Role of HYdrogen peroxide in the esTablisHment of the Medicago truncatula-Sinorhizobium meliloti symbiosis – RHYTHMS

When molecular oxygen is exposed to electron-transferring chemical reactions, it can be converted to various highly reactive chemical forms, collectively designated 'Reactive Oxygen Species' (ROS). This results in the sequential reduction to superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH•). In plants, ROS are unavoidable by-products of biochemical pathways such as glycolysis and photosynthesis. In some circumstances, plants appear to purposefully generate ROS as signalling molecules to control various processes. In this framework, there is now compelling evidence that H2O2 is an important signal in plants. The production of ROS and particularly hydrogen peroxide (H2O2) has been evidenced during the Sinorhizobium meliloti - Medicago symbiotic interaction. Furthermore, bacterial mutants with impaired capacities to scavenge H2O2 exhibit modified nodulation phenotypes. This is particularly the case of a mutant over-expressing a hydroperoxidase, acting as a sink for H2O2, which strongly suggests that a threshold level of H2O2 is required for a harmonious nodule development. Thus, H2O2 appears to play a signalling role in the symbiotic interaction, which goes beyond a trace of an incompatible reaction. Thus, the overall goal of the project is to characterise the signalling role of H2O2 in the symbiotic process. To answer the questions 'how is H2O2 generated?' and 'what are the consequences of its production?' the project will be organised in two main objectives. Objective I is to define the spatiotemporal characteristics of H2O2 formation and the system(s) that generate it during symbiosis. Firstly, this will allow the identification of the Medicago truncatula specific 'H2O2 signature' in the symbiotic process through cell biology approaches (fluorescent probes, new H2O2 specific protein probe, confocal microscopy). Secondly, the H2O2-producing system(s) will be identified in focusing on NADPH oxidase and peroxidases. Once identified, the putative candidates will be characterised via a functional validation by inactivating their in planta expression (RNAi). Objective II is to determine the role of H2O2 on gene expression in both symbiotic partners. At the bacterial level, H2O2 involvement as a signal molecule under non stress conditions will be established. Thus, a comparative microarray analysis will be performed using S. meliloti wild-type cells grown in presence or absence of an H2O2 concentration threshold, that doesn't induce oxidative stress markers. This type of analysis will be also carried out on S. meliloti mutants with modified H2O2 degrading activities and exhibiting a modified symbiotic phenotype (katB++ and katB/katC strains). A functional analysis of candidates genes will be performed trough the construction of deletion/insertion or overexpression mutants. Taken together, this will provide new insights into the signalling effect of H2O2 production on the microorganism during plant-microbe interactions that have yet to be investigated from the bacterial partner side either during pathogenic or symbiotic interactions. At the plant level, the transcriptome analysis of inoculated or not M. truncatula plants exposed to various H2O2 treatments will be performed in order to identify H2O2 and symbiosis regulated genes. Moreover, the transgenic lines used for the characterisation of the H2O2-producing system(s) will also give us the opportunity to identify the H2O2 regulated genes in a symbiotic context. Putative candidates will be characterised via a functional validation by RNAi thecnology. This will allow to define the signalling role of H2O2 in the plant partner, during its interaction with S. meliloti. This project considers both symbiotic partners and is based on a multidisciplinary approach, combining physiology (definition of symbiotic phenotype), biochemistry (analysis of the peroxidase activities), genetics (transgenic and mutants M. truncatula; S. meliloti mutant strains), cell biology (in vivo H2O2 imaging) and molecular biology (transcriptome analysis). It involves two teams with complementary skills. Finally, this project proposes to characterise a completely new role for H2O2 as a signalling molecule in plants, i.e. its 'positive role' in the M. truncatula – S. meliloti symbiosis. The research programme will lead to the identification of key players of the H2O2 derived signal: the H2O2 generating system(s) and the H2O2 targets in both partners. In identifying the spatiotemporal characteristics of H2O2 production, it will define how H2O2 governs specific signals that optimise the symbiotic interaction. This will lead to important breakthroughs not only in the symbiotic field but also in that of compatible plant pathogen interaction and more generally in that of plant development. In this respect, it is expected to open new fields of investigation.