From health to sickness; the metabolomics transition associated with plant disease and defense.

As plants are firmly rooted to the ground, they are open to attack by a wide variety of nasties such as insects, fungi, bacteria and viruses. Fascinatingly, despite this constant invasion of privacy, disease is an exception due to the deployment of an extraordinary complex and highly effective network of synergistic defensive strategies -collectively known as basal defense or non-host resistance. Many of these defensive barriers are the combination of actions of one or more metabolites - small chemicals. Metabolites are synthesised by the plant and act in a multitude of ways - as signals, as sedatives or even as toxins - to individually or cooperatively prevent pathogen ingress. When pathogens, such as bacteria, do successfully invade a plant, they themselves employ a variety of strategies to further suppress or evade host defenses and manipulate host metabolism to provide nutrients for their multiplication. In sum, they turn their intercellular environment into a cosy apartment with food and nutrients online. They achieve this 'success' by actively injecting proteins into the plant cell via what is basically a syringe-like structure known as a type III secretion system. These 'effector' proteins manipulate host transcription (expression of genes which are the template for protein synthesis) and by definition protein expression, to orchestrate a complex network of signalling events. Occasionally, one or more of these effector proteins is recognised and triggers an alarm signal that both locally and systemically induces immunity, leading to disinfection of the plant. Our studies underpinning this proposal have used a technique known as transcriptional profiling to look at how the expression of all plant genes is modified by various pathogen challenges. Analysis of the very complex expression patterns revealed families of genes specifically involved in basal defense and disease. In particular, we found the successful pathogens induced gene expression patterns modified from those seen in unsuccessful attempts to infect. These data represent a significant milestone in our understanding of plant defense as many of these genes encode proteins which themselves produce or modify metabolites that might enhance or interfere with plant immunity. Through a combination of modern technologies we now wish to discover the chemicals that coordinate defense. We will attempt to look at all the small molecules / a procedure known as metabolomics or metabolite profiling / and identify which ones change in quantity following specific treatments. Profiling can be targeted (to identify known compounds) or non-targeted, measuring the pattern of changes. New technologies allow us to undertake large scale metabolic profiling to identify differences between plant tissues treated in different ways or undergoing different reactions. This way we can identify metabolites that differ, even though we may not actually be able to identify the metabolite from the preliminary screen. By using this technology in combination with mutants which cannot fully activate specific defense pathways, we will obtain clues to how the important compounds protect or destablise resistance and maybe even identity the signal molecules that start the whole process of defense. In short, we aim to identify metabolite 'signatures' specifically associated with various aspects of defense. The long-term outcome of this work will be better understanding of the small molecules (i) recruited for plant defense and (ii) those metabolites associated with successful infections. These results will inform future strategies aimed toward manipulating plant responses to develop broad spectrum immunity to pathogens.

Our interests lie in the extent and manner in which basal defenses are deployed and how microbial pathogen effectors, delivered through the type III secretion apparatus, overcome or suppress basal defenses and redirect normal host metabolism to facilitate pathogen multiplication. Experiments underpinning this project used variants of the Pseudomonas syringae pv. tomato DC3000 and its host Arabidopsis thaliana to dissect the dynamics of basal defense at the level of transcription and determine how this evolutionary conserved process is modulated by the activities of pathogen effector proteins. Further work has investigated the role of red-ox signaling on establishing the 'defense zone' of cells surrounding an expanding lesion, which both contains the pathogen and triggers local acquired resistance. Reprogramming of secondary metabolism is often associated with transcriptional regulation and, as detailed in our proposal, extensive informatics analyses of our transcript profiling studies have enabled insight into some probable metabolic pathways which are modulated. These data now provide the opportunity to study, the major biochemical changes that contribute to pathogenicity and defense in a co-ordinated manner using both targeted and unbiased metabolomic approaches. A particular focus of this study are the flux of metabolites to the plant cell wall during the rapid expression of basal defense, alterations in red-ox chemistry and changes in plant hormones, phenylpropanoids and carbohydrate metabolism which may enhance bacterial virulence. This study will provide the first opportunity to correlate how modulation of the defence transcriptional network is translated into metabolic perturbations. As no one metabolite profiling methodology can be totally comprehensive we propose a hierarchical approach to analyse spatial and temporal aspects of metabolome differences during interactions between Arabidopsis, or selected mutants and different Pseudomonas strains using Met-Ro and in house facilities. Met-RO will be used to produce highly accurate and quantitative NMR-MS fingerprints of baseline samples to capture any gross metabolome signals reflecting differences between samples. ESI-MS and GC-tof-MS fingerprinting will be performed in parallel to produce a comprehensive overview of the pathogen modified metabolomes. Where ESI-MS, NMR-MS and GC-tof-MS fingerprinting suggested robust differences, then some of the sample replicates will be used to generate high resolution ESI-MS fingerprints using LC-ESI-FTICR-MS/MSn analysis in Data Dependent Scan mode and both ionisation modes. This analysis will provide a 'bench mark' for the total diversity of individual metabolites for each host-microbe combination and guide further analyses using HPLC and soft ionisation. Advanced Met-Ro data analysis employing Decision Tree analysis and Genetic Algorithms (coupled to Multiple Linear Regression, PLS or LDA) will be used to mine out metabolite peaks and NMR signals that are discriminatory between samples representing different temporal phases of defense or disease for further investigation. Wye has a particular focus on (i) targeted metabolite analysis based on prediction derived from analysis of transcriptomic studies and (ii) temporal changes in cell wall metabolites during disease and defense responses. Data resulting from NMR-MS and GC-TOF-MS analyses will also used to guide further analyses by LC-SPE-NMR-MS approaches (MetRo). In addition to GC-tof-MS fingerprinting and LC-ESI-FTICR-MS/MS(n), Aberystwyth will use LC-coulometry to profile red-ox changes during the HR and development of local acquired resistance. Informatics will enable generation of predictive models of the metabolomic signature of a diseased plant and dynamic changes associated with the infection interface and cell wall alterations. Moreover, the datasets generated here will underpin a more comprehensive systems biology approach to infection and defense.