Osmotic and pH Stress-Responsive Two-Component System, OmpR/EnvZ, Modulates Type III Secretion, Biofilm Formation,...

Acidovorax citrulli, the causal pathogen of bacterial fruit blotch of cucurbits, relies on a functional type III secretion system (T3SS) for pathogenicity. Two-component systems (TCSs) are primary signal transduction mechanisms for bacteria to detect and adapt to various environmental conditions. However, the role of TCS on regulating T3SS and other virulence factors in response to environmental stimuli is still poorly understood in A. citrulli. Here, we report the identification of a conserved TCS, OmpR/EnvZ, involved in hypersensitive response (HR) induction in Nicotiana benthamiana by screening a transposon-insertion library in the group II strain xjL12 of A. citrulli. Transcription analysis confirmed that OmpR_Ac/EnvZ_Ac was upregulated in response to elevated osmotic pressure, low and high pH conditions, and host environment. Deletions of envZ_Ac, ompR_Ac, or both envZ_Ac and ompR_Ac in A. citrulli attenuated virulence to melon seedlings and mature leaf tissues, and delayed HR in N. benthamiana. OmpR_Ac was activated by EnvZ_Ac and directly bound to the promoter region of hrpG, a major regulator of T3SS. This binding activated hrpG transcription and promoted T3SS assembly in T3SS-inducing medium, XVM2. Additionally, the OmpR_Ac/EnvZ_Ac mutants of A. citrulli displayed reduced swimming motility due to impaired flagella formation, but also had enhanced biofilm formation and exopolysaccharide production. OmpR_Ac/EnvZ_Ac regulation of these virulence factors in A. citrulli depended on its own conserved phosphorylation sites. This work illuminates a signalling pathway for regulating the T3SS and provides insights into the OmpR/EnvZ-mediated virulence regulatory network in A. citrulli.