Functional Characterization of Key Residues in Regulatory Proteins HrpG and HrpV of Pseudomonas syringae pv. tomato DC3000

The plant pathogen Pseudomonas syringae pv. tomato DC3000 uses a type III secretion system (T3SS) to transfer effector proteins into the host. The expression of T3SS proteins is controlled by the HrpL σ factor. Transcription of hrpL is σ54-dependent and bacterial enhancer-binding proteins HrpR and HrpS coactivate the hrpL promoter. The HrpV protein imposes negative control upon HrpR and HrpS through direct interaction with HrpS. HrpG interacts with HrpV and relieves such negative control. The sequence alignments across Hrp group I-type plant pathogens revealed conserved HrpV and HrpG amino acids. To establish structure–function relationships in HrpV and HrpG, either truncated or alanine substitution mutants were constructed. Key functional residues in HrpV and HrpG are found within their C-terminal regions. In HrpG, L101 and L105 are indispensable for the ability of HrpG to directly interact with HrpV and suppress HrpV-dependent negative regulation of HrpR and HrpS. In HrpV, L108 and G110 are major determinants for interactions with HrpS and HrpG. We propose that mutually exclusive binding of HrpS and HrpG to the same binding site of HrpV governs a transition from negative control to activation of the HrpRS complex leading to HrpL expression and pathogenicity of P. syringae.