Unravelling Plant Systemic Immunity; a genetic and metabolomic dissection of systemic acquired resistance

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Plants defend themselves against various pathogens by the induction of both local and systemic responses. In classical gene-for-gene interactions, R proteins recognition usually leads to a localised hypersensitive response and pathogen resistance. The majority of such interactions also result in systemic immunity to subsequent infection by a range of normally virulent pathogens. Once initiated, this systemic acquired resistance (SAR) is broad spectrum and durable. A mechanistic understanding of SAR requires the comprehensive analysis of local signal generation, its transmission, subsequent perception, and activation of signalling networks in distant tissues. A necessary prerequisite is the capability to temporally dissect these sequential events in a genetically manipulable pathosystem. We have achieved this initial goal using a combination of genetic mutants, real-time imaging and RNA blot analyses. We have also analysed global gene expression patterns of immunised and non-immunised systemic tissue at the optimal inductive timepoint. These data have revealed the coordinate up-regulation of specific primary and secondary metabolic pathways involved in jasmonate, phenylpropanoid and glucosinolate biosynthesis. Independent genetic studies with defence mutants further implicate a role for jasmonates in (i) primary signal generation and (ii) orchestrating the early metabolic reprogramming that underpins SAR. In this project, we will first define targeted metabolite profile signatures of leaves induced and non-induced for SAR. This approach will employ LCMS- and GCMS- based analysis of jasmonate and its intermediates, phenylpropanoids and glucosinolates. In parallel, we will use mutants in (i) key regulatory steps of the tryptophan biosynthetic, phenylpropanoid and glucosinolate pathways and (ii) mutants in various steps in JA biosynthetic or signal response pathways to determine their contribution to perception and elaboration of systemic immunity to both biotrophic and necrotrophic pathogens. A subset of mutants showing differential responses will be further analysed through integrated transcriptions and metabolomics. Our goal is to dissect both the JA-dependent and independent signalling pathways underlying systemic immunity and to associate those pathways with particular modes of infection. To reveal more about the upstream long-distance signalling molecules that initiate SAR, we will undertake comparative metabolomic analysis of phloem exudates derived from avirulent- and virulent-challenged and unchallenged leaves in a range of selected genetic backgrounds. Using GCMS and novel nano-LCMS methods, datasets derived from this global unbiased screen will be subjected to state-of-the art informatics data-mining to generate candidate compound lists. These datasets will additionally be used to interrogate emerging database, eg. MeT-RO, as and when these resources become publicly accessible. Biological significance of candidate signal molecules and pathways will be tested via graft-based complementation of mutants which are compromised in generation of the mobile SAR signal. The outcomes of this work will include (i) clarification of the interacting roles of multiple secondary metabolite pathways in generating SAR responses to diverse pathogen classes, (ii) improved definition of genes and candidate molecules involved in long-distance signal transmission and (iii) inputs into a revision of models for SAR. In addition, the comparisons of transcript and metabolite profiles will provide new insights into the utility of transcriptomics at predicting actual changes in levels of functional gene products.