Environmental cues controlling the pathogenicity of

Abstract

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.

In 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).

Ralstonia solanacearum es una bacteria Gram-negativa que causa una enfermedad de plantas conocida como marchitez bacteriana. El espectro de plantas huéspedes afectadas es amplio, incluyendo algunas especies con relevancia económica, como por ejemplo el tomate, la patata, el pimiento, la berenjena y el plátano. El objetivo a transversal de la investigación desarrollada en esta tesis de doctorado es la determinación del programa genético utilizado por R. solanacearum durante diferentes etapas de colonización de las plantas. Describimos un novedoso sistema - pRC, basado en el uso de inserciones específicas y estables en un punto específico del cromosoma bacteriano. Proponemos el uso de nuestra versátil colección de plásmidos suicidas para el estudio de la actividad de promotores de genes de patogenicidad y virulencia, la sobreexpresión y purificación de proteínas efectoras y la complementación de genes. El uso del sistema de pRC en cualquier cepa de R. solanacearum permitirá la estandarización de los estudios genéticos realizados en el campo de investigación. En esta tesis también se describe la utilización un reportero luminiscente, integrado en el genoma de la bacteria para visualizar y cuantificar en tiempo real la actividad de promotores de genes de patogenicidad durante la infección. Esta estrategia nos permitió determinar el momento y el lugar exacto de la planta donde se expresan los genes bacterianos. Nuestro principal hallazgo fue que el sistema de secreción de tipo III se transcribe a lo largo del proceso infeccioso y no sólo en las primeras etapas de colonización. Junto con los dos artículos publicados en revistas internacionales, se incluyen también dos manuscritos, que describen el progreso actual de dos otros proyectos. En el primer manuscrito se describe un nuevo proceso de regulación de la expresión de hrpB, cuando R. solanacearum se cultiva en presencia de las células vegetales. Este trabajo es parte de una colaboración con Stéphane Genin, del “Laboratoire des Plantes Interacciones Microorganismos” (LIPM, INRA-CNRS, Castanet Tolosan, Francia). El segundo proyecto describe el uso del sistema pRC para descifrar la capacidad de infección de la cepa UW551 a temperaturas bajas. Este trabajo es parte de una colaboración con el grupo de investigación de Caitilyn Allen (Universidad de Wisconsin - Madison, Wisconsin, EE.UU.)