Experimental Evolution of a Plant Pathogen into a Legume Symbiont

Abstract / Rhizobia are phylogenetically disparate a- and b-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.Author Summary / 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