Proteome Analysis Of Xanthomonas Oryzae Pv. Oryzicola Under Phosphate Deficiency Stress And Study Of Function Of The Key Genes In Its Phosphate Metabolism

Xanthomonas oryzae pv.oryzicola(Xoc)is an important phytopathogenic bacterium,which can cause bacterial leaf streak of rice(BLS),and results in a huge loss of the total rice productivity.BLS is prevalent in tropical and subtropical areas,and is also a important rice disease in southern of China,especially in Guangxi and Guangdong.The pathogenic ability of Xanthomonas oryzae pv.oryzicola is closely related with environment in addition to pathogenic factors.Xoc can be colonized on the host rice by adapting to the environment,especially changing nutrients.Inorganic phosphate(Pi)is an essential nutrient element of Xoc,which plays an important role in signal transduction and energy metabolism.Its uptake is mainly accomplished by Pst system,which is controlled by pho BR.pho R regulates the activity of pho B through phosphorylation and dephosphorylation.pho B can active or inhibits expression of downstream genes.At present,there are many researches on functional genes related to the infection and pathogenicity of Xoc,but there are rare reports on phosphate metabolism regulation of Xoc.In this study,Xoc GX01 strain was taken as the research object,which is isolated from Guangxi.Xoc GX01 under phosphate-dificient condition were analyzed by i TRAQ proteomics,and the function of phosphate-regulated pho BR was studied.1.In this study,using the isobaric tags for relative and absolute quantification i TRAQ proteomics approach to identify differentially expressed proteins in phosphate-rich and phosphate-dificient conditions.A total of 252 differentially expressed proteins(DEPs)are identified.212 proteins were up-regulated and 40 proteins were down-regulated.Differential proteins mainly involve ribosomal protein synthesis,two-component regulatory systems,cell membranes,phosphorylation,arginine biosynthesis,signal transduction and so on.They were related to flagella,type Ⅳ pilus,type Ш secretion system,adhesin and so on.The key proteins of DEPs were analyzed with Centiscape tool.Pho B,which is related to the phosphate-specific system,was found in the key proteins and was up-regulated.By comparing BLASTP with the pho regulator of E.coli,it was found that at least 22 genes in Xoc GX01 are homologous to the pho regulator of E.coli,and among the differential proteins,proteins related to pho regulators include XOCgx＿0834 and Pdx H(related to phosphate conversion).Pho U and Pst C(related to phosphate-specific systems).XOCgx＿2155 related to triglyceride and porin Opr O＿P.2.Based on the double exchange method,the key domain of pho B-REC domain was deleted,named pho B(ΔREC).In the mutant library of the research group,pho B,pho R and pho BR deletion mutants were verified.They were name DMpho R,DMpho B and DMpho B,respectively.The recombinant plasmid was introduced into the wild type to construct the overexpression strains GX01(p Jpho R)and GX01(p Jpho B).Using Xoc GX01 as a control,the biochemical phenotype and pathogenicity of mutants and overexpressing strains were tested.The test results are as follows: extracellular proteases,extracellular polysaccharides,biofilms and pathogenicity of GX01(p JCpho R)and GX01(p JCpho B)were not significantly different from the wild type.Compared with Xoc GX01,all mutants significantly reduced extracellular protease activity.The extracellular polysaccharides of DMpho R decreased,while the extracellular polysaccharides of DMpho B and DMpho BR showed no significant changes.The swimming characteristics of DMpho R,DMpho B and DMpho BR are consistent with the wild type.All mutants reduced the ability of forming biofilm and pathogenicity.And the complementary strains of each mutant are constructed by the trans-functional complementary method,and all can be replenished to the wild type.It shows that pho BR can affect the ability of Xoc GX01 to infect the host through the extracellular protease,biofilm and so on.3.By measuring the growth curves of each mutant under phosphate-rich and phosphate-dificient condition,respectively.The growth of DMpho R,DMpho B and DMpho BR is slower than that of Xoc GX01,indicating that pho BR is important for Xoc GX01 to survive in the environment.By q RT-PCR analysis,Xoc GX01 increased the expression of pho B,pho R,pho U,pst S under phosphate deficiency stress.FIMO software analysis and comparison with the differential expression proteins.It was found that the target is related to pathogenic factors such as T3 SS and flagella,and under phosphate-deficient conditions,Pho B involved in binding DNA,translation,modified protein and so on in Xoc GX01.In summary,this study explored the expression of differential proteins of Xoc GX01 under the conditions of sufficient and dificient phosphate through the above-mentioned method to analyze the proteins related to phosphate regulation.A functional study was conducted on the two-component regulator pho BR where the key gene pho B which can regulate phosphate is located.It was found that changing phosphate concentration can affect the colonization of Xoc GX01 on the host.By changing the environmental nutrient and regulating the virulence of pathogenic bacteria,the infection ability of pathogenic bacteria can be reduced,which provides a theoretical basis for clarifying the mechanism of phosphate regulation of Xanthomonas,which is conducive to further research on the mechanism of rice-pathogen interaction,and provides method and strategy for control of rice disease.