The Mechanism Underlying Broad-host-range Adaptation Of Sinorhizobium Sp. NGR234

The rhizobium-legume interaction is characterized by its ability of forming nitrogen-fixing nodules. Nodules can be categorized into either indeterminate or determinate nodules based on whether a persistent meristem is present or not. The adaptation mechanisms of rhizobium in these two kinds of nodules have been intensively studied. However because of host specificity, most systematic studies in this field were performed on different bacteria and their corresponding hosts. In this study, by using broad host rhizobium Sinorhizobium sp. NGR234and its indeterminate nodule host Leucaena leucocephala and determinate nodule host Vigna unguiculata, we aimed to study the adaptive mechanisms of a rhizobium in these two different nodules. RNA-seq was used to uncover transcriptomic differences between bacteroids in determinate nodules and those in indeterminate nodules. Genetic methods were then used to study the symbiotic role of representatives of differentially expressed genes. This study would provide further insights into the adaptation mechanisms of rhizobium in determinate and indeterminate nodules.In contrast to exponentially growing free-living bacteria, in bacteroids most genes on two plasmids were significantly up-regulated whereas the majority of chromosomal genes were down-regulated. Bacteroids from two legumes recruited several common cellular functions such as cbb3oxidase, thiamine biosynthesis, nitrate reduction pathway (NO-producing), succinate metabolism, PHB (poly-3-hydroxybutyrate) biosynthesis and phosphate/phosphonate transporters. However, different transcription profiles between bacteroids from two legumes were also uncovered for genes involved in the biosynthesis of exopolysaccharides, lipopolysaccharides, T3SS (type three secretion system) and effector proteins, cytochrome bd ubiquinol oxidase, PQQ (pyrroloquinoline quinone), cytochrome c550, pseudoazurin, biotin, phasins and glycolate oxidase, and in the metabolism of glutamate and phenylalanine.Based on these transcriptomic results,19deletion mutants were constructed for corresponding differentially expressed genes. Nine mutants showed defects in bacteroids persistance in V. unguiculata nodules and these genes encode Phasin1, Phasin2, ATP-dependent carboxylate-amine ligase, Hypothetical protein, PRC-barrel domain protein, UspA, cytochrome bd-Ⅱ, and RibH respectively. Among these nine mutants, five and four mutants were impaired in infecting nodules of Cajanus cajan and Lablab purpureus, respectively; one mutant showed defects in bacteroids persistence in L. purpureus nodules. However no significant differences were observed between wild type strain and18/19mutants when they were inoculated on L. leucocephala. Thus we hypothesized that, in contrast to V. unguiculata, C. cajan and L. purpureus, L. leucocephala might provide rhizobia with more carbon and energy sources that could support normal symbiotic process when the above mentioned mutants were inoculated. Among these four lemume hosts, L. leucocephala and C. cajan formed indeterminate nodules whereas V. unguiculata and L. purpureus formed determinate nodules. Taken together, these findings suggest that the symbiotic role of the same gene of NGR234showed noticeable variations, when forming symbiosis with different legume hosts, in terms of the essentialbility for different symbiotic stages (infection or bacteroids differentiation). And these symbiotic phenotypes have no correlation with nodule types (indeterminate or determinate nodules).All strains closely clustered with NGR234in the genome based phylogenetic tree can form nitrogen-fixing nodules with Glycine max/Glycine sojae, whereas NGR234can not. By constructing and screening a Tn5mutant library of NGR234, seven mutants were found to be able to form nitrogen-fixing nodules on G. sojae (1-1.6nodules per plant), though10%-30%of plants inoculated with these seven mutants did not have effective nodules. These results imply that multiple mutation events or some foreign genes might be needed by NGR234for establishing more stable and effective symbiosis with G. sojae. Interestingly, in this mutant library, a mutant of the gene encoding a LysR regulator formed effective nodules on C. cajan, L. leucocephala and V. unguiculata, but was impaired in infecting its primary host L. purpureus. Most homologs of this LysR regulator were present in pathogenic bacteria, indicating that NGR234could use certain mechanism similar to those pathogens to infect L. purpureus and that the coding gene of this regulator could have been laterally transferred to NGR234.