A Study On The Role Of ? Factors In Xanthomonas Campestris PV.Campestris Virulence

Bacteria have developed various mechanisms to accurately modify their behavior in response to changes in their environmental soundings to facilitate growth,survival and infection.One important class of global regulators/signaling systems are the sigma???factors which are typically master transcriptional regulators of gene expression.Sigma factor is an important part of RNA polymerase,they are responsible for identifying specific promoter sequences and improving the ability of RNA polymerase to identify promoters,with bacteria possessing multiple sigma factors that are used coordinately to regulate the expression of genes involved in diverse functions,including temperature,osmotic pressure,acid and alkali,heavy metal stress responses,etc.The ? factors can be classified into two structurally and evolutionarily distinct families:the ?54 family and the ?70 famrly.Xanthomonas campestris pv.campestris?Xcc?is the causative agent of black rot disease in vegetable brassica crops worldwide.In this study,the role of ? factors in the regulation of virulence genes was investigated,which will further clarify the pathogenic mechanism of Xcc.Using a bioinformatic approach?SMART,Simple Modular Architecture Research Tool,http://smart.embl-heidelberg.de?,we identified fifteen open reading frames?ORFs?encoding 2 putative ?54?XC₁311?rpoN1?and XC₂251?rpoN2??and 13 putative ?70 factors within the genome of Xcc strain 8004.The ?70 family can be further divided into group 1,group 3 and group 4,XC3806?RpoD?belongs to group 1,XC3843?RpoH?and XC2281?FliA?belong to group 3,while XC0556?RpoE9?,XC1193?RpoE6?,XC1474?RpoE5?,XC2566?RpoE4?,XC2905?RpoE3?,XC2934?RpoE7?,XC2974?RpoEl?,XC3099?RpoE10?,XC3383?RpoE2?,and XC3864?RpoE8?belong to group 4,also known as ECF?extracytoplasmic function??70 factors.As an approach to identify the potential role of each ? factor in the physiology of Xcc,we constructed mutations in each of the ORFs as well as generating collective mutations to charify their functionalities.As a result,we obtained mutants of 13 of the 15 ORFs under the conditions tested?in the nutrient rich medium NYG and the optimum growth temperature 28??,with the exceptions of XC₃806 and XC₃843.This hints that XC₃806 and XC₃843 are essential for viability and survival of Xcc in the tested conditions.This was confirmed when an extra copy of the target gene was introduced into Xcc cells and expressed in trans allowing its chromosomal homologue to be deleted.The pathogenicity of ? factors is detected by leaf cutting method on the host plant Chinese radish?Raphanus sativus?Manshenhong.The results showed that the lension length caused by mutant ArpoEl is significantly lower than that by the wild type strain Xcc 8004.The results of in planta growth demonstrated the number of colonies of ArpoEl in leaf was significantly less than that of wild type.These results indicated that rpoEl is required for full virulence of Xcc in the host plant Chinese radish.The qualitative hypersensitive response?HR?and quantitative HR induced by the mutants were also determined in the pepper ECW-10R?Capsicum annuum cv.ECW-10R?.As a result,mutant ?rpoE1 triggered a delayed and weakened HR compared to the wild type,and the HR-induction capability of ArpoEl could be restored by rpoE1 in cis complementation,which suggested that rpoEl contributes to HR in the pepper ECW-10R.Since RpoE1 affects HR,and HR is an important symptom characteristically induced by type III secretion system?T3SS?encoted by hrp?hypersensitive response and pathogenicity?,whether RpoE1 regulates the expression of hrp associated genes should be investigated.As a result,promoter reporter analyses and qRT-PCR revealed that RpoEl positively regulates the expression of hrpX?the key regulatory gene of hrp?,the hrp cluster genes,and two T3SE?type ? secreted effectors T3SE?genes XC₀241 and XC₁553,but does not affect the expression of hrpG?another key regulatory gene of hrp?.The constitutively expressed hrpX in ArpoEl strain could bypass the requirement of RpoEl for the expression of the hrp operons and the effector genes.This was achieved by introducing the recombinant plasmid pR3X constitutively expressing hrpX into the rpoEl deletion mutant ArpoE1.The expression levels of the hrp operons and the effector genes XC₀241 and XC₁553 in the obtained recombinant strain ArpoEl/pR3X were determined by qRT-PCR.The results showed that constitutive expression of hrpX could bypass RpoEl for the expression of the genes tested.HR induction capability of strain ArpoEl/pR3X was also tested.The results displayed that theArpoEl/pR3X strain could produce wild-type HR symptoms.The above data demonstrated that the ?70 factor RpoE1 influences the expression of hrp and type ? effector genes via HrpX.In addition,promoter reporter analyses and qRT-PCR confirmed that the expression of rpoEl was not regulated by HrpG,but induced by minimal media and plant signals.To dissect the molecular mechanism of RpoE1 activating the expression of hrpX,we compared the global transcriptional profile of rpoEl deletion mutant??rpoE1?with the wild-type by RNA deep sequencing?RNA-seq?.The transcriptome analysis results showed that 131 genes were found to be differentially expressed;of which,21 are up-regulated,and 110 are down-regulated,respectively.To confirm the transcriptome analysis results,semi-quantitative RT-PCR?sqRT-PCR?was employed to analyze the randomly selected genes.The results indicated that the transcriptome data are credible.The promoter region of the regulon genes regulated by RpoE1 was surveyed by bioinformatics analysis.Importantly,we found that a conserved motif "GNWWTTYYGMWKTT?N?A/T/C/G?,W?A/T?,Y?C/T?,M?A/C?,K?G/T??" are present in promoter region of some RpoEl regulated genes,including hrpX.The study of random point mutation of hrpX promoter indicated that the expression of hrpX activated by RpoE 1 is dependent on this conserved motif.The results of the subsequently site-directed mutagenesis of hrpX promoter further verified that RpoEl activating hrpX expression is dependent on this conserved motif.These results hint that RpoE1 might specificly bind to this conserved motif and hence activate the expression of hrpX.Therefore,we then detected the binding of RpoE1 with the promoter region of hrpX through in vivo and in vitro analysis.The expression of hrpX in the strain 8004/pJrpoE1 was detected by qRT-PCR.The results showed that overexpression of rpoEl could enhance the expression of hrpX significantly in vivo.The results of in vitro transcription assays demonstrated that RpoE1 could promote the transcription of hrpX.The Chromatin Immunoprecipitation assay?ChIP?assay showed that the hrpX promoter region could be eluted together with RpoEl protein,indicating that the hrpX promoter region could be combined with RpoEl protein in vivo.The results of in vitro EMSA?Electrophoretic mobility shift assay?experiment showed the mobility of hrpX promoter decreased significantly after adding RpoE1 protein,indicating that RpoE1 could bind specifically to the promoter region of hrpX.The response of the 10 ECF a factors in Xcc 8004 to diverse environmental soundings was tested in this study.The tolerance of heavy metal ions,sodium chloride,SDS,phenol,H2O2,and different alkali stress were tested.It was found that the 10 ECF ? factors deletion mutant A10 was involved in resistance to heavy metal ions,SDS,phenol,and alkali stress.And then,the single gene complementary strains of ?10 were detected to determine the role of each ECF sigma factor in environmental stress.The results showed that rpoE1 and rpoE4 are involved in resistance to Cu2+;rpoE1,rpoE3,rpoE4,and rpoE7 are involved in resistance to Cd2+;rpoE1,rpoE2,rpoE3,rpoE8,and rpoE10 are involved in resistance to Zn2+;rpoE1,rpoE2,rpoE5,rpoE7,rpoE8,and rpoE10 are involved in resistance to SDS;rpoE1 and rpoE10 are involved in resistance to phenol;rpoE1,rpoE2,rpoE3,rpoE4,rpoE5,rpoE6,rpoE7,rpoE9,and rpoE10 are involved in resistance to alkali stress.In conclusion,a systematic functional analysis of the ? factors in Xcc 8004 was conducted in this study.We confirmed that there are two ?factors appeares essential for viability and survival in Xcc 8004,one of the ECF ? factor XC₂974?rpoE1?affects the pathogenicity and HR,RpoEl regulates the expression of hrpX and T3SS positively,the control site of RpoE1 regulating hrpX was determined as"GNWWTTYYGMWKTT",RpoE1 protein could promote the transcription of hrpX and binds to the hrpX promoter,and different ?factors are involved in diverse stress tolerance.