When the rhizosphere is nitrogen‐starved, legumes and rhizobia (soil bacteria) enter into a symbiosis that enables the fixation of atmospheric dinitrogen. This implies a complex chemical dialogue between partners and drastic changes on both plant roots and bacteria. Several recent works pointed out the importance of rhizobial surface polysaccharides in the establishing of the highly specific symbiosis between symbionts. Exopolysaccharides appear to be essential for the early infection process. Lipopolysaccharides exhibit specific roles in the later stages of the nodulation processes such as the penetration of the infection thread into the cortical cells or the setting up of the nitrogen‐fixing phenotype. More generally, even if active at different steps of the establishing of the symbiosis, all the polysaccharide classes seem to be involved in complex processes of plant defense inhibition that allow plant root invasion. Their chemistry is important for structural recognition as well as for physico‐chemical properties.

Rhizobia are phylogenetically disparate α- and β-proteobacteria that have achieved the environmentally essential function of fixing atmospheric nitrogen in symbiosis with legumes. Ample evidence indicates that horizontal transfer of symbiotic plasmids/islands has played a crucial role in rhizobia evolution. However, adaptive mechanisms that allow the recipient genomes to express symbiotic traits are unknown. Here, we report on the experimental evolution of a pathogenic Ralstonia solanacearum chimera carrying the symbiotic plasmid of the rhizobium Cupriavidus taiwanensis into Mimosa nodulating and infecting symbionts. Two types of adaptive mutations in the hrpG-controlled virulence pathway of R. solanacearum were identified that are crucial for the transition from pathogenicity towards mutualism. Inactivation of the hrcV structural gene of the type III secretion system allowed nodulation and early infection to take place, whereas inactivation of the master virulence regulator hrpG allowed intracellular infection of nodule cells. Our findings predict that natural selection of adaptive changes in the legume environment following horizontal transfer has been a major driving force in rhizobia evolution and diversification and show the potential of experimental evolution to decipher the mechanisms leading to symbiosis. Most leguminous plants can form a symbiosis with members of a group of soil bacteria known as rhizobia. On the roots of their hosts, some rhizobia elicit the formation of specialized organs, called nodules, that they colonize intracellularly and within which they fix nitrogen to the benefit of the plant. Rhizobia do not form a homogenous taxon but are phylogenetically dispersed bacteria. How such diversity has emerged is a fascinating, but only partly documented, question. Although horizontal transfer of symbiotic plasmids or groups of genes has played a major role in the spreading of symbiosis, such gene transfer alone is usually unproductive because genetic or ecological barriers restrict evolution of symbiosis. Here, we experimentally evolved the usually phytopathogenic bacterium Ralstonia solanacearum, which was carrying a rhizobial symbiotic plasmid into legume-nodulating and -infecting symbionts. From resequencing the bacterial genomes, we showed that inactivation of a single regulatory gene allowed the transition from pathogenesis to legume symbiosis. Our findings indicate that following the initial transfer of symbiotic genes, subsequent genome adaptation under selection in the plant has been crucial for the evolution and diversification of rhizobia.

We report the first complete genome sequence of a β-proteobacterial nitrogen-fixing symbiont of legumes, Cupriavidus taiwanensis LMG19424. The genome consists of two chromosomes of size 3.42 Mb and 2.50 Mb, and a large symbiotic plasmid of 0.56 Mb. The C. taiwanensis genome displays an unexpected high similarity with the genome of the saprophytic bacterium C. eutrophus H16, despite being 0.94 Mb smaller. Both organisms harbor two chromosomes with large regions of synteny interspersed by specific regions. In contrast, the two species host highly divergent plasmids, with the consequence that C. taiwanensis is symbiotically proficient and less metabolically versatile. Altogether, specific regions in C. taiwanensis compared with C. eutrophus cover 1.02 Mb and are enriched in genes associated with symbiosis or virulence in other bacteria. C. taiwanensis reveals characteristics of a minimal rhizobium, including the most compact (35-kb) symbiotic island (nod and nif) identified so far in any rhizobium. The atypical phylogenetic position of C. taiwanensis allowed insightful comparative genomics of all available rhizobium genomes. We did not find any gene that was both common and specific to all rhizobia, thus suggesting that a unique shared genetic strategy does not support symbiosis of rhizobia with legumes. Instead, phylodistribution analysis of more than 200 Sinorhizobium meliloti known symbiotic genes indicated large and complex variations of their occurrence in rhizobia and non-rhizobia. This led us to devise an in silico method to extract genes preferentially associated with rhizobia. We discuss how the novel genes we have identified may contribute to symbiotic adaptation.