The Biological Function Research Of Transcription Regulators Sar And Gar In Xanthomonas Oryzae Pv.Oryzae PXO99A

Xanthomonas oryzae pv.oryzae as a kind of plant pathogenic bacteria,can cause leaf blight disease and have a seriously reduction of rice production in 1980s.As planting different resistant clutivars of rice in a large area,the harmful of Xanthomonas oryzae pv.oryzae have significantly reduced,but there still have a little of new pathovars,these pathovars also can cause pathogeny to resistant rice.Therefore,studying Xanthomonas oryzae pv.oryzae pathovor PX099A will provide a strong reference value for other plant and animal pathogenic bacteria,as well as prevent the occurrence of plant diseases.There have many pathogenic factors in plant pathogenic bacteria,they can play a role in pathogeny by a complex regulate network.At present,there are many studies related to regulate pathways of pathogenic factors,such as two-component signal transducing system(TCS)?Quorum sensing(QS)signaling pathway?Second messenger cycle diguanylate(c-di-GMP)signaling pathway.Transcriptional regulators can be found in these pathways,because they can bind with the promotor of target gene by specific DNA-binding domain and regulate the expression of downstream gene.Studying the biological function of transcriptional regulators and seeking its molecular targets can consummate the research of the pathogenesis in pathogenic bacteria and control the occurrence of rice diseases.Our study group early identified a transcription regulator can positively regulate PX099A pathogeny,also confirmed another transcription regulator can negatively regulate growth and the ability of resisting hydrogen peroxide of PX099A.They have be respectively named as Sar(Secretion associated transcription regular)and Gar(Growth associated transcription regular).We will lucubrate these two transcription regulators for finding the specific biological functions.First,we verify that Sar can positively regulate the pathogeny of PX099A,and determinate the transcriptome of Sar.We find that Sar can regulate the expression of 501 genes,and we use these regulated genes to build a promoter sublibrary,then utilize Sar protein to interaction with promoter sublibrary,we find there have 76 promoter can interact with Sar protein.The prediction of discovering conserved motif in these promotors,we find three conserved motifs.That illustrate its target sequence is conserved.According to the interaction results and transcriptome results,we can find that Sar can regulate a variety of secretion systems to influence the pathogeny of PX099A.Sar positively regulate three genes(raxA,raxB,raxST)of type I secretion system(TlSS)and bind with the raxST promotor.Sar negatively regulate three gene clusters of type IV secretion system(T4SS)and can interact with the promotor of two gene clusters.Because Sar protein can bind with the promotor of hrpG and hrpX,but it can't bind with the promotor of T3SS gene cluster,and some studies reported that hrpG,hrpX can directly regulate T3SS.So,Sar positively regulate type III secretion system(T3SS)by regulating hrpG and hrpX.Bacterio Two-Hybrid Screening verify Sar protein can interact with HrpG protein.We draw a pattern about Sar regulate T3SS genes by regulating hrpG and hrpX.In addition,Sar positively regulate two gene dusters of type VI secretion system(T6SS)and negatively regulate one gene cluster of T6SS.At the same time,to verify the influence of environmental adaptability in PX099A by T6SS through Sar regulating,we carry out coculture experiments in different bacteria.We selected two characteristic secreted proteins related to T6SS for gene knockout.PX099A,?sar,three knockout mutants and PX099A(pHMI),Xcc8004,E.coli XL1-Blue are respectively mixed in a ratio of 10:1 to coculture.We find that two Hcp protein have no competitive advantage in intraspecific competition,but in interspecific and intergeneric competition,Hcp2 is in favour of competing,but Hcp1 is disadvantage of competing;The trends of competition in PX099A and ?sar are consistent,we guess that the different function of two kinds of Hcp protein balance the effect of Sar to the environment adaptability of PX099A.Furthermore,we also verify Gar can negatively regulate growth and the ability of resisting hydrogen peroxide of PX099A.We also determinate the transcriptome of Gar and find that Gar can regulate the expression of 78 genes,including some membrane related genes.Utilizing Sar protein to interaction with promoter sublibrary,we find 75 promotor of interacting with Gar,also including some membrane related genes.We suppose that Gar may regulate growth and the ability of resisting hydrogen peroxide of PX099A by regulating membrane related genes,but there have not find a direct evidence.In the future,we will observe bacteria population shape by scanning electron microscope and resistance experiment to find the deep reason about this phenomenon.About all of these,we find the factors that the effects of PX099A pathogeny by Sar.Sar can regulate a variety of secretion systems related with pathogeny,through regulating the functions of secretion systems to achieve the purpose of pathogeny.These state that Sar is a global regulator factor and indispensable in the pathogenic process of infecting rice.In addition to,Gar can negatively regulate growth and the ability of resisting hydrogen peroxide of PX099A,that need a further experiment to explore.