Characterization of secreted peptides involved in the response to biotic stress in Arabidopsis – STRESS-PEPT

Plant responses to biotic aggressions involve a great diversity of molecules including regulatory proteins and hormones. Among these actors, small secreted peptides, also named peptide phytohormones or phytocytokines, may directly interact with pathogens or act in signalling and cell-to-cell communication. They are produced from non-functional precursors through a maturation process, making characterization difficult only on the basis of their gene sequences. Only a small fraction of the genes liable to encode these secreted peptides has been described and their impact and diversity appears to be seriously underestimated. The main goal of STRESS-PEPT is to better understand the plant responses to biotic stress at the peptidome level and to characterize new molecular actors involved in defence mechanisms. Based on our experience and previous results, we plan to develop and apply a multidisciplinary genome-wide approach combining bioinformatics, differential transcriptomics and peptidomics to identify new secreted peptides in Arabidopsis and to describe their contribution to plant responses to different representative biotrophic and necrotrophic pathogens (oomycete, fungus and bacterial elicitor). The proposed methodology is organized in three complementary tasks:

(i) An original bioinformatics pipeline will be used to screen the Arabidopsis thaliana genome in order to identify genes encoding precursors of secreted peptides, to cluster and classify them in gene families by homology and phylogenetic profiling and to predict the putative mature peptides through a sensitive conserved motif searching method ; these predictions will be integrated to RNAseq transcriptomics analyses applied on Arabidopsis in presence or absence of the different pathogens in order to tag the fraction of the secreted peptide precursor genes that are transcriptionally regulated by pathogen aggression(s).

(ii) The same biological samples will be used to prepare extracts of peptides, from apoplastic fluids and total extracts, through an original protocol optimized for efficient mass spectrometry (MS) analysis: a LC-MS/MS based peptidomics approach will be applied on the different peptide extracts for the identification and the differential quantification of the secreted peptides and to characterize their post-translational modifications.

(iii) A selection of promising peptides, based on transcriptomics and peptidomics data, will be made for functional analyses including assays on knock-out mutants and overexpressing transgenic lines. Synthetic peptide treatments will be performed in order to understand and validate their role in plant-pathogen interactions.

The STRESS-PEPT consortium gathers bioinformaticians, molecular biologists, biochemists and plant pathologists expert of each studied pathosystem as well as a proteomics platform. The already established collaborations between these partners will ensure close cooperation and synergy throughout the 4 year project, and ensure obtaining results of high interest for the plant biology community. All the data generated will be stored and organized for efficient querying in a relational database publicly available at the end of the project. STRESS-PEPT will lead to significant advances in the discovery of new secreted peptides which are key players in danger sensing and the modulation of immune responses. According to their conservation among plant species, these peptides might be used in innovative strategies aiming at jointly optimizing plant quality and resistance, and should open new opportunities for sustainable crop management.