Study On The Function Of Early Symbiotic Genes In Mesorhizobium Huakuii Based On Transcriptome Analysis

Mesorhizobium huakui 7653R is a kind of Gram-negative bacteria belonging to?-Proteus,which can survive freely in the soil and form nitrogen-fixing nodules in the root of Astragalus sinicus,a leguminous plant.In order to better understand the early symbiotic steps in the A.sinicus-M.huakuii symbiotic interaction,RNA-Seq was performed to study the differential transcriptomes of 7653R early nodule bacteroids and free-living cells.Bioinformatics analysis was further used to analyze the potential early symbiotic genes.The roles of these potential symbiotic genes during N-fixing symbiosis were explored by analyzing the phenotype of their mutant strain.RNA-Seq was performed to investigate the differential transcriptomes of early nodule bacteroids and free-living cells.The results are as follows:6605 expressed genes were detected in both early nodule bacteroids and free-living cells.Comparative analysis of gene expression levels found that 397 genes were differentially expressed,of which 363 were upregulated and 34 were downregulated in the early nodule bacteroids compared to in free-living cells.Through GO classification and KEGG pathway enrichment analysis,all of this differentially expressed genes were classified.The results showed most up-regulated differential genes involved in the early nodule bacteroids involved in bacteroid differentiation and nodule development.15 genes related to early symbiotic nitrogen fixation were selected from the up-regulated differentially expressed genes for further functional study.Mutation in these selected genes was generated by homologous recombination.Through the plant pot experiment,these mutant strains were inoculated with A.sinicus.4 weeks post inoculation,the leaves of 15 mutant plants were yellow in different degrees.Nitrogen fixation activity was further measured using the acetylene reduction method.The results showed that plants inoculated with 15 mutant strains had a significantly lower nitrogen fixation capacity than plants inoculated with the wild-type 7653R strain.fmo A encodes flavin-containing monooxygenases(FMOs)which catalyzes the NADPH-dependent oxygenation of numerous foreign chemicals.The fmo A mutant displayed decreased antioxidative capacity under higher concentration of H₂O₂and cumene hydroperoxide(CUOOH),indicating that Fmo A plays an important role in response to the peroxides.The fmo A mutant is not only severely impaired in rhizosphere colonisation,but also shows a 65%reduction in nitrogen-fixation ability.Real-time quantitative PCR showed that the fmo A gene expression is significantly up-regulated in the root nodule.The structural organization of 4-week-old nodules of A.sinicus is studied by thin-sectioning and scanning electron microscopy techniques.Microscopic analysis of fmo A mutant nodules showed that they were spherical rather than elongated.Moreover,bacterial size and membrane incrassation indicated they had undergone premature senescence,poly-b-hydroxybutyrate(PHB)was also observed in the fmo A mutant bacteroids.abi E encodes an antitoxin protein,which belongs to bacterial type IV toxin-antitoxin system.Mutant in abi E gene had no significant effect on the growth of M.huakuii in AMS minimal medium,but mutation changed its resistance ability of different antibiotics.The plants inoculated with the abi E mutant showed a 77%reduction in nitrogen-fixation ability,although the number of nodules infected by abi E mutant was not significantly different from that infected by the wild type 7653R.Moreover,mutant in abi E gene seriously affects the colonization and competitive nodulation of M.huakuii in the rhizosphere.Ultrastructural analysis of the nodule by transmission electron microscopy revealed that nodule cells infected by M.huakuii abi E mutant contained a few normal bacteroids and numerous senescences.These results showed that abi E gene plays an important role in environmental adaptation and symbiotic nitrogen fixation of M.huakuii.tep A gene encodes a potential ABC-type bacteriocin exporter protein.Mutant in tep A gene had no significant effect on the growth of M.huakuii in AMS minimal medium.In order to study the symbiotic phenotype of tep A mutant,Astragalus sinicu were inoculated with M.huakuii tep A mutant strain formed smaller abnormal nodules,and the number of nodules infected by tep A mutant was more than 3 times that of nodules infected by the wild type 7653R.The tep A mutant showed a 96%decrease in acetylene reduction activity.Ultrastructural analysis of the nodule by transmission electron microscopy revealed that nodule cells infected by tep A mutant consisted of vacuolation,senescing bacteroids and dissociations.Furthermore,mutant in tep A gene had a negative effection on the colonization and competitive nodulation of M.huakuii in the rhizosphere.These results showed that tep A gene plays an important role in bacteroid differentiation and symbiotic nitrogen fixation of M.huakuii.