Structure Diversity Of Known Symbiosis Signals And Screening Of New Symbiosis Genes In Fast-growing Rhizobia Nodulating Soybean

Glycine max (soybean) is one of the most important legume crops, and its symbiosis with rhizobia plays an important role in the agricultural field. Until now, the most well known soybean microsymbionts in the world are from Bradyrhizobium, while Sinorhizobium fredii is the dominant microsymbiont of soybean in Chinese alkaline-saline soils. As S. fredii grows faster, making the production of inoculants easier, and exhibits higher competitive nodulation ability than Bradyrhizobium strains in the alkaline-saline soils, S. fredii has been expected to be a better soybean inoculant in the alkaline-saline soils than Bradyrhizobium. Thus, knowing more about the symbiosis mechenism between S. fredii and soybean will bring more opportunities to develop the sustainable agriculture.The Nod factors and Exopolysaccharides released by rhizobia are known as important signals between the early rhizobium-legume interaction. To know the structural deversities of Nod factors and EPS produced by soybean microsymbionts, the structures of these metabolites from S. fredii (CCBAU45436, CCBAU25509), S. sojae (CCBAU05684) and Sinorhizobium sp. (CCBAU05631) have been analyzed by ESI-MS/MS and NMR. It was shown that when grown in presence of Naringenin, the four rhizobial strains produced a common composition of Nod factors. These strains have been producing mostly tetramer (DP4) chitine backbones substituted by a methylfucose and a C18:1 fatty acid. Here we are naming this metabolite as Nod-Ⅳ (C18:1, Me-Fuc). The second most abundant metabolite is the Nod-Ⅴ (C18:1, Me-Fuc), which is the corresponding pentamer. When cultured in the medium contains Tryptone and Yeast-extract, the low molecular weight exopolysaccharides released by four strains shared the common sugar compositions and similar 1H NMR spectrum. Two samples of S. fredii CCBAU45436 and S. sojae CCBAU05684 have been submitted to complete NMR studies, in order to assess the identical structures. As a result, the repeating unit structure of S. fredii or S.sojae Exopolysaccharides is composed of glucose, galactose, gulucuronic acid in the ratios 5:1:2 or 6:1:2, which are bearing one pyruvate. All of these results above show that the fast growing soybean microsymbionts in this study produced the conserverd structures and compositons of Nod factors and Exopolysaccharides. Although there is much difference among the genomes of four strains, these conserved symbiosis signal may result from the selection pressure by soybean during the long evolutionary symbiosis mechenism.To obtain more new symbiosis gene in S. fredii, a large STM mutant library that contained 25500 mutants from 17 specific tagged pools was constructed. Three mutants with impaired nodule occupancy were obtained in three batches’ screening. Furthermore, the soybean inoculated by ptsP: Tn5-Tag4 showed nitrogen-deficiency phenotype. The gene knock-out and complementary experiments proved that lacking of ptsP resulted in the less production of PHB and low nitrogen fixation ability. The protein encoded by ptsP is the EINtr, which is composed of a GAF domain and three PEP-utilizer domains. The mutant lacking GAF domain showed the similar phenotypes with wild type strain in the symbiosis process, which indicated that GAF domain is not necessary for the symbiotic functions of EINtr. Moreover, AptsP didn’t grow well on L-Alanine as sole carbon source, which indicated that EINtr encoded by ptsP is also related to Carbon catabolic pathway. Thus, EINtr may be an important member in intergrating Carbon and Nitrogen catabolic pathways.