Research On The Mechanism Of Tat System And Host-induced XanA In Regulating Virulence In Xanthomonas Oryzae Pv. Oryzae

Xanthomonas oryzae pv.oryzae?Xoo?that causes the bacterial blight of rice is an important pathogenic bacterium in world's rice production.The prevalence of Xoo may lead to a reduction in rice yield by 10-20%,or even above 50%and total crop failure in some severe cases.Xoo invades the plants through hydropore or wounds,then absorbs nutrients,spreads and proliferate in vascular bundles of rice,and produces a large amount of exopolysaccharides?EPSs?that block the vascular bundles.As a result,brownish or grayish white spots will appear on the leaves.Studies have shown that Xoo invasion highly depends on the protein secretion systems or the expression of virulence-related genes.Type II secretion system and the relevant extracellular enzyme,type ? secretion system and the secreted effectors,type V secretion system and the secreted adhesion factors have been proved to play important roles in enhancing the virulence of Xoo.In addition,a growing number of high-throughput transcriptome analyses have discovered the induced expressions of some important virulence-related genes in the Xoo-rice system.The functions of these genes are closely associated with the survival,proliferation and synthesis of virulence factors of Xoo in rice.Our preliminary study has shown that mutation in the Tat system,a twin-arginine protein transport system,can greatly reduce the virulence of Xoo.However,the molecular mechanism of the Tat system regulating Xoo virulence is not fully understood yet.We have also discovered virulence factor xanA using proteomic method,whose sustained expression is induced by host in the Xoo-rice system.However,little is known about the molecular mechanism of host-induced expression of xanA and its regulation of virulence of Xoo.Therefore,the purpose of the present study was to analyze how the Tat system and its substrate protein regulate the virulence of Xoo in rice;we also discussed the mechanism of host-induced expression of xanA in Xoo and its regulation of Xoo virulence.?i?Xoo strain PXO99A was used for the study.Comparative extracellular proteomics analysis data?PXO99A-VS-?tatC?were used to screen for 10 TDTPs?Tat-dependent translocation proteins?whose extracellular content in the AtatC mutant decreased by five folds.A total of 77 TDTPs were identified by three Tat prediction prograns,namely,PRED-TAT,TATFIND V1.4 and TatP V1.0.Protein function annotation indicated that these 87 TDTPs were widely involved in the oxidation-reduction system,ion uptake and balance,transport and metabolism of carbon source,cell membrane synthesis and environmental adaptation.Systematic mutation and virulence-related phenotypes were analyzed in 15 TDTPs.PXO₀2203,PXO₀3477 and PXO₀2523 were found to be the novel virulence-related genes.In addition,several TDTPs played important roles in invasion,expression of virulence factors,and adaptation to adversity in PXO99A.The above results implied that the Tat system may influence virulence and adaptation to adversity of the pathogenic bacteria by controlling the biological behaviors of TDTPs.?ii?We demonstrated that xanA was a virulence gene whose expression could be induced in both the in vitro Xoo-rice system and inside the rice using Western Blot,quantitative fluorescence assay,and GUS staining.Through the prokaryotic expression and biochemical function analysis of the XanA protein,it was found that XanA possessed glucose phosphomutase activity in Xoo and regulated the dynamic balance between glucose-1-phosphate and glucose-6-phosphate.The combined use of genomic,transcriptomic and proteomic techniques and virulence phenotype analysis indicated that xanA regulated virulence by influencing the utilization of carbon source by Xoo,EPS syntthesis,biofilm synthesis,flagellar motion and chemotaxis.FruA,CheY and FlhF might be the important mediators of the above regulatory mechanism.In addition,bacterial one-hybrid experiment and gene expression analysis revealed that carbon sources such as xylose and galactose widely present in rice induced the expression of xanA to varying degrees.xanA itself received regulation by the AraC family transcriptional regulators such as PXO₀6036.Taken together,the AraC family transcriptional regulators may play important roles in virulence regulation by xanA and host-induced expression of xanA in nCe.To conclude,15 substrate proteins for the Tat system and their pathogenic related functions in Xoo were identified.The potential mechanism of the Tat system regulating the virulence and adaptation to adversity of XOo was discussed.Virulence phenotype analysis,proteomic and transcriptomic analyses were used to reveal the novel pathways of xanA regulating the virulence and synthesis of virulence factors in Xoo.It was preliminarily found that the earboin sources in rice were important reasons for host-induced expression of xanA.The AraC family transcriptional regulators also played a regulatory role in the host-induced expression of xanA.The research shed light on the virulence mechanism of Xoo and Xoo-rice interaction and provide important clues for formulating countermeasures against the bacterial blight of rice by blocking the virulence pathway.