Bacterial biotechnology of the plant pathogen Ralstonia solanacearum

Ralstonia solanacearum is a soil-borne pathogen with an unusually wide host range. It has been one of the pioneering bacterial species studied as a plant pathogen. Genetic analyses conducted in this organism have established that the type III secretion system (T3SS) is the major virulence determinant.

The type III protein secretion machinery delivers bacterial effector proteins into host cells, in an engaging example of information transfer between evolutionary distant organisms. Sequencing of the complete genome of the bacterium recently led to the discovery of a number of candidate genes for type III effectors. The T3SS genes and those encoding its associated effectors form a regulon that responds to bacterial contact with plant cells.

This research project is oriented towards the characterisation of novel R. solanacearum effectors so as to unravel their molecular function and their contribution to disease. The process includes a) study of the expression profile of candidate genes, b) disruption of each structural gene and analysis of the resulting phenotype regarding pathogenicity, c) demonstration of effector translocation into plant cells, d) determination of the sub-cellular localisation of bacterial effectors inside the plant and e) identification of molecular targets in the eukaryotic host.

Although much is known about the biochemical features of virulence gene products, all regulation studies have been performed in vitro cultures and the genes actually expressed in planta at different stages of the disease have not been determined. The aims of this project are a) generate a set of tools for promoter probing in Ralstonia, b) identify the plant signal/s that cause transcription of the T3SS and c) establish the genetic program deployed by the pathogen during infection. Deciphering the molecular mechanisms governing these functions is a first step towards the rational design of new strategies for plant protection.

Understanding the function of R. solanacearum pathogenicity determinants in plants may be difficult because of the redundancy in the effector repertoire of the bacterial strain and the presence of plant resistance genes. Expression of type III effectors of Ralstonia in the budding yeast S. cerevisiae overstep these limitations. This research project aims to: design a yeast system for the expression and detection of Ralstonia bacterial type III effector proteins identify through gain-of-function and loss-of-function screenings the proteins that Ralstonia type III effectors target to cause cell death study by yeast two hybrid the molecular interactors of Ralstonia type III effectors in the host plant cell. Using yeast as a tool to express Ralstonia type III effectors could be useful to the study of pathogenicity determinants delivered into the host cell by any other pathogen.