Molecular Mechanism Of Rhizosphere Chemotaxis And Colonization Of Symbiotic Rhizobia To Sesbania Rostrata

Soil salinization is an important factor causing the deterioration of coastal soil quality,and it is also an important research field under the framework of global environmental change research.Coastal saline-alkali land is an important reserve land resource in China.Reasonable development and utilization of coastal saline-alkali land is conducive to alleviate the shortage of land resources in China and ensuring national food security.The development of salt-tolerant plants is an important strategy to solve soil salinization.Sesbania rostrata is a legume plant that can interact with nitrogenfixing rhizobia to form nodules on its roots or stems.In addition to the plant itself,rhizosphere microorganisms,including Azorhizobium caulinodans,also play an important role in the process of relieving soil salt pressure of plant.These rhizosphere microorganisms can promote plant growth in saline soil and increase crop yield.Exploring the plant-microbe interaction,especially the interaction between A.caulinodans and S.rostrata symbiosis system,is useful for improving the physical and chemical properties of coastal saline soil,improving fertility,and forming a systematic saline soil microbe-phytoremediation technology.In this study,we explored the regulation of the chemotactic behavior of A.caulinodans,as well as the role of chemotactic system in the process of A.caulinodans chemotaxis towards and colonization to the rhizosphere of Sesbania,the main research result is as follows.(1)Plant exudates have significant chemotactic effects on rhizosphere bacteria.After identifying the A.caulinodans chemotaxis towards three amino acids and five organic acids in S.rostrata exudates.We identified Tlp H and Tlp A1 from the 43 chemoreceptors of A.caulinodans,which are involved in the chemotactic response to the above-mentioned amino acids and organic acids,respectively,and found that histidine,arginine,aspartic acid,and oxalic acid directly or indirectly regulate the chemotactic behavior of A.caulinodans.(2)A.caulinodans has multiple Che Y proteins that are ubiquitous in alpha proteobacteria,but compared with alpha proteobacteria Sinorhizobium meliloti,it has an additional CheZ protein,and this CheZ protein is different from that in E.coli.Bacterial chemotaxis experiments and phosphate transfer experiments identified that CheZ of A.caulinodans has an important role on the regulation of bacterial chemotaxis.The experiment of tethered bacteria revealed that unlike S.meliloti whose flagella can only rotate clockwise,the flagella of A.caulinodans can rotate both clockwise and counterclockwise,and CheZ plays an important role in controlling the direction of flagella rotation.(3)The mechanism and dynamic of CheZ location in A.caulinodans.Fluorescent protein fusion and cell sublocalization observations revealed that the CheZ of A.caulinodans could be localized to the cell poles,but the achievement of polar localization of CheZ in A.caulinodans is independent of Che A.In addition,the polar localization of CheZ is affected by the environmental oxygen concentration and nitrogen fixation state.There might be a possible cross-linking between CheZ phosphatase activity and intracellular sublocalization.(4)CheZ also participates in the early adhesion and colonization process of the interaction between A.caulinodans and S.rostrata.The ?cheZ mutant has significant advantages in competitive adhesion and colonization.However,the early adhesion advantage of the ?cheZ mutant failed to transfer to the final nodulation.After tracking the long-term colonization of wild type and ?cheZ through Lac Z labeling,it was found that the advantages of ?cheZ gradually disappeared due to the inability to freely diffuse during the long-term colonization process.The abnormal performance of ?cheZ mutants in early adhesion stems from the role of CheZ on biofilm and extracellular polysaccharide synthesis.The analysis of polysaccharide gene expression showed that the synthesis of extracellular polysaccharides in A.caulinodans is regulated by CheZ,and CheZ and Che A antagonize each other in these processes.Although ?cheZ has defects in the process of symbiosis and nodulation with Sesbania,the ?cheZ mutant has a better effect on the plant growth promoting effect of wheat than the wild type.(5)By removing calcium ions or adding calcium chelating agents to the culture medium,the necessity of calcium for A.caulinodans biofilms formation was proved.The promotion and maintenance of calcium ions on the biofilm of A.caulinodans are mainly achieved by cross-linking extracellular DNA and extracellular proteins.Studies have also found that calcium ions not only promote the formation of biofilms,but also promote the movement of A.caulinodans and the colonization of Sesbania.In a word,in this study,we tried to explore the relationship between A.caulinodans and Sesbania from how Sesbania exudates attract A.caulinodans,how A.caulinodans regulate its chemotactic behavior,root surface adhesion,biofilm formation,and host colonization,etc.These works have further enriched our understanding of how to establish a connection between A.caulinodans and the Sesbania,and provide a scientific rationale for the better application of the Sesbania-A.caulinodans system on coastal saline-alkali soils.