P-154 - Diguanylate cyclase domain proteins control virulence and interbacterial competition through targeting distinct two-component systems in Agrobacteria

Agrobacterium is a plant pathogen causing crown gall disease by transferring T-DNA and effectors through the type IV secretion system to the host. For niche competition, Agrobacterium also deploys the type VI secretion system (T6SS) to deliver toxin effectors for interbacterial competition. Diguanylate cyclases (DGCs), which synthesize the prokaryotic second messenger c-di-GMP, regulate processes like biofilm formation, virulence, and competition. Our study identifies two membrane-associated DGC domain proteins, DcvA and DcvB, that negatively regulate T-DNA transfer and tumor formation by inhibiting the promoter activities of virulence genes. DcvB, but not DcvA, promotes biofilm formation while repressing T6SS-mediated interbacterial competition. Catalytic analysis reveals that the regulation of DcvB is cyclase activity-dependent, whereas DcvA inhibits virulence through its membrane region without the DGC domain. VirA/VirG and ChvG/ChvI are crucial for virulence activation. Using the constitutively active response regulator, we show that DcvA inhibits virulence downstream of VirA, while DcvB suppresses both virulence and interbacterial competition downstream of ChvG. Further analysis identifies additional DGC proteins targeting similar or distinct regulatory pathways. These findings illustrate functional redundancy in DGC proteins and distinct regulatory strategies Agrobacterium uses to regulate virulence and competition, reflecting bacterial adaptation in plant environment.