Rôle des peptides spécifiques des nodosités de Medicago dans la différentiation terminale des bactéroïdes fixateurs d?azote – nsPEPbac

Plants are able to develop symbiotic associations with many microorganisms. Among the most economically and ecologically important ones are the interactions of legume plants with nitrogen-fixing bacteria known as rhizobia. This symbiosis leads to the formation of new organs, the root nodules, inside which rhizobia enzymatically reduce atmospheric nitrogen to ammonium that is transferred to the plant. Nodules house inside their symbiotic cells millions of nitrogen-fixing rhizobia, called bacteroids. Bacteroids are differentiated bacteria adapted to symbiotic life and nitrogen fixation. In the legume Medicago truncatula, bacteroids undergo during their differentiation several morphological and cellular modifications such as membrane permeabilization and genome endoreduplication, supporting an important cell enlargement. Bacteroids also lost capacity for reproduction and are thus terminally differentiated bacteria. We demonstrated before, in the frame of the ANR grant Nod-anti-mic (ANR blanc 2005), that in M. truncatula the symbiotic nodule cells produce hundreds of nodule-specific antimicrobial peptides (nsPEP) which induce the microbial symbiont Sinorhizobium meliloti in this terminal differentiated state. We found that nsPEP are transported to the bacteroids via the secretory system of the cells and that some of the nsPEP are localized in the cytosol of the bacteroids indicating that they have intracellular bacterial targets. They can induce bacteroid differentiation in planta and pure peptides can induce in vitro features of bacteroids on S. meliloti. However, the molecular mechanisms underlying the activity of these plant peptides on the endosymbiont are unknown. The central questions that this proposal attempts to answer are therefore the following: With which bacterial functions nsPEP interfere’ And what is the diversity of these functions’ Are nsPEP acting exclusively as inhibitors of cellular functions or do they have also inducing or signalling roles during bacteroid differentiation’ Since the symbiotic cells produce several hundreds of them, nsPEP have potentially multiple effects on the endoysmbiotic bacteria. Regulation of bacterial cytokinesis is among their predicted actions but also interference with the synthesis of surface structures or the central carbon/nitrogen metabolism to improve the efficiency of nitrogen fixation. Possibly they are also in part responsible for the oxidative stress that is known to affect bacteroids. This project is obviously of relevance for the legume-Rhizobium symbiosis and biological nitrogen fixation by legumes. It will help us to understand how bacteroid differentiation is achieved by nsPEP. But it will be equally of significance for understanding how antimicrobial peptides (AMP) in general function and it will make also possible to evaluate if the nsPEP are of interest for therapeutic applications as novel antibiotics. The work plan consists in a proteome approach to identify the peptides which are associated with the early and late stages of bacteroid differentiation and which are localized in the cytosol of bacteroids or which are attached to bacteroid membranes. The activity of major intracellular peptides will be studied on rhizobia. Targets of candidate peptides in S. meliloti will be identified through systems-level approaches. Transcriptome analysis will identify S. meliloti genes induced by the nsPEP. Genetic screens of mutagenized populations and testing mutants in candidate genes will identify mutants with altered sensitivity to the nsPEP. S. meliloti proteins interacting directly with nsPEP will be identified biochemically. Overlaying these datasets with available gene-connectivity maps will lead to the identification of gene-networks in S. meliloti that are affected by the nsPEP. These will be experimentally confirmed.