The Regulation Mechanism Of Transcription Regulator Easr In Xanthomonas Oryzae Pv.oryzae PXO99~A

Xanthomonas oryzae pv.oryzae as an important plant pathogenic bacteria,can cause leaf blight disease.Bacterial blight was the second most serious disease of rice after rice blast and caused a seriously reduction of rice production in last century.Although bacterial blight has been greatly controlled by resistance breeding,in recent years,new pathogenic races have been isolated,which makes the prevention and control of bacterial blight become seriously again.There are many pathogenic factors in Xanthomonas,including extracellular polysaccharide,adhesin,secretory systems,effector proteins and so on.They can play a role in pathogeny by complex regulatory networks.The pathways that have been well studied in Xanthomonas are related to pathogenic factors as two-component conduction system(TCS),the second messenger c-di-GMP signaling pathway,quorum sensing(QS)system and secretion regulation.In these processes,transcription factors are involved in transcriptional regulation,which specifically binds to DNA and initiates the expression of target genes.Xanthomonas oryzae pv.oryzae PXO99~Ais a model strain for studying the interaction between pathogen and host.Its genome sequence has been completed in 2008.A large number of potential transcription factors have been predicted.In our research,transcription factors were studied from the whole genome layer.Mutants were obtained by gene knockout.Pathogenicity and related phenotypes of rice tests of rice were explored to all these mutants.52 mutants were obtained through the homologous double-crossover.A few genes for example PXO＿RS04675,PXO＿RS19435,PXO＿RS08960 have not been constructed yet,speculation they are necessary for growth.Through pathogenicity test in rice leaves,we found that the pathogenicity of the mutants,PXO?RS06520,PXO?RS02870,?eas R were significantly declined(40%,90%and 50%,respectively).PXO＿RS02870 is the global transcriptional regulator clp(c AMP-regulatory protein)which has already been reported that it related to pathogenicity.PXO＿RS06520 as a Fur family transcription factor,is required for regulation and absorption of Fe.We also found a new gene that named it as eas R(environmental adaption switch regulator).By sequence alignment,we found that EasR is highly conserved in Xanthomonas.It contains winged HTH domain and ROK family domain,is a potential transcriptional regulator required for virulence.Firstly,gene complement of?eas R can restore virulence to wide type levels.Secondly,we analysis the transcriptome data find that it can regulate the expression of414 genes,mainly related to type III secretion system(T3SS),type VI secretion system(T6SS),type IV pili and so on.Among them,239 genes are up-regulated and175 genes are down regulated.We find 37 of the down regulated genes are related to type III secretion system.And we further explored the EasR and T3SS key factors Hrp G、Hrp X by overlap analysis of their down regulated genes,showed that eas R mutant covering 81%of that compaired with hrp G mutant,92%of hrp X mutant,and constitutive overexpression of hrp G in eas R mutant can restore the pathogenic phenotype.In addition,bacterial two hybrid technology find that EasR protein and Hrp G protein have strong interaction,proved that EasR formed a protein complex with Hrp G then initiates the expression of transcription factor hrp X,thus activating the T3SS to function and causing host rice disease.We also find that?eas R has a great survival advantage when compete with Escherichia coli in vitro.T6SS is confirmed in competition with other bacteria and can secrete effector proteins against target cells to obtain survival advantage.So we analysis the two gene clusters of T6SS and use the bacterial one hybrid technique find that EasR strongly interacts with T6SS,proved that EasR can regulate T6SS and exhibit competitive advantage.Other phenotype detection find that?eas R movement decreased,tolerance to ROS reduction,and extracellular quorum sensing signal DSF production decline detection.Interestingly,we find that EasR can negatively regulate type four fimbriae(T4P),bacterial one hybrid verification eas R direct regulate T4P,while high expression of pili can be used as the receptor during phage infection,so we detect the sensitivity of the phage,find eas R mutant becomes more sensitive to phage,however further exploration need to explain the relationship between them.We have find some targets by bacterial one hybrid,at the same time,bacterial two hybrid find EasR can interact with a variety of proteins,the most classic is the c-di-GMP associated proteins.In summary,we find that EasR is a new global regulatory factor,EasR plays a pathogenic function during the infection of rice through type III secretion system.In vitro non pathogenic environment,EasR regulates the type VI secretion system in order to enhance its competitive capacity.Further study of EasR will help us to understand the ecological adaptability of Xanthomonas better.