Lipides du plasmalemme et signalisation des interactions biotiques chez les végétaux – PANACEA

Plants are exposed to a great number of pathogenes, only a small proportion cause diseases. Indeed, plants defend themselves against microorganisms by triggering a wide range of mechanisms. A better understanding of the molecular basis of defence is not only of high scientific interest, but also an essential priority to develop environment-friendly agricultural processes. Evidences for signaling events common to most of incompatible interactions have been found: recognition by plant receptors of elicitors secreted by the microorganism, modification of ionic fluxes, occurrence of intense oxidative burst... Many additional data evidencing particular signaling events have been published; however they were often obtained on many different models. The present challenge is to integrate as many as possible signaling elements on a single model, so as to get a global and integrative understanding of a given signaling process. In plants, studies have demonstrated the critical role of lipid in regulating multiple processes of plant growth and development, signal transduction, mediating hormone effects and cell responses to environmental stimuli. However, the precise composition of the molecular species of all these various lipids involved in such signaling processes remains mostly unknown, in term of relative amount, fatty acid composition, sn position, membrane from which they originate. Moreover, a complete understanding of the dynamic of lipid and membrane protein network is far to be achieved, with regard to kinetic and recruitment or exclusion of enzymes in PM. Finally, several biological models have been used which prevents integration of these data in a consistent knowledge of the network. Over the last 10 years, another aspect of membrane biology raised from biological, biochemical and biophysical studies: the existence of membrane domains, called 'membrane rafts, MR'. Biological membranes should no longer be considered as homogeneous bilayers composed of lipids and proteins. Indeed, 'discrete islands' with specific location and composition, especially in the PM. MR allow the segregation of active components in membranes and are involved in many cellular processes. This organizational hierarchy of lipid and protein constituents in PM mediates many life processes involving perception and early transduction of external stimuli. These processes allow the adaptation of the organism to cope with pathogen threats and adverse environment conditions. Related to the present project, animal MR are able to recruit and/or exclude enzymes and lipids involved in the transduction of stimuli, and play a role of platform controlling the response. Two French teams, partners 1 and 2, develop a strong collaboration to address this question in plants, being the first to characterize membrane rafts from plant PMs. They evidenced the peculiar proteic and lipid content of plant MR, by a strong enrichment in sphingolipids together with free sterols and sterol conjugated. They also demonstrated the high proportion in MR of proteins linked to signaling in response to biotic stress. Recently, preliminary studies conducted by partner 1 suggested that MR are concentrating "signaling phospholipids". Taken together, all these data were supporting the hypothesis of the involvement of PMs domains in the set up of plant defence mechanisms, and particularly the possible role of lipids enriched in these domains as signaling mediators.. The present project aims to decipher the involvement of PM lipids in the early signaling process triggered by cryptogein, and to analyze how the PM dynamics is itself an actor of signalisation via modification of the lateral segregation of its components. The single biological model of elicitation of BY-2 cells challenged with the cryptogein, secreted by Phytophthora was chosen beacause (i) it one of those in which the highest number of signaling events have been described making easier the integration of data in a general network , (ii) partner 2 has a long experience in studying the early signaling events triggered on cells by cryptogein The four main axes of the study are very complemenary, to tackle this complex question as a whole -A comparative description of lipid fluctuation in the PM and MR of cells upon cryptogein elicitation -In vivo determination of the enzyme activities involved in lipid metabolism upon elicitation. Effects of inhibitors of these enzymes on marker events of cryptogein signaling. Study of the variation of activities of enzymes involved in lipid metabolism upon elicitation and their repartition between MR and non-rafts phase of the PM - Development of imaging techniques allowing the visualization of the lipidic and proteic components identified in 1 and 2, to correlate biochemical data to in vivo reality 4- Functional validation by altering the expression of genes corresponding to enzymes identified in 1, 2, to demonstrate their involvment in cryptogein signaling