| Soybean is an important crop and can establish symbiotic nitrogen-fixing system with rhizobium.Promoting the application of rhizobium inoculant can reduce the amount of chemical fertilizer usage and environmental pollution. Different rhizobia can exhibit variance in symbiotic performance on the same soybean cultivar. The aim of this study is to reveal the genetic mechanism underlying the variance of rhizobial symbiotic ability, and guide the practice of engineering rhizobia of improved symbiotic performance.Sinorhizobium fredii CCBAU 45436, Sinorhizobium sp. CCBAU 05631 and Sinorhizobium sojae CCBAU 05684 belong to different phylogenetic lineages. Their genome sizes are different (45436, 6.91 Mb; 05631, 6.26 Mb; 05684, 6.09 Mb), and they have contrasting symbiotic performences with tested host plants such as commercial soybean cultivar JD17, wild soybean and pigeon pea. The symbiotic ability of 45436 is much stronger than that of 05631 and 05684, and the symbiotic ability of 05684 is the weakest.The variances in genome content of three strains were analyzed in this study by comparative genomics. The 15 gene clusters of 45436 were individually deleted, and among the resultant mutants,the deletion mutant of nitrate-reduction gene cluster showed symbiotic defects on JD17, wild soybean and pigeon pea. The nitrate-reduction gene cluster is also present in the genome of 05631, and knocking out this gene cluster resulted in impaired symbiotic performance of 05631 on wild soybean and pigeon pea. However, the genome of 05684 doesn't have the 15 gene clusters. In the nitrate-reduction gene cluster, the nap operon (encoding nitrate reductase) other than the nir (encoding nitrite reductase) and nor (encoding nitric oxide reductase) is essential for 45436 to establish symbiosis with host plants. The gene necessary for 05631 to build symbiotic relationship with host plants is not nap, nir, or nor, but hemNl (encoding oxygen-independent coproporphyrin III oxidase). There are two copies of hemN in the genomes of 45436, 05631 and 05684. The hemNl deletion mutants of 45436 and 05631 formed red nitrogen-fixing nodules on wild soybean plants, which however showed nitrogen deficient symptoms due to the decreased glutamine synthetase activity in nodules. These ineffective nodules showed a significant decrease of the glutamine synthetase concentration and an elevated nitrification level of glutamine synthetase. Wild soybean plants inoculated with the hemNl and hemN2 mutants of 05684 formed white nodules and pseudo-nodules without nitrogenase activity. The hemN1 gene function can be mutually complemented in the pairs of 45436-05631, and 05631 -05684. The hemNl of 45436 cannot complement the symbiotic phenotype of 05684 hemNl mutant, while the hemNl of 05684 can restore the symbiotic phenotype of 45436 hemNl mutant. The functions of hemNl and hemN2 were differentiated, and the hemN2 gene was not able to complement the symbiotic phenotype of the hemN1 mutant. Taken together, nap and hemNl exert lineage-dependent contributions to the optimization of Sinorhizobium symbiosis with their host plants,05631 and 05684 were not compatible with the JD17, forming pseudo-nodules. In order to reveal the determinant of the compatibility between the two strains and the JD17, both Tn5 random insertion mutant libraries and spontaneous mutants evolved in an experimental evolutionary experiment were subjected to the selective pressure of JD17. As a result,34 mutants of 05631 were found as compatible microsymbionts on JD17, forming effective nitrogen-fixing nodules. But no variants compatible with JD17 were obtained from 05684. There are 27 Tn5 mutants and 7 spontaneous mutants among the 34 mutants of 05631, and the mutated genes by Tn5 insertion included those encoding type III secretion system (T3SS), ABC transporter and so on. Reverse genetics revealed that only pV0155 insertion mutation in the T3SS gene cluster (including rhcC2 and ttsl) can induce red nitrogen-fixing nodules on JD17 plants. Fifteen Tn5 mutants and spontaneous mutants were selected for genome sequencing, and the results showed that insertion mutations within the T3SS gene cluster dominantly mediated by IS(insertion sequence) transposition accounted for the adaptive evolution of 05631 compatibility with JD17. These compatible variants still kept stable symbiosis ability with wild soybean, and their symbiotic phenotypes were similar to that of the wild type, indicating that mutation of genes encoding T3SS expanded the host range of 05631. Based on the findings of our team during the same period,inactivation of T3SS in 05684 by reverse genetics can lead to effective nitrogen-fixing symbiosis with JD17. Therefore, it could be the relatively lower evolutionary potential that restricted 05684 from the adaptive evolution of symbiotic compatibility with JD17 under the same experimental condition as 05631. |
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