Environmental cues controlling the pathogenicity of "Ralstonia solanacearum" on plants / Señales ambientales que determinan la patogenicidad de "Ralstonia solanacearum" en plantas

Ralstonia solanacearum is a soil-borne beta-proteobacterium that causes wilting disease on a wide range of plants with economic importance like tomato, potato, pepper, eggplant and banana. Each year, bacterial wilt pose important threats to agriculture by producing significant economic losses to small-scale producers in developing countries and, lately, the geographical distribution of the pathogen is spreading to temperate regions of the globe. The long-term aim of the work developed was the determination of the genetic program used by R. solanacearum during plant colonization and at the different stages of disease, in order to provide a biologically relevant understanding of the repression/activation regulatory switches controlling R. solanacearum pathogenicity. We noticed that new molecular tools for functional genetic studies adapted to R. solanacearum were needed, because the widely used mutants obtained bytransposon mutagenesis contain gene disruptions rendering, in some cases, bacteria with affected virulence, pathogenicity and unable to multiply inside susceptible plants. In addition, a common issue in R. solanacearum studies was the difficulty to trans-complement gene disruptions. So far, the only alternative available was the use of plasmids, which provided a means of overexpression rather than stoichiometrical complementation, Moreover, the use of antibiotics to maintain plasmids during plant infection is not an option due to the complexity of the system.
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\nIn this thesis we developed a novel system – pRC, after Ralstonia chromosome –, based in targeted and stable insertions in a precise and permissive location of the bacterial chromosome. We proposed the use of our versatile set of suicide plasmids for the study of transcriptional output (promoter probing) during plant infection, effector overexpression and purification, and monocopy gene complementation in any R. solanacearum strain. The use of the pRC system in any strain will allow the standardization of the genetic studies made in the field. We also investigated gene activities in planta. To that end, we successfully applied a luminescent reporter in the bacterial chromosome to visualize and quantify in real time the activity of pathogenicity-related promoters. We fused the promoter regions controlling two major virulence determinants to the luxCDABE reporter and followed light emission at different stages of plant infection. This strategy allowed us to establish both the timing and the exact location in the plant where these bacterial genes were expressed. Our main finding was that the T3SS is active throughout plant infection and not only at the first colonization stages. It is likely that during plant infection many overlapping signals are perceived by the bacteria, adding complexity to the gene regulatory model proposed in the literature. Together with the two articles published in peer-review journals, two additional drafts, describing the current progress of two other projects are also provided. The first draft reports a novel regulatory feedback loop governing hrpB expression when R. solanacearum is grown in the presence of plant cells. This work is part of a collaboration with Stéphane Genin (Laboratoire des Interactions Plantes Micro-organismes (LIPM, INRA-CNRS, Castanet Tolosan, France).The second draft reports the use of the pRC system to decipher “cool-adaptation” on strain UW551. This work is part of a collaboration with Caitilyn Allen research group (University of Wisconsin – Madison, Wisconsin, USA).

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