constitutively expressed in healthy plants but repressed in response to infection by necrotrophic fungi. Together, these results suggest that OCP3 is an important factor for the COI1-dependent resistance of plants to infection by necrotrophic pathogens.

Acquired resistance is an important component of the complex disease resistance mechanism in plants, which can result from either pathogen infection or treatment with\synthetic, resistance-inducing compounds. In this study, Arabidopsis, a tractable genetic system, is shown to develop resistance to a bacterial and a fungal pathogen following 2,6dichloroisonicotinic acid (INA) treatment. Three proteins that accumulated to high levels in the apoplast in response to INA treatment were purified and characterized. Expression of the genes corresponding to these proteins was induced by INA, pathogen infection, and salicylic acid, the latter being a putative endogenous signal for acquired resistance.

The effect of the abiotic stress hormone abscisic acid on plant disease resistance is a neglected field of research. With few exceptions, abscisic acid has been considered a negative regulator of disease resistance. This negative effect appears to be due to the interference of abscisic acid with biotic stress signaling that is regulated by salicylic acid, jasmonic acid and ethylene, and to an additional effect of ABA on shared components of stress signaling. However, recent research shows that abscisic acid can also be implicated in increasing the resistance of plants towards pathogens via its positive effect on callose deposition.

We have examined the role of the callose synthase PMR4 in basal resistance and b-aminobutyric acid-induced resistance (BABA-IR) of Arabidopsis thaliana against the hemi-biotrophic pathogen Pseudomonas syringae and the necrotrophic pathogen Alternaria brassicicola. Compared to wild-type plants, the pmr4-1 mutant displayed enhanced basal resistance against P. syringae, which correlated with constitutive expression of the PR-1 gene. Treating the pmr4-1 mutant with BABA boosted the already elevated levels of PR-1 gene expression, and further increased the level of resistance. Hence, BABA-IR against P. syringae does not require PMR4-derived callose. Conversely, pmr4-1 plants showed enhanced susceptibility to A. brassicicola, and failed to show BABA-IR. Wild-type plants showing BABA-IR against A. brassicicola produced increased levels of JA. The pmr4-1 mutant produced less JA upon A. brassicicola infection than the wild-type. Blocking SA accumulation in pmr4-1 restored basal resistance, but not BABA-IR against A. brassicicola. This suggests that the mutant's enhanced susceptibility to A. brassicicola is caused by SA-mediated suppression of JA, whereas the lack of BABA-IR is caused by its inability to produce callose. A. brassicicola infection suppressed ABA accumulation. Pre-treatment with BABA antagonized this ABA accumulation, and concurrently potentiated expression of the ABA-responsive ABI1 gene. Hence, BABA prevents pathogen-induced suppression of ABA accumulation, and sensitizes the tissue to ABA, causing augmented deposition of PMR4-derived callose.

We report on constitutive subtilisin3 (csb3), an Arabidopsis mutant showing strikingly enhanced resistance to biotrophic pathogens. Epistasis analyses with pad4, sid2, eds5, NahG, npr1, dth9 and cpr1 mutants revealed that the enhanced resistance of csb3 plants requires intact salicylic acid (SA) synthesis and perception. CSB3 encodes a 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase, the enzyme controlling the penultimate step of the biosynthesis of isopentenyl diphosphate via the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway in the chloroplast. CSB3 is expressed constitutively in healthy plants, and shows repression in response to bacterial infection. We also show the pharmacological complementation of the enhanced-resistance phenotype of csb3 plants with fosmidomycin, an inhibitor of the MEP pathway, and propose that CSB3 represents a point of metabolic convergence modulating the magnitude of SA-mediated disease resistance to biotrophic pathogens.