A microaerobically induced small heat shock protein contributes to <i>Rhizobium leguminosarum</i> / <i>Pisum sativum</i> symbiosis and interacts with a wide range of bacteroid proteins

During the establishment of the symbiosis with legume plants, rhizobia are exposed to hostile physical and chemical microenvironments to which adaptations are required. Stress response proteins including small heat shock proteins (sHSPs) were previously shown to be differentially regulated in bacteroids induced by <i>Rhizobium leguminosarum</i> bv. viciae UPM791 in different hosts. In this work, we undertook a functional analysis of the host-dependent sHSP RLV_1399. A <i>rlv_1399-</i>deleted mutant strain was impaired in the symbiotic performance with peas but not with lentil plants. Expression of <i>rlv_1399</i> gene was induced under microaerobic conditions in a FnrN-dependent manner consistent with the presence of an anaerobox in its regulatory region. Overexpression of this sHSP improves the viability of bacterial cultures following exposure to hydrogen peroxide and to cationic nodule-specific cysteine-rich (NCR) antimicrobial peptides. Co-purification experiments have identified proteins related to nitrogenase synthesis, stress response, carbon and nitrogen metabolism, and to other relevant cellular functions as potential substrates for RLV_1399 in pea bacteroids. These results, along with the presence of unusually high number of copies of <i>shsp</i> genes in rhizobial genomes, indicate that sHSPs might play a relevant role in the adaptation of the bacteria against stress conditions inside their host.IMPORTANCEThe identification and analysis of the mechanisms involved in host-dependent bacterial stress response is important to develop optimal <i>Rhizobium</i>/legume combinations to maximize nitrogen fixation for inoculant development and might have also applications to extend nitrogen fixation to other crops. The data presented in this work indicate that sHSP RLV_1399 is part of the bacterial stress response to face specific stress conditions offered by each legume host. The identification of a wide diversity of sHSP potential targets reveals the potential of this protein to protect essential bacteroid functions. The finding that nitrogenase is the most abundant RLV_1399 substrate suggests that this protein is required to obtain an optimal nitrogen-fixing symbiosis.