Regulation Of Virulence In The Plant Pathogen Pseudomonas Syringae Pv. Tomato Dc3000
The type III secretion system (T3SS) is required for virulence of the gram-- negative plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) in tomato and Arabidopsis. The alternative sigma factor HrpL directly regulates expression of T3SS genes by binding to a short DNA sequence designated as the "hrp promoter". The ability of DC3000 to colonize plants, subdue multiple layers of plant defense and multiply in plant tissues relies on the activities carried out by the many T3SS regulon members (known collectively as hrp genes). Efforts to identify genes involved in pathogenicity were initiated over three decades ago. However, HrpL binding to hrp promoters has never been directly demonstrated and it is unclear if the list of HrpL--regulated genes is complete. The first goal of the research described here was to systemically and exhaustively identify HrpL--binding sites and likely hrp promoters in the DC3000 genome. Employing chromatin immuno--precipitation, coupled with high-- throughput sequencing (ChIP--Seq) and transcription start site analysis (modified RNA--Seq), we found twenty sites representing novel hrp promoters. Using deletion analysis, we attempted to determine if the genes downstream from a subset of these promoters could be linked to virulence. However, the deletions did not affect the hypersensitive response or in planta growth of the resulting strains. Interestingly, many new HrpL regulon members appear to be unrelated to the T3SS (based on their annotations), and orthologs for some of these can be identified in non--pathogenic bacteria. The connection of these new HrpL regulon members to virulence is not obvious. The HrpL regulon is activated as a result of a chain of events, most of which are not well understood. It is known that RpoN, which controls the transcription of hrpL in DC3000, is required for virulence in several bacterial species. Motivated by the hypothesis that genes are coordinately regulated in order to serve a strategic purpose (e.g., virulence), our second goal was to look for other genes activated by RpoN in parallel with hrpL. RpoN ([sigma]54) requires specialized enhancer--binding proteins (EBPs) in order to activate transcription. This arrangement presumably allows the cell to respond to environmental signals by modifying the transcription of particular genes. Using ChIP--Seq and RNA--Seq, we identified candidate RpoN--dependent genes as well as genes that were differentially expressed under hrp--inducing conditions. This initial survey includes more than 200 likely RpoN--regulated genes involved in flagella biosynthesis, energy metabolism, nitrogen metabolism, transport and binding proteins, and small noncoding RNAs, as well as putative regulatory proteins and EBPs. Among the genes that were differentially regulated between hrp--inducing and repressing conditions, more than one dozen appear to be regulated by RpoN and are therefore potentially important in functions related to plant association or virulence.
Pseudomonas syringae; Type III secretion; virulence; sigma factor; enhancer; HrpL; RpoN; regulation; transcription
Myers, Christopher R; Perry, Keith Lloyd; Helmann, John D; Collmer, Alan Raymond
Ph.D. of Plant Pathology
Doctor of Philosophy
dissertation or thesis