An integrated Msr-antioxidase-host gene circuit maintains redox homeostasis in legume-rhizobium symbiosis

Methionine sulfoxide reductases (Msrs) play a critical role in oxidative stress resistance; however, their functions in rhizobium-legume symbiotic nitrogen fixation (SNF) are not well understood. In this study, we systematically characterized four Msrs (MsrA1, MsrB1, MsrA2, MsrB2) from Mesorhizobium huakuii 7653R, a symbiotic partner of Astragalus sinicus . Sequence and phylogenetic analyses confirmed the presence of conserved catalytic domains and revealed genus-specific clustering of these Msrs. Expression profiling demonstrated distinct patterns: msrA1 and msrB1 were transiently induced during early symbiotic infection, whereas msrA2 and msrB2 exhibited biphasic upregulation at both early infection and nodule maturation stages. Notably, msrA1 responded specifically to H 2 O 2 , and all msr genes were induced by sodium hypochlorite in a concentration-dependent manner. Phenotypic analyses of overexpression (OE) and deletion (Δ) strains indicated that Msrs modulate key bacterial physiological traits. Deletion mutants showed impaired motility, reduced biofilm formation, and decreased activities of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase), accompanied by elevated intracellular superoxide anion and H 2 O 2 content. In contrast, msrs overexpression enhanced oxidative stress resistance but suppressed bacterial growth. In symbiotic assays, overexpression of msrA1 , msrA2 , or msrB2 resulted in leaf chlorosis, reduced nodule number, and impaired nitrogen fixation efficiency, while msrA2Δ and msrB2Δ mutants affected nodulation without compromising plant vigor. Further investigation revealed that Msrs regulate host root antioxidant responses and the transcription of symbiotic-related genes ( AsNIN , AsNPL2 ) and defense-related genes ( AsFLS2 , AsPR10 ). Bacterial two-hybrid assays identified physical interactions between Msrs and chaperone proteins (GroEL1/2/3), antioxidant enzymes (SodA/B, KatE/G), and the LysR-type transcriptional regulator LsrB, suggesting the formation of an integrated redox regulatory network. Collectively, our findings demonstrate functional specialization of Msrs in M. huakuii 7653R, mediating oxidative stress resistance, bacterial physiology, and host–symbiont crosstalk. We propose a “Msr – antioxidant enzyme – host gene” regulatory model that maintains redox homeostasis during SNF. This study provides novel insights into the roles of rhizobial Msrs and offers potential targets for engineering high-efficiency nitrogen-fixing strains.