A Competitive Index Assay Identifies SeveralRalstonia solanacearumType III Effector Mutant Strains with Reduced Fitness in Host Plants

Ralstonia solanacearum, the causal agent of bacterial wilt, is a soil bacterium which can naturally infect a wide range of host plants through the root system. Pathogenicity relies on a Type 3 Secretion System which delivers a large set of approx. 75 Type 3 Effectors (T3E) into plant cells. On several plants, pathogenicity assays based on quantification of wilting symptoms failed to detect significant contribution of R. solanacearum T3E in this process, thus revealing the collective implicationeffect of T3E in pathogenesis. We developed a mixed infection-based method with R. solanacearum to monitor bacterial fitness in plant leave leaf tissues as a virulence assay. This accurate and sensitive assay provides evidence that growth defects can be detected for T3E mutants: we identified twelve genes contributing to bacterial fitness in eggplant leaves and three of them were also implicated in bacterial fitness on two other hosts, tomato and bean. Contribution to fitness of several T3E appears to be host-specific, and we show that some known avirulence determinants such as popP2 or avrA do provide competitive advantages on some susceptible host plants. In addition, this assay revealed that the efe gene, which directs the production of ethylene by bacteria in plant tissues, and hdfB, involved in the biosynthesis of the secondary metabolite 3-hydroxy-oxindole, are also required for optimal growth in plant leaves leaf tissues.