SYMPA

The project aims at evolving a plant pathogen, Ralstonia solanacearum into a legume (Mimosa) symbiont and elucidating the underlying genetic basis of the lifestyle change. Our experimental approach is based on a 'design, then evolve' strategy, which consists in i) the construction of an ancestral strain with symbiotic potential, by transfer of the symbiotic plasmid pRalta of the Mimosa symbiont Cupriavidus taiwanensis into R. solanacearum GMI1000 and ii) progressive evolution into a Mimosa symbiont by serial passages on plants. Short (15-day) and long (45-day) regimes of selection on plants will be assayed as these may shape bacterial evolution differently. Preliminary experiments have demonstrated the feasibility of the project as nodulating and infective derivatives of GMI1000(pRalta) have already been obtained by mutagenesis or in planta selection. Endpoint clones, as well as their progenitors, will be phenotypically characterized and the best (or more different) symbiotically-improved clones identified. The genome of 9 clones having acquired nodulation, intracellular infection and possibly nitrogen fixation ability will be fully re-sequenced by Roche (454) Genome Sequencer FLX technology (Genoscope as service supplier) and compared to that of the ancestral strain GMI1000(pRalta). Sequence discrepancies will be analysed in an expert manner (filtration of false positives, re-sequencing of PCR products…) so that to track all genuine genomic alterations having occurred in the course of the experimental evolution. A dedicated MicroScope interface of the Mage platform will be implemented with new facilities that will help visualisation and analysis of acquired mutations. A phenotype-genotype correlation analysis will be performed to identify the mutations responsible for improved symbiotic capacities. Their role in shaping symbiosis will be ultimately assessed by molecular genetics. Mutations appearance in all ancestral populations from the best evolved lineage will be analysed, so that to get access to the kinetic and dynamic parameters of the evolutionary process. Finally, all data will be integrated into a comprehensive view of this lifestyle change. Deciphering the molecular events that underlie the evolution of a phytopathogenic bacterium into a legume symbiont should shed new light on our understanding of legume symbiosis determinism, including the control of nodulation, intracellular infection, the evolution of rhizobia and symbiosis-pathogenesis relationships. In addition, the present project should be a case study of a major ecological transition towards a complex function, and have significance in the biodiversity, environmental microbiology and bacterial evolution fields.