| Citrus canker is an important bacterial disease caused by the Gram-negative bacterium Xanthomonas citri subsp.citri（Xcc）.Xcc attacks host plants by means of a number of virulence factors such as extracellular polysaccharides and mobility.In the genome of Xcc,vemR gene encodes an atypical response regulator with only N-terminal signal receiving domain and lack of C-terminal output domain.Thereby,it is unable to regulate its targets through binding to promoter region DNA.Those atypical response regulators are widely distributed in plant pathogenic Xanthomonas,but its regulatory mechanism in association with specifically binding its targets remains unclear.Our laboratory reported that theΔvemR derived from Xcc 29-1 showed reduced virulence,cell mobility,and less extracellular polysaccharides production.In the Xcc genome,fleQ-vemR-rpoN2 is expressed as an operon,which is under conrol of a promoter upstream of rpoN2 gene.VemR and RpoN2 jointly regulate the flagellum gene flgG.Based on the conclusions,this thesis aimed to explore the regulatory mechanism mediated by FleQ-VemR-RopN2 complex,which will help to know the regulatory mechanism executed by VemR;to find the acting target of copper-based chemicals by analyzing the effect of copper on FleQ-VemR-RopN2 interaction.The main results of this thesis are as follows:（1）The transcriptional regulator FleQ exerts negative regulatory role in virulence and extracellular polysaccharides,and positive regulation in cell mobility.A deletion mutantΔfleQ was generated from wild-type Xcc 29-1through recombinant suicide plasmid to construct.Compared with wild type,theΔfleQ caused more server canker symptom in citrus.The mutant reduced its motility on the semi-solid medium.In addition,the deletion mutation of fleQ gene affects the growth rate in culture medium and citrus plant tissues.（2）FleQ regulates cell motility by positively modulating the transcription of flgG gene.Fluorescence quantitative Real-time PCR（qRT-PCR）was performed to detect the expression of flagellar genes dependent on RpoN2.It was found that the flagellar body gene flgG was significantly down-regulated in fleQ mutant.The promoter GUS fusion analysis showed that flgG promoter activity was reduced.However,FleQ was unable to bind to flgG promoter as revealed by yeast one-hybrid experiment.（3）VemR physically interacts with both FleQ and RpoN2.The yeast two-hybrid experiment proved that VemR interacts with both FleQ and RpoN2,while FleQ did not interact with RpoN2.This demonstated that FleQ-VemR-RpoN2 protein complex is formed through the intermediate connector VemR.（4）Copper inhibited the interaction of VemR with both FleQ and RpoN2.Based on the results of yeast two-hybrid experiment,applying of 0.01 mM CuSO4suppressed the interaction.This suggested that VemR/FleQ/RpoN2is targteted by copper chemicals.（5）VemR negatively regulated fleQ-vemR-rpoN2 operon expression.The promoter GUS fusion revealed that the fleQ-vemR-rpoN2 operon promoter activity was increased inΔvemR mutant.This demonstrated that the dereased virulence and extracellular polysacharride were caused by the accelerated expression level of fleQ,which showed negative regulatory role. |
