Type III Effector suppression of basal defense and activation of ABA signalling.

Plants deploy of an extraordinary complex network of synergistic defensive strategies, collectively termed basal defense or non-host resistance. Many of these defensive barriers are metabolites which act individually or cooperatively prevent pathogen ingress. However, bacteria do successfully invade a plant. They achieve their success from activities of a variety of virulence factors, of a chemical or proteinaceous nature. We know bacteria inject a collection of 30-40 proteins into the plant cell and these 'effector' proteins manipulate host transcription and by definition protein expression, to orchestrate a complex network of signaling events. Broadly speaking, these signaling events have two outcomes, (i) suppression or evasion of host defenses and (ii) manipulation of host metabolism to provide nutrients for pathogen multiplication. We are interested in the molecular mechanisms underpinning these invasion strategies. That is, how do pathogens overcome plant defenses using such a small collection of proteins. These are major issues as disease can account for over 20% of crop yields worldwide. To initially address this issue we used a technique of global gene profiling that allowed us to quantitate the expression of all the genes in the plant at any single time point. We examine a time-delimited infection series. With the appropriate controls and extensive bioinformatics analyses and literature research we were able to highlight a large number of genes that could be associated with the ability of the pathogen to overcome defense. This approach led us to identify a set of genes that were previously known to protect plants from water stress. Many of these genes were implicated in the biosynthesis and response pathways of the plant hormone abscisic acid (ABA). We further investigated these leads using a combination of mutants in ABA signaling pathways and direct hormone measurement using mass spectrometry. Our research led us to the overwhelming conclusion that infectious bacteria manipulate plant ABA hormone pathways to promote virulence. As water availability is strongly correlated with successful bacterial pathogenesis, this strategy provides a powerful means of co-opting established host stress signalling pathways in infected tissues to promote virulence. We now seek further funding to build on these current unpublished to establish a mechanistic insight into the role of bacterial effectors in suppressing plant defense and establishing the disease state. These data compel us to re-evaluate the interaction of different hormones in the infection process. As plant hormone signaling is probably ultimately controlled through a few central components, perturbation of one pathway may adversely effect the biological activity of another. In other words, it may be compensatory changes in other hormones that are actually causal to the observed effect. From an industrial perspective it is important to accurately quantitate what impact modifying plant hormonal pathways will have on crop productivity. ABA is a well characterised stress hormone; therefore The project will investigate hormonal responses to pathogens in mutants, and develop gene profiling experiments with infected plants that are insensitive or hypersensitive to ABA. In parallel experiments we will generate transgenic plants expressing (i) reporter fusions designed to allow us to visualize the dynamics of the infection process. We will also generate transgenic plants expressing a reporter that is normally suppressed by bacterial effectors. These plants will be used in a genetic screen to identify key regulators involved in suppression of basal defense. The long-term outcome of this work will be better understanding of how pathogens successfully overcome host defenses. These results will inform future strategies aimed toward manipulating plant responses to develop broad spectrum immunity to pathogens.

This proposal comprises a multidisciplinary work programme that requires integrating a range of various techniques. We have, or are in the process of acquiring* all the necessary skills and access to equipment required to undertake these experiments. We have experience in designing experiments for microarray and metabolomic analyses due to either previous research (P13864), or current projects (BB/D007046/1). This aspect of the research is not trivial, as we are working with two biological entities, pathogen and plant. Additionally, we have considerable experience in the organisation of large-scale pathogen infection screens (BB/D007046/1), as required in components of this proposal. We have published on microarray analyses and are conversant with the necessary analysis programmes. As outlined in the proposal, we are in the process of developing a plant stress response database using very powerful analysis platform. The molecular component of this project is routine. We undertake qRT-PCR studies in BB/C514115/1. We have experience in making transformation constructs and substantial experience in high volume transformation, seed handling and ontology phenotyping through our participation in the Agrikola FW5 project. Central to the project is acid hormone analysis. We have recently developed a protocol for rapid aqueous extraction of ABA, JA and SA. These are routinely analysed by LC/MS using the Multiple Reaction Monitoring mode and negative ion Turboion Spray source on an Applied Biosystems Q-TRAP 2000. Quantitation is based on appropriate MRM ion pairs for labelled and endogenous ABA, SA and JA. * We have limited experience in confocal imaging, but for the last 6 months have had unlimited access to a new Lecia multiphoton confocal.