UC Davis

Plant-pathogenic bacteria in the Ralstonia solanacearum species complex (RSSC) cause disease in a wide variety of plant hosts, troubling agriculture in tropical and subtropical climates worldwide. Bacteria in the RSSC enter plants through the roots and colonize the xylem until bacterial biomass physically blocks fluid flow, resulting in often-fatal bacterial wilt. Many of the traits that contribute to the virulence of the RSSC have been studied extensively, but relatively little is known about the Type VI secretion system (T6SS). Well-characterized in model Gram-negative bacteria, the T6SS is a multicomponent apparatus that forcibly ejects sharp multimeric projectiles, potentially piercing the envelope of other nearby cells. These projectiles bear T6SS effector proteins which degrade important cellular substrates, such as lipids and DNA, potentially killing the target cell. Previous studies implicate the T6SS in RSSC virulence and indicate that the T6SS is present in genomes from each of the three constituent species of the RSSC. In this dissertation, I further investigated the T6SS in the RSSC through various functional and phylogenetic approaches. I show that the T6SS is represented among most, but not all, RSSC genomes, and is notably absent from most genomes in the major RSSC clade known as phylotype III, as well as from several genomes of strains with atypical, vectored modes of transmission. Ralstonia genomes with a T6SS have an expanded arsenal of T6SS effectors compared to non-pathogenic relatives, suggesting the pathogenic lifestyle of Ralstonia may influence T6SS evolution. Comparing data from various genome-scale transposon mutant screening experiments, I demonstrate that several Ralstonia T6SS immunity (rsi) genes, which function as antitoxins for specific T6SS effectors, were necessary for fitness specifically when colonizing living plant hosts. Follow-up experiments with two T6SS immunity mutant strains, Δrsi5IBSBF1503 and Δrsi9IBSBF1503, showed an unclear competitive fitness disadvantage against wild-type, suggesting differences in the assay conditions may have contributed to the rsi fitness defects observed in the transposon mutant screens. This work suggests that the T6SS plays an important, if not essential, role in the pathogenic lifestyle of RSSC bacteria, and lays the groundwork for continued study of the significance of the T6SS for the causal agent of bacterial wilt.