Experimental evolution of a plant pathogen into a legume symbiont

Abstract

Rhizobia are phylogenetically disparate alpha- and beta-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.

Figure 1. Nodulation of M. pudica by C. taiwanensis LMG19424, symbiotically evolved clones CBM356, CBM212, and CBM349, and mutant chimeric Ralstonia CBM125 and CBM664.

(A) Nitrogen-fixing nodules formed by C. taiwanensis LMG19424. (B) Fix⁻ nodules formed by CBM212 on M. pudica. (C) Nodulation kinetics of the evolved clones and the mutants. (D) Number of nodules harvested at 14 days postinoculation and number of bacteria isolated per nodule. The number of in planta bacterial generations is estimated at 20 per nodule for CBM212 and CBM349 and 10 per nodule for CBM356.

Figure 2. hrcV inactivation allows chimeric Ralstonia to nodulate and to enter root hairs via infection threads (ITs).

(A) Inoculation with the chimeric strain CBM124-gfp resulted only in microcolony formation within curled hairs (no IT formation). (B–D), CBM125-gfp strains (hrcV) formed ITs in root hairs (B) and were located in intercellular spaces within nodules (C and D).

Figure 3. hrpG inactivation allows intracellular invasion of nodule cells.

(A–C) CBM124ΔhrpG massively invaded plant cells intracellularly. A few bacteria were found in intercellular spaces ([B] arrow). Intracellular bacteria (bacteroids) were surrounded by a peribacteroid membrane ([C] black arrowhead) forming typical symbiosomes. Vesicles containing osmophile material ([C] white arrowhead) were often seen.