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

Reactive oxygen species (ROS) play a crucial role in various stages of legume-rhizobium 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 source of hydrogen sulfide (H₂S) under our conditions, its absence significantly disrupted symbiotic nodule development, redox homeostasis, infection capacity, and nitrogen fixation efficiency in soybean. We identified host plasma membrane-localized NADPH oxidase (RbohB) as a pivotal regulator that activates immune responses during nodule development. Notably, 3MST localized to the nucleoid and cytoplasmic membrane and was secreted during nodulation, where it interacted with RbohB and persulfidated Cys791 to suppress NADPH oxidase activity. This 3MST-mediated regulation maintained symbiotic redox balance and promoted nodule development. Genetic analysis of soybean involving RbohB overexpression, RNA interference and site-directed mutagenesis at Cys791 supported the model, linking the 3MST-RbohB interaction to effective rhizobial colonization and improved plant growth. Collectively, our findings shed light on a rhizobium-host redox pathway in which a rhizobial sulfurtransferase modulates RbohB via persulfidation to promote nodulation.