A functional kinomics approach to dissecting signalling pathways in plant PAMP-triggered immunity

Plants get sick with diseases just like animals do. But there are differences in the way the immune systems of plants and animals work. We all know that if we come down with a runny nose and a sore throat, once we recover, we won't get sick to the same bug twice. This is because our immune system has a memory - it learns how to recognise the bug that made us sick, which stops it infecting again. This is called acquired immunity. Plants don't have acquired immunity, but they do have recognition systems to detect pathogens. The way this works is that instead of recognising individual characteristics of pathogens, plants recognise generalities - like being able to distinguish people from trees, but not individual faces. Something is known about how plants detect pathogens, but what happens next is a bit of a mystery. In general terms, we can talk about detecting and responding to a pathogen as an exercise in relaying signals - what scientists call 'signal transduction'. So, the presence of the pathogen is one signal, and this signal must be relayed around particular parts of the plant cell to coordinate the immune response through various cellular responses. How does this work? We know something about pathogen recognition, where parts of the bug are detected by so called 'receptor' proteins that sit in the plant cell wall. Often, the receptors are linked to a type of switch called a protein kinase. The protein kinase activates a series of relays (or a 'signal transduction pathway', in the jargon), which may contain more protein kinases. These activate the cellular responses which coordinate the output of the signal, in this case the defence response. So, we know that protein kinases are a very important part of this signalling process. Here, we propose to remove each protein kinase from the cell, and see how this affects cellular responses and immunity. We can do this using genetic techniques which silence the expression of each gene responsible for the existence of each kinase. Some of these kinases will have general or specific roles in the signalling process, and we hope to identify them using this strategy. This will provide a lot of new, important information on the signalling pathways that underlie plant immunity. Also, we know that pathogens have their own tricks to disrupt signal transduction. We have already identified many of the proteins that pathogens make to do this. Some of these proteins will 'target' plant protein kinases, by binding to them and inactivating them. We propose an additional screen to identify pathogen proteins that bind to the protein kinases discovered here. This will provide important information on how the pathogen is able to infect the plant by stopping signal transduction.

Plants detect pathogens on two levels. Cell surface receptors respond to characteristic pathogen molecular components known as PAMPs (for pathogen-associated molecular pathogens) and elicit defence responses that have been termed PAMP-triggered immunity (PTI). Plants also possess a secondary surveillance system that responds to the presence of specific effectors, and induces strong defences that have been termed elicitor-triggered immunity (ETI). PAMP-triggered immunity (PTI) is an important but poorly understood defence mechanism in plants. Cellular phenomena associated with PAMP perception include rapid ion fluxes across the plasma membrane including the uptake of calcium ions (Ca2+), a burst of reactive oxygen species (ROS), rapid and widespread protein phosphorylation events, and alterations in gene expression. Protein phosphorylation mediated by protein kinases plays a cardinal role in PAMP-mediated signalling. We will investigate roles for protein kinases in PTI. We will build a complete library of the protein kinase genes of tomato and potato (the 'kinome'), and silence them individually in N. benthamiana. We will assay silenced plants for loss of known cellular responses associated with pathogen defence, and loss of PTI. The results will identify important molecules that mediate PTI, and help to delineate pathways downstream of PAMP perception. In addition, phytopathogenic bacteria deposit effector proteins into the host cytoplasm to promote parasitism using a specialised type-III secretion pathway. We will conduct a secondary screen for bacterial type-III effector proteins that target the novel protein kinases identified in this screen. Overall, the data will provide important insights into signalling pathways triggered by the pathogen, and how the pathogen responds to counteract this.