Projet ANR-06-BLAN-0095 – NewNod : Identification of new molecular determinants involved in the early steps of symbiotic interactions between plants and bacteria
Programme : Programme blanc (BLANC) 2006
Année de financement : 2006
Coordinateur du projet : CNRS DELEGATION REGIONALE RHONE AUVERGNE (CNRS DELEGATION REGIONALE RHONE AUVERGNE)
Partenaires : Centre national de la recherche scientifique; Institut de recherche pour le développement; Institut de recherche pour le développement
Montant : 300000.0 euros
Date de début : 2006
Durée : 36 mois
Résumé : Plants have developed root symbioses with bacteria that allow them to thrive in nitrogen-poor sites and initiate ecological successions. This entails complex molecular signaling to induce organogenesis. Lipo-chito-oligo-saccharides (LCOs) synthesized by rhizobia and coded for by nod genes have been shown to be perceived by dedicated plant root kinases that in turn induce in plant root cells a specific response that leads to nodule formation. This is the case in all rhizobia characterized so far but the universality of the paradigm has been recently reassessed as a consequence of the study of two symbiotic bacteria : the actinobacterium Frankia and the photosynthetic Bradyrhizobium. In both instances several full genomes have recently become available, yet have failed to show the presence of the common nod genes. The aim of the present project is to understand what are the bacterial and plant determinants that in these two models permit the symbiotic interactions.  Molecular determinants of signaling in photosynthetic Bradyrhizobium and Frankia. Genetic determinants involved in symbiosis in Frankia (Fr) and in photosynthetic Bradyrhizobium (Br) will be identified through two complementary approaches. The first one, global, will be a transcriptomic approach (DNA array, Fr and Br) and by screening of a Tn5 mutants library on their inability to induce nodulation (Br). The second approach, targeted, will be focussed on genes having functions that are homologous to those of nodC or lectin-coding genes (Fr), alpha-glucan branching enzymes (LCOs mimicking) , a chalcone synthase, type IV secretion systems (Br). Moreover, we plan to identify the signal-molecules potentially involved in the first recognition steps (based on the data obtained from the mutagenesis step) and to establish their chemical structure.  Molecular determinants of signaling in Casuarina, Alnus and Aeschynomene. In the case of Casuarina and Alnus, we plan to screen EST libraries prepared from Frankia infected roots harvested at different steps of infection (50 000 per species). Annotation of ESTs and comparison of the different libraries will allow us to identify putative candidate genes. We will also perform microarrays experiments based on these different EST libraries to assess changes in transcript profiles during early symbiotic interaction. A targeted approach will be used in parallel. We plan to clone Casuarina genes homologous to those which in legumes are involved in signaling pathways. More particularly, we will investigate and address the role of genes like LysM/NFR, DMI1, DMI3, NSP or DMI2/SymRK. To validate the function of these candidate genes, we aim to: (i) study the effect of their silencing by RNAi using the « hairy root » genetic transformation technique; (ii) analyze the spatiotemporal expression pattern (qRT-PCR, promoter/reporter gene fusion); (iii) complement Medicago and/or Lotus mutants. A targeted approach similar to the one described above for Casuarina will be implemented on Aeschynomene. The results obtained in this project should lead to the identification of new signaling pathways governing interactions between plant and bacteria and thus contribute to understanding the evolutionary origin of root symbioses.