The Mechanism Research About Glycosyltransferase PssF Affecting Sinorhizobium Meliloti-alfalfa Symbiosis

Nitrogen is one of the most important elements in organism and is also an important limiting factor for agricultural production.With the increasing in population,the demand of the nitrogen in agricultural is increasing.The nitrogen compound is mainly to be absorbed by organisms from the environment.While the crop production has been improved because of synthetic nitrogen fertilizer,the irrational use of nitrogen fertilizer and artificial synthesis process pollution to the environment.Biological nitrogen fixation especially legume-rhizobia symbiotic nitrogen fixation is precisely regulated by plants and microorganisms and does not pollute to the environment.So it is a kind of energy-saving,environmental protection and efficient nitrogen fixing pathway.Therefore,it is an effective means to improve the efficiency of plant nitrogen fixation by studying the mechanism of legume-rhizobium symbiotic system.Previous studies have shown that bacterial surface polysaccharides especially extracellular polysaccharide can not only effect the infection ability and host specificity of rhizobial,but also affect the competitive nodulation process.However the mechanism is not particularly clear.In Sinorhizobium meliloti exo/exs and exp genes are involved in extracellular polysaccharide synthesis that influences host symbiotic process,while there are mainly the pss genes that are also existed in Rhizobium leguminosarum impacting the process.Sinorhizobium meliloti-alfalfa symbiosis is a natural model for research in legume and rhizobia interaction.We would like to known whether there are other ways to affect the extracellular polysaccharide synthesis so that the symbiotic process is affected.In addition,we try to explore the symbiosis mechanism abot the extracellular polysaccharide impacting the process.In this paper,we studied the mechanism of pssF gene in the symbiotic process by the model of S.meliloti 1021-alfalfa.The physiological status of the bacteria will affect the symbiotic process,So we have studied the growth characteristics the movement of the pssF gene mutant.We found that the mutant strain is not affected in growth and the movement activity.The salt tolerance of the rhizobial wasn't affected,while the content of secondary metabolites exopolysaccharides was significantly increased in the mutant strain(both the low molecular weight and high molecular weight).In this study the effect of extracellular polysaccharide on the adhesion of the rhizobia and the formation of infection thread was studied.When inoculated of alfalfa 1-2 days by wild type and pssF gene mutant,the mutant will cause root hair deformation delayed and the expression of defense gene MsPR10 in the alfalfa root inoculated of the mutant in 30 min to 48 hours is higher than inoculated of the wild-type which is the result to the change of the plant defense capability.The infection thread numbers in alfalfa root of 5 and 7 days post-inoculated mutant with Lacz label is far less than inoculated the wild-type,while the infection thread structure is unchanged.The binding experimental result shows that the binding ability of root hairs in pssF gene mutant is much lower than the wild type,which suggesting that pssF gene may through reducing the rhizobial binding ability lead to the decrease of the infection thread numbers.The decrease of infection thread numbers possibly lead to the decrease of the numbers of nodules,but there was no significant difference in the of 28 days nodule numbers by inoculation of the wild-type and the mutant strains respectively.This result suggested that the knockout of pssF gene in Sinorhizobium meliloti 1021 increased the symbiotic efficiency due to the low utilization rate of the infected threads.The nitrogen fixation ability experiment results showed that the pssF gene mutant could increase the nitrogen fixation ability in 28 days nodules.And the competitive experiments was found that the proportion of the nodule formed by inoculated of the mutant strain was about 2 times higher than the wild type,which showed that the mutant was more competitive than the wild type.These results showed that the symbiotic ability of the mutant strain was better than that of the wild type in symbiosis process.In order to further study the mechanism of pssF gene affecting the symbiosis process,PssF protein is purified in vitro.PssF protein is speculated to the glycosyltransferase and belongs to the GT2 family of the superfamily GT-A by bioinform atics analysis.The conserved DxD structure is the catalytic active site of the enzyme.In this study,The donor of PssF protein was identified as UDP-glucose by detection of the reaction residual amount by HPLC.Unfortunately,the recipient of PssF was not found.In conclusion,S.meliloti 1021 glycosyltransferase PssF directly or indirectly inhibit symbiotic efficiency and nitrogenase activity through reducing the exopolysaccharides content.This result reinforces us the understanding about the mechanism of extracellular polysaccharides affecting the symbiotic process and providing the basis for development of the effective rhizobia.