Functional Studies on Xanthomonas citri Type VI Secretion System and Gene Regulation by Alternative Sigma Factors

Abstract

Bacterial type VI secretion systems (T6SS) are multiprotein complexes that translocate protein effectors and/or toxins to target cells, playing major roles in interspecies competition, virulence and environmental adaptation in several bacteria. Xanthomonas citri subsp. citri is the causative agent of citrus canker disease, infecting all citrus crops. Previous studies by our research group have demonstrated that the X. citri T6SS is required for resistance to predation by the soil amoeba Dictyostelium discoideum, which potentially have important impact on X. citri persistence and dissemination in the environment. This project will further characterize the molecular mechanisms involved in resistance to amoeba predation and the signaling pathways that promote activation of the T6SS in X. citri. For that, we will perform in vivo translocation assays and yeast two-hybrid assays to identify proteins secreted by the T6SS. Characterization of two X. citri alternative sigma factors named EcfK and EcfL has shown that EcfK is required for transcriptional activation of T6SS genes, while EcfL regulates a small set of genes of unknown function. Bacterial alternative sigma factors are important regulators of environmental responses in bacteria. To further characterize these regulators, we will identify the signaling pathway that activates EcfK and the function of EcfL in X. citri physiology. To reach these goals we will use several bacterial genetics and biochemical approaches, including phenotypic analysis of knock-out strains, transcriptional fusions to reporter genes and biochemical characterization of proteins of interest. (AU)