Persulfidation of host NADPH oxidase RbohB by rhizobial 3-mercaptopyruvate sulfurtransferase maintains redox homeostasis and promotes symbiotic nodulation in soybean.

Reactive oxygen species play a crucial role in various stages of the legume-rhizobia symbiosis, from initial nodulation signaling to nodule senescence. However, how rhizobial redox-related proteins regulate symbiotic nodulation in legumes remains largely unknown. By combining transcriptomics, proteomics, and biochemical and molecular genetics, we investigated the role of the Sinorhizobium fredii Q8 enzyme 3-mercaptopyruvate sulfurtransferase (3MST). Although 3MST was not the primary enzyme responsible for hydrogen sulfide (H2S) production under our conditions, its absence significantly impaired symbiotic nodule development, redox homeostasis, infection capacity, and nitrogen-fixation efficiency in soybean. We identified a host plasma membrane-localized NADPH oxidase, respiratory burst oxidase homolog B (RbohB), as a key regulator of immune activation during nodule development. Notably, 3MST was secreted during nodulation and localized in the nucleoid and cytoplasmic membrane, where it interacts with and persulfidates RbohB at Cys791, thereby suppressing the NADPH oxidase activity of RbohB. We observed that 3MST-mediated persulfidation of RbohB maintains symbiotic redox balance and promotes nodule development. Genetic analyses in soybean, including RbohB overexpression, RNA interference, and site-directed mutagenesis at Cys791, further supported this observation, linking the 3MST-RbohB interaction to effective rhizobial colonization and improved plant growth. Taken together, these findings uncover a rhizobia-initiated symbiotic regulatory mechanism by which a rhizobial sulfurtransferase modulates soybean RbohB via persulfidation to limit NADPH oxidase activity and promote nodulation.