Functional Analysis Of Genes Involved In C-di-GMP Signaling Pathway In Azorhizobium Caulinodans Symbiosed With Coastal Salt-tolerant Plant Sesbania Rostrata

Coastal saline-alkali land has advantages in the hydrothermal conditions and has become a key area in the development and utilization of saline-alkali land.Effective improvement and utilization of such land has far-reaching significance for the economic development and ecological construction of coastal areas.Planting salt-tolerant plants can improve the soil structure and microbial activity of coastal saline-alkali soil,and fundamentally improve saline-alkali soil.Rhizobium-legume symbiosis system is one of important bio-improvement systems.A thorough study of the symbiosis mechanism can provide theoretical basiss for the bio-improvement model of coastal saline-alkali land.Sesbania rostrata is a leguminous plant with salt and waterlogging tolerance and has become pioneer plant for improving coastal saline-alkali land.Azorhizobium caulinodans is a versatile nitrogen fixer that can fix nitrogen in the symbiotic interaction with Sesbania rostrata and also in a free-living state.As a second messenger,c-di-GMP is commonly found in the bacteria and regulates multiple cellular functions including motility,extracellular polysaccharide production and virulence,etc.The establishment of A.caulinodans-S.rostrata symbiosis involves the motility and chemotaxis ability,the colonization ability and the reactive oxygen species-resistant ability of A.caulinodans.However,the role of second messenger c-di-GMP in the establishment has not yet been studied.In this study,the c-di-GMP signaling pathway of A.caulinodans ORS571 was analyzed and the results were as follows:c-di-GMP metabolism genes of A.caulinodans ORS571 was analyzed by using bioinformatics,and there were 37 genes encoding GGDEF/EAL domain present in ORS571 genome.Three genes（AZC₃085,AZC₃226 and AZC₄658）that encoding GGDEF-EAL composite proteins were selected for functional analysis.Only mutantΔ4658 showed impaired motility,increased EPS production and biofilm formation,and elevated competitiveness in competitive nodulation assay.The deletion of AZC₄658 also led to increased intracellular c-di-GMP level.The results suggested that A.caulinodans ORS571 has a vast number of c-di-GMP metabolism related genes and may posses a complex metabolic network.In addition,some c-di-GMP metabolism related genes may have redundant function.Chp1,encoded by gene AZC₀308,was identified as a PDE.There were three separate domains in Chp1:a PAS domain,a GGDEF domain and an EAL domain.Enzymatic activity assay showed that Chp1 had only phosphodiesterase activity,and PAS domain of Chp1 functioned as a regulatory domain by binding heme.?chp1mutant showed elevated intracellular c-di-GMP concentrations and increased EPS production.The result of qRT-PCR showed that EPS synthesis related genes were higher expressed in?chp1 mutant,suggesting that the c-di-GMP regulation of EPS synthesis in A.caulinodans ORS571 occurs at least partially at the transcriptional level.Mutant impaired in EPS synthesis was also constructed,and the result of H₂O₂sensitivity assay indicated that EPS may protect A.caulinodans ORS571 against detrimental H₂O₂.?chp1 mutant displayed stronger competitiveness in root colonization and nodulation of S.rostrata,while the nitrogen fixation efficiency was the same as wild type.Together,c-di-GMP regulated EPS may play roles in the root colonization,and the the following nodulation process by conferring survival advantage on ORS571 against H₂O₂ that released during nodule formation.Cbp1,encoded by gene AZC₃349,was identified as a c-di-GMP effector.Cbp1 could specifically bind c-di-GMP and c-di-GMP binding led to a significant conformational change in Cbp1.Mutation of key residue Arg320 led to complete loss of c-di-GMP binding ability of Cbp1.As a chemoreceptor,loss of Cbp1 led to the impaired chemotaxis ability of ORS571.The complementary experiment further confirmed that the c-di-GMP binding ability of Cbp1 was essential for Cbp1 mediated chemotaxis.With impaired chemotaxis ability,?cbp1 mutant showed decreased competitiveness in root colonization and nodulation of S.rostrata.The results suggested that the c-di-GMP signaling pathway cooperates with the chemotaxis pathway to regulate the establishment of A.caulinodans-S.rostrata symbiosis.Two single PilZ domain proteins encoded by AZC₁615 and AZC₁616 were shown to regulate the motility of ORS571 in an opposite way,and the investigation of underlying mechanism is underway.In conclusion,the c-di-GMP signaling pathway of A.caulinodans ORS571 was investigated systematically,and a PDE and a c-di-GMP effector were identified.The analysis revealed that c-di-GMP could regulate A.caulinodans-S.rostrata symbiosis through different mechanisms,which contributing to further understanding of the complex c-di-GMP signaling pathway and the establishment of A.caulinodans-S.rostrata symbiosis.