The extracellular Loop2 domain of the rhizobia outer membrane protein MhOpa22 mediates symbiotic nodulation and nitrogen fixation

The Mesorhizobium huakuii opacity protein (MhOpa22) is essential for effective nodulation and nitrogen fixation in rhizobium-legume symbiosis. Astragalus sinicus AsGLP1 interacts with MhOpa22, but the molecular mechanisms underlying the ability of MhOpa22 to mediate symbiosis remain elusive. This study demonstrated that the Loop2-3 domains of MhOpa22 interact with AsGLP1 in planta. Inoculating A. sinicus with different M. huakuii loop deletion mutants revealed that the Loop2 mutant gives rise to fewer root nodules and decreased nodule nitrogenase activity relative to inoculation with the wild-type strain. The Loop2 mutation also significantly affects reactive oxygen species production and the coordinated expression of defense and symbiosis genes in host plant roots during early symbiosis. Furthermore, MhOpa22 was confirmed to be a membrane protein that exerts its function via outer membrane vesicles (OMVs). Taken together, these results provide molecular insights into the function of the outer membrane protein MhOpa22 and its critical extracellular Loop2 domain during symbiosis. MhOpa22 inhibits host defense responses through OMVs during early symbiosis and plays an essential role in rhizobial infection and nodule formation.