Function of c-di-GMP Synthase Rsp1208 of Ralstonia solanacearum Strain GMI 1000

Bacterial wilt is a devastating soil-borne vascular disease caused by Ralstonia solanacearum. As a conserved second messenger in bacteria, c-di-GMP orchestrates multiple pathogenic phenotypes of plant bacterial pathogens. However, the functional characteristics of core c-di-GMP metabolic genes in R. solanacearum remain to be fully elucidated.

This study aimed to investigate the functions and regulatory mechanisms of c-di-GMP metabolism-associated genes in R. solanacearum GMI 1000, clarify the impacts of key genes on bacterial physiological phenotypes and pathogenicity, refine the c-di-GMP signaling regulatory network, and to provide a theoretical foundations for screening novel control targets and developing green management strategies against bacterial wilt.

Using R. solanacearum GMI 1000 as the material, RT-qPCR was performed to compare the transcriptional profiles of 24 putative c-di-GMP metabolic genes under routine culture and simulated infection conditions. For the most significantly downregulated gene Rsp1208, gene deletion, complementation, enzyme active-site mutagenesis and overexpression strains were constructed via homologous recombination and electroporation. Key physiological phenotypes including growth kinetics, motility, biofilm formation and exopolysaccharide (EPS) yield were systematically quantified. Transcriptional levels of motility- and EPS synthesis-related genes were analyzed by RT-qPCR. Intracellular c-di-GMP content and in vitro diguanylate cyclase activity were detected using LC-MS/MS and thiazole orange fluorescence assay, respectively. Pathogenicity assays on tomato seedlings were conducted using the root-wounding inoculation method.

Under simulated infection conditions, all 24 c-di-GMP metabolic genes were transcriptionally downregulated to varying degrees, among which Rsp1208 showed the most extreme downregulation. The protein encoded by Rsp1208 harbors both GGDEF and EAL domains. Deletion of Rsp1208 significantly increased bacterial motility by 26.57% and EPS production by 85.92%, reduced biofilm formation by 75%, decreased intracellular c-di-GMP levels markedly, and extremely enhanced pathogenicity on tomato. The complemented strain restored wild-type phenotypes, whereas the overexpression strain exhibited attenuated motility, mildly elevated biofilm formation and significantly weakened pathogenicity. Transcriptional levels of motility-related genes (flhC, fliA, fliM, fliC) and EPS synthesis genes (xpsR, epsB) were drastically upregulated in the Rsp1208 deletion mutant. Mutation of the GGDEF active site abolished the c-di-GMP synthetic activity of Rsp1208, while EAL site mutation exerted no significant effect.

Rsp1208 functions as a c-di-GMP diguanylate cyclase through its GGDEF domain, regulating intracellular c-di-GMP homeostasis in R. solanacearum, mediating phenotypic remodeling of motility, biofilm formation and EPS production, and ultimately governing the pathogenicity of the pathogen. These findings deepen the mechanistic understanding of R. solanacearum pathogenesis and provide critical theoretical support for the development of green control technologies against bacterial wilt.