Regulation of the hrp Type III Secretion System in Pseudomonas syringae pv. tomato DC3000

Pseudomonas syringae pv. DC3000 is a gram-negative bacterium that infects the model plant Arabidopsis thaliana. Pathogenicity is achieved via secretion of effector proteins into the host cytoplasm through a Type III Secretion System (T3SS). In Ps. DC3000 the T3SS (and associated effector proteins) are dependent on HrpL for their transcription. hrpL transcription is sigma54-dependent and requires two co-dependent enhancer binding proteins, HrpR and HrpS (HrpRS), for activation. HrpRS are regulated by two hrpL-dependent proteins, HrpV and HrpG, where HrpV negatively affects HrpRS activity and HrpG relieves this repression. Here the mechanism of HrpV and HrpG’s action on HrpRS activity was tested in vivo and in vitro; and the molecular determinants of HrpV and HrpG functionality were characterised by in silico and mutational analysis. Whole-gene deletion mutants of hrpV and hrpG in Ps. DC3000 revealed complications associated with inserting marker cassettes in transcriptionally-antagonistic orientations. Truncation mutants of HrpV and HrpG showed that C-terminal helices in both proteins play a functional and/or structural role. Alanine mutants indicated a structural role for residues 108-110 in HrpV and a functional role for residues 111-113 in HrpG (NQR motif). HrpV was demonstrated to form a dimer and represses the activity of HrpRS (in vivo and in vitro) but not via inhibiting ATPase activity or destabilising open promoter complexes. HrpG was shown to destabilise the HrpV dimer (potentially via the C-terminal NQR motif). Findings from this study were used to propose a revised model of hrpL regulation in which an active dimer of HrpV binds two adjacent HrpS subunits of a HrpRS hexamer to “lock” the hexamer in an unfavourable conformation. HrpG acts by destabilising the HrpV dimer into an inactive monomeric form. Additional experiments to confirm this model include creating an artificially linked dimer of HrpV and testing its repressive action on HrpRS in the presence or absence of HrpG