| Xanthomonas campestris pathovar campestris(Xcc)is the causal agent of black rot disease,one of the most destructive diseases of cruciferous crops worldwide.This pathogen can infect almost all members of the crucifer family(Brassicaceae).Over the past several decades,Xcc has been used as a model bacterium for studying molecular mechanisms of bacterial pathogenicity.This pathogen recruits two important pathogenicity systems:T3SS(type ? secretion system)encoded by hrp(hypersensitive response and pathogenicity),and rpf(regulation of pathogenicity factors)response to DSF(diffusible signaling factor)signal.T3SS plays crucial roles in the pathogenicity of many animal and plant pathogenic bacteria.These pathogenic bacteria recruit the T3SS to translocate type ? secretion effector(T3SE)proteins into host cells,causing pathogenicity in susceptible hosts,or inducing hypersensitive response(HR),a disease-resistant phenomenon on the resistant hosts and nonhost plants.T3SS of Xanthomonas is encoded by hrp cluster(XC3001-XC3025),dependent on two key regulators HrpG(XC3077)and HrpX(XC3076)and one sensor HpaS(XC3670).HpaS is identified as a cognate sensor kinase for the two-component response regulator(RR)HrpG which is predicted to be an OmpR family's RR,and HrpX is an AraC-type transcriptional activator.The expression of hrp genes is induced in nutrition infertile medium or in plants.However,the stimuli signal from the environment and in plant remains unknown.It has been demonstrated that HpaS,HrpG and HrpX form a regulatory cascade.HpaS senses unknown stimuli signal and auto-phosphorylated,and then activates HrpG by transferring phosphate from histidine residue of HpaS to aspartate residue of HrpG through phosphorelay.HrpG positively regulates the expression of hrpX and HrpX then activates the expression of hrpA to hrpF operons and the repertoire of T3SEs genes.In addition,it is reported that HpaR,HpaRl,HpaR2,HpaP,RsmA,and Zur of Xcc are involved in hrp regulation.In addition to the hrp/T3SS patho-system,the regulation of virulence factors in Xcc is also dependent on the rpf/DSF system,which is involved in the regulation of synthesis of extracellular plant cell wall-degrading enzymes and extracellular polysaccharide(EPS);production of DSF;alterations in biofilm formation,and mobility,and so on.The rpf/DSF system recruits the sensor kinase RpfC and the response regulator RpfG forming the two component signal transduction regulatory system(TCSs),and the global transcriptional regulator Clp(cAMP receptor-like protein).It is reported that cyclic di-GMP(c-di-GMP)binds to Clp,thus preventing binding of Clp to the promoters of target genes that include those encoding extracellular enzymes and EPS biosynthesis.In high bacterial population reached a sufficient cell density cultured in rich medium,RpfC senses the high concentration of environmental DSF signal,auto-phosphorylated,and then activates RpfG by transferring phosphate from histidine residue of RpfC to aspartate residue of RpfG through phosphorelay.RpfG harbors HD-GYP domain and participates in the degradation of c-di-GMP.The activity of Clp,the key regulator controlling the expression of other virulence genes,is regulated by the turnover of c-di-GMP.Success infection of pathogens lies on the cooperation of varies of patho-systems and virulence factors.However,the relation-ship between the rpf patho-system and the hrp patho-system is few reported.In this study,the reporter strain 8004/pL6hrpXsacB harboring the reporter plasmid pL6hrpXsacB in which the promoter of hrpX was in frame fused with sacB,a suicide gene sensitive to sucrose in Xcc,was employed to screen the candidate regulators positively activating the expression of hrpX through construction of mutant library and screen in MMX plus 5%sucrose.As a result,a mutant(named as XB003)was obtained.The transposon insertion sites in XB003 were further mapped by plasmid rescue and sequencing.The sequencing data indicated that the mutation in XB003 lies in the ORF XC2333.Interestingly,the ORF XC2333 is the rpfC gene,which encodes the key TCS sensor kinase in rpfDSF patho-system.It hints that RpfC positively activates the expression of hrpX.To validate the above result,we examined the transcript level of hrpX and hrpG in the deletion mutant of rpfC(named as ?rpfC)grown in minimal medium XCM1 compared to the wild type by quantitative real-time PCR(qRT-PCR).The results showed that the transcript level of hrpX in the rpfC mutant was significantly lower than that in the wild type(p<0.01 by t test),while the transcript level of hrpG in the rpfC mutant was not significantly affected when compared to the wild type(P>0.05 by t test).To further verify the above result,the expression of hrpG and hrpX was surveyed in the minimal medium XCM1,in XCM1 plus host pant(Chinese radish Manshenhong)tissues extraction,and in host plant(Chinese radish Manshenhong).The results showed that the knockout of rpfC reduced the expression of hrpX significantly,both in XCM1 and XCM1 plus pant tissues extraction(p<0.01 by t test),while the expression of hrpG has no significant difference(P>0.05 by t test).Interestingly,in host plant(Chinese radish Manshenhong),the expression of hrpG and hrpX were all significantly reduced in the rpfC mutant(p<0.01 by t test).Combined above results,it indicates that RpfC positively activates the expression of hrpX both in the minimal medium and in host plant,and RpfC positively activates the expression of hrpG only in host plant.It is speculated that RpfC activates hrpX via different mechanism in different culture condition.Xcc 8004 harboring avirulence protein AvrBs1 could induce T3S specific hypersensitive response(HR)in ECW-10R(Capsicum annuum cv.ECW-10R)which is a non-host plant of Xcc and harbors corresponding resistant gene Bs1.Since RpfC positively activates the expression of hrpX both in the minimal medium and in host plant,whether RpfC affects HR should be invertigated.We checked the ability of the rpfC deletion mutant and the trans-complementary strain to induce HR in ECW-10R by the qualitative and quantitative HR assay.The results showed that 8 hours after inoculation,no significant HR phenotype was observed for the mutant strain ArpfC,while typical HR for the wild type strain Xcc 8004 was observed.However,all the strains except the negative control?avrBs1 produced typical HR symptoms 16 h and 24 h after inoculation.These results were further substantiated using an electrolyte leakage assay.Both mutants showed significantly decreased electrolyte leakages at 8,16,and 24 h after inoculation compared with the wild type,but showed much stronger electrolyte leakages than the HR-null mutant ?avrBs1(the negative control).The trans-complementary strain could restore the phenotype to the wild type at 24 h after inoculation.Taken together,these results revealed that knockout of rpfC reduced the relayed HR significantly.It demonstrates that RpfC is important for Xcc to stimulate HR on plants.To dissect the molecular mechanism of RpfC activating hrpX,the transcriptome of the mutant strains ArpfC,ArpfG,and Aclp were determined through RNA deep-sequencing(RNA-seq).Through data analysis,compared with wild type,a total of 527 RpfC-regulated genes were identified,in which 328 were down-regulated and 199 up-regulated by RpfC;a total of 610 RpfG-regulated genes were identified,in which 277 were down-regulated and 333 up-regulated by RpfG;a total of 376 Clp-regulated genes were identified,in which 137 were down-regulated and 239 up-regulated by Clp.Importantly,despite of hrpG,the expression of hrpX,all the hrp cluster genes,and 9 reported T3S effector genes in ArpfC mutant were significantly lower than that in wild type strain.However,the expression of the response regulator RpfG and Clp was not affected in the tested condition.While in ?rpfG,only 10 of the hrp genes and 8 reported T3SS effectors were significantly down-regulated by RpfG.The expression of hrpG,rpfG,and clp were not affected in ArpfG.As for Clp,hrpX,23 of the hrp cluster genes,and 9 reported T3S effector genes were significantly down-regulated by Clp.The expression of hrpG was not affected in Aclp.These results indicated RpfC and Clp positively activated the expression of hrpX,the hrp cluster genes,and most reported T3S effector genes,but not hrpG in minimal medium MMX,while RpfG only activated partial hrp cluster genes and T3S effector genes and didn't activate hrpX.In rich medium,RpfC regulates extracellular enzymes and EPS biosynthesis by Clp through the turnover of c-di-GMP.Whether RpfC activates hrpX still recruits this strategy?To verify this hypothesis,the concentration of c-di-GMP in Xcc 8004,ArpfC,and ArpfG in rich medium NYG and minimal medium MMX was determined respectively.The results showed that,the concentration of c-di-GMP in MMX is about 10 times lower than that in NYG,and disruption of rpfC and rpfG significantly increased the concentration of c-di-GMP both in NYG and MMX,and the concentration of c-di-GMP in ArpfG is even higher that that in ArpfC(p<0.01,t test),and hinting that activating of hrp/T3SS by RpfC might perform not through the turnover of c-di-GMP.Clp commonly binds to the promoter of target genes that include those encoding extracellular enzymes and EPS biosynthesis.There is normally so called Clp box(Clp binding sites,CBSs)in promoter region of the target genes regulated by Clp.The typical motif of CBSs is 5'-AAATGTGA-TCTAGA-TCACATTT-3',Bioinformatics analysis of promoter region of hrpX was peformed.We found that 11 putative Clp CBSs are present in promoter region of hrpX,in wich CBS-1 shares the highest rating ratio of 0.7.The gel electrophoresis mobility shift assay(EMSA)was employed to determine whether Clp binds directly to the promoter region of hrpX labeled with fluorescein(FAM).The results showed that Clp with CBS-1 yielded shift significantly,while CBS-2 and CBS-3 not.It indicated that Clp binds to the promoter of hrpX CBS-1 with high affinity.To further verify the contribution of CBS-1 to the regulation of Clp,CBS-1 and another two putative CBSs(CBS-2 and CBS-3)were disrupted by fusion PCR directed site-mutagenesis.Interestingly,when CBS-1 is mutated,Clp didn't regulate the expression of hrpX any more.The disruption of another two CBSs didn't affect the regulation of Clp to hrpX.Taken together,it demonstrated that Clp might activate the expression of hrpX through directly binding to CBS-1 of hrpX promoter.Since RpfC and Clp activate hrpX but not hrpG in MMX,and RpfG doesn't regulate hrpX,how RpfC bypasses RpfG and activates hrpX by Clp?To identify the unknown response regulators(RR)downstream of RpfC,the Bacterial two-hybrid system(B2H)was employed.We aimed to screen the annotated response regulators in Xcc 8004 genome,and completed 65 RR up to now.The results showed that RpfC might interact with RpfG,XC0198,XC0816,and XC3059.However,GUS assay showed that XC0816 and XC3059 didn't regulate the expression of hrpX and hrpG,although XC3059 affect HR,XC0816 doesn't affect HR,XC0816 and XC3059 are essential for full virulence of Xcc 8004.It hints that RpfC activates hrpX probably not through XC0816 and XC3059.The unknown response regulators downstream of RpfC might be affected by RpfC in translation level or in post-translation level.To further identify the unknown response regulators downstream of RpfC,the proteomics iTRAQ was used to identify the proteomic differential displays of the mutant strains ?rpfC,?rpfG,?rpfF,and ?clp,compared with the wild type Xcc 8004.The results showed that 433 proteins were differentially expressed in?rpfC,382 in ?rpfG,445 in ?rpfF,and 944 in Aclp.HrpX is down expressed in ?rpfC,?rpfG,and ?clp.The expression of c-di-GMP degradation related proteins including 11 HD domain proteins,2 HDOD domain proteins,14 EAL domain proteins,and 29 c-di-GMP synthesis related proteins with GGDEF domain were analyzed.The results showed that XC0249 harboring GGDEF and cNMP_binding domain was up expressed in ?rpfC,RpfG with HD domain was down expressed in ?rpfC,XC1582 with GGDEF and EAL down expressed in ?rpfC,?rpfG,?rpfF,and Aclp.These genes should be investigated for dissecting the molecular mechanism of hrpX activated by RpfC in future. |
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