SAR PEPTIDE SIGNAL · Peptide-mediated signalling in plant disease resistance

Final Activity Report Summary - SAR PEPTIDE SIGNAL (Peptide-mediated signalling in plant disease resistance)

Plants use complex recognition and response mechanisms to protect themselves from pathogen attack. Major resistance (R) genes specify recognition of pathogens carrying the corresponding avirulence genes leading to the rapid activation of a battery of inducible defences including reinforcements of the cell wall, synthesis of phytoalexin antibiotics and deployment of pathogenesis-related (PR) proteins such as chitinases and glucanases. This battery of induced defences is often accompanied by the collapse of challenged plant cells in the hypersensitive response (HR), which results in a restricted lesion clearly delimited from surrounding healthy tissue. T-DNA activation tagging provides a means of generating dominant, gain-of-function mutations to uncover genes that affect resistance even where there is considerable redundancy and/or cross talk within and between signalling pathways. Using this approach, in the host laboratory has been identified an Arabidopsis gene, CDR1 (Constitutive Disease Resistance 1), over-expression of which leads to enhanced resistance to bacterial pathogens. CDR1 encodes an aspartic protease that releases an endogenous peptide elicitor of salicylic acid-dependent inducible resistance-related responses including PR induction, micro-oxidative bursts and micro-HRs. The aim of this project was to get insights about the role of CDR1 in the complex reiterative signal networks underlying local and systemic acquired resistance with the following objectives: 1. How CDR1 is integrated within the disease resistance signal network To position CDR1 relative to possible downstream signal components, we used transgenic plants containing CDR1 under the control of the dexamethasone inducible TA promoter (TApr::CDR1) that has been characterised. We then crossed TApr::CDR1 plants with the following key mutant/transgenic phenotypes: npr1-1, eds1-1, pad4-5, dir1-1, rboh D, rboh F, rboh D/F and sid 2-2. DAB staining in TA:CDR1/rbohF plants suggests that rbohF and not rbohD is the NADPH oxidase involved in CDR1-dependent H2O2 production. Pathogenesis-related genes has been analysed in the crosses to identify the downstream of CDR1-dependent gene induction. The results obtained suggest that CDR1-dependent PR1 induction seems to be SA dependent. Furthermore, to clarify if CDR1-D dwarf phenotype and disease resistance is fully dependent on SA and/or NPR1, CDR1-D plants have been crossed with sid2-2 and npr1-1. Analyses on these plants are in progress. 2. Nature of the peptide signal generated by CDR1 action I have developed an analytical method for metabolite profiling of apoplast and petiole exudates with an initial focus on jasmonic acid and other oxylipins. This method has allowed me analyse the AOS products of apoplast and petiole exudates of key mutants using gas chromatography-mass spectrometry (GC-MS). Experiments have been done to compare lipid and oxylipin profiles in the apoplastic fluid from control and TApr::CDR1 plants and likewise the petiole (phloem) exudates from leaves of these plants. However, no differences have been detected between these samples. Also, profiles obtained from leaves of wild type plants inoculated with avirulent P. syringae or not, have been obtained but no difference has been found. Probably possible changes in oxilipin profile from our samples (petiole exudates and apoplasts) were below the detection limit of the MS. Then, to determine the nature of the peptide signal generated by CDR1 action we decided to apply an alternative strategy by comparison of the apoplast proteome from control and induced tissues. A band about 15 KDa that cross react with CDR1 antibody has been observed in WT plants (extracts and apoplasts) after 24 h pathogen induction (Pst, Pst AvrRpm1). Plants overexpressing CDR1 with a His tag will be used to concentrate the peptide of interest and facilitate its identification.

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