Modulation of Quorum Sensing as an Adaptation to Nodule Cell Infection during Experimental Evolution of Legume Symbionts.

Over millions of years, changes have occurred in regulatory circuitries in response to genome reorganization and/or persistent changes in environmental conditions. How bacteria optimize regulatory circuitries is crucial to understand bacterial adaptation. Here, we analyzed the experimental evolution of the plant pathogen Ralstonia solanacearum into legume symbionts after the transfer of a natural plasmid encoding the essential mutualistic genes. We showed that the Phc quorum sensing system required for the virulence of the ancestral bacterium was reconfigured to improve intracellular infection of root nodules induced by evolved Ralstonia. A single mutation in either the PhcB autoinducer synthase or the PhcQ regulator of the sensory cascade tuned the kinetics of activation of the central regulator PhcA in response to cell density so that the minimal stimulatory concentration of autoinducers needed for a given response was increased. Yet, a change in the expression of a PhcA target gene was observed in infection threads progressing in root hairs, suggesting early programming for the late accommodation of bacteria in nodule cells. Moreover, this delayed switch to the quorum sensing mode decreased the pathogenicity of the ancestral strain, illustrating the functional plasticity of regulatory systems and showing how a small modulation in signal response can produce drastic changes in bacterial lifestyle. IMPORTANCE Rhizobia are soil bacteria from unrelated genera able to form a mutualistic relationship with legumes. Bacteria induce the formation of root nodules, invade nodule cells, and fix nitrogen to the benefit of the plant. Rhizobial lineages emerged from the horizontal transfer of essential symbiotic genes followed by genome remodeling to activate and/or optimize the acquired symbiotic potential. This evolutionary scenario was replayed in a laboratory evolution experiment in which the plant pathogen Ralstonia solanacearum successively evolved the capacities to nodulate Mimosa pudica and poorly invade, then massively invade, nodule cells. In some lines, the improvement of intracellular infection was achieved by mutations modulating a quorum sensing regulatory system of the ancestral strain. This modulation that affects the activity of a central regulator during the earliest stages of symbiosis has a huge impact on late stages of symbiosis. This work showed that regulatory rewiring is the main driver of this pathogeny-symbiosis transition.