Rhizobial HmuS pSym as a heme-binding factor is required for optimal symbiosis between Mesorhizobium amorphae CCNWGS0123 and Robinia pseudoacacia.

Nitrogen-fixing root nodules are formed by symbiotic association of legume hosts with rhizobia in nitrogen-deprived soils. Successful symbiosis is regulated by signals from both legume hosts and their rhizobial partners. HmuS is a heme degrading factor widely distributed in bacteria, but little is known about the role of rhizobial hmuS in symbiosis with legumes. Here, we found that inactivation of hmuS _pSym in the symbiotic plasmid of Mesorhizobium amorphae CCNWGS0123 disrupted rhizobial infection, primordium formation, and nitrogen fixation in symbiosis with Robinia pseudoacacia. Although there was no difference in bacteroids differentiation, infected plant cells were shrunken and bacteroids were disintegrated in nodules of plants infected by the ΔhmuS _pSym mutant strain. The balance of defence reaction was also impaired in ΔhmuS _pSym strain-infected root nodules. hmuS _pSym was strongly expressed in the nitrogen-fixation zone of mature nodules. Furthermore, the HmuS _pSym protein could bind to heme but not degrade it. Inactivation of hmuS _pSym led to significantly decreased expression levels of oxygen-sensing related genes in nodules. In summary, hmuS _pSym of M. amorphae CCNWGS0123 plays an essential role in nodule development and maintenance of bacteroid survival within R. pseudoacacia cells, possibly through heme-binding in symbiosis.
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