RNA synthesis by the Brome mosaic virus RNA-dependent RNA polymerase and regulation of virulence in the plant pathogenic bacterium Ralstonia solanacearum

RNA viruses are represented by many medically and agriculturally important pathogens including human immunodeficiency virus, hepatitis C, and cucumber mosaic virus. RNA replication is a requirement for pathogenesis of these viruses and my work is aimed at understanding the mechanistic details of this process. Brome mosaic virus (BMV) is a model plus-strand RNA virus useful for studies of viral RNA replication. Each of the BMV genomic and subgenomic RNAs contains a conserved 3' end that forms a tRNA-like structure that is both required and sufficient to direct initiation of minus-strand synthesis. I determined that sequences 5' of the tRNA-like structure can modulate minus-strand synthesis. Thorough characterization of the sequences required for the initiation of minus-strand synthesis led to the identification of a stem-loop within the conserved tRNA-like structure that is required and sufficient to interact with RdRp. When this stem structure is amended with an initiation sequence it can faithfully direct RNA synthesis. This work has led to a better understanding of minus-strand synthesis and has allowed the construction of a functional minimal minus-strand promoter.; In addition, I also studied regulation of genes required for virulence in R. solanacearum, one of the world's most devastating bacterial plant-pathogens. A major virulence factor in R. solanacearum are the abundant exopolysaccharides (EPS). My research centered on EpsR, which when present in higher than normal amounts, dramatically decreases the amount of EPS produced from otherwise wild-type cells. EpsR has homology to proteins in the two-component family of transcriptional regulators whose ability to regulate gene expression is modulated by phosphorylation. EpsR can act as both a positive and negative regulator of EPS genes depending on its copy number and state of phosphorylation. Furthermore, EpsR's activity is likely mediated via phosphorylation-dependent binding to the promoter region of the major EPS operon in R. solanacearum.