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.