Ralstonia solanacearum is one of the world's most important phytopathogenic bacteria and is also is one of the model systems for elucidating how Gram-negative bacteria cause plant disease. The collective importance of the more than 100 exoproteins (EXPs) secreted by the R. solanacearum type II and type III systems has been established, but the identity and detailed role of individual proteins in the secretome has only been assessed superficially. The overall goals of this project are to carry out a proteomic analysis of the R. solanacearum secretome and a genetic investigation of the role multiple EXPs play in making this organism such an effective phytopathogen. A comprehensive evaluation of several proteomic methods will precede a large-scale characterization and identification of EXPs and their corresponding genes in the genomic sequence of strain GMI1000. Bioinformatic analyses and comparative genomics will be used to characterize the possible nature, function and importance of the genes encoding the EXPs. The contribution to pathogenesis of individual EXPs and groups of EXPs will be determined by constructing unmarked, site-directed deletion mutants and testing them for colonization of tomato plants. Results of this project will provide insight into how EXPs function in R. solanacearum pathogenesis, and also will be relevant to understanding the biology of other plant and animal pathogens and of environmental bacteria, such as the closely-related Ralstonia and Burkholderia species. Assembling a large catalog of secreted proteins and their predicted amino acids sequences will provide a working data set for determining if there are common structural characteristics of secreted proteins that can be used to predict which genes in genomic DNA sequences encode EXPs.
