Xanthomonas campestris pv. campestris (Xcc) is a vascular pathogen that causes black rot in host. It is an important model strain for studying the interaction between the phytopathogen and plants. In Xcc, global transcription regulator HpaR1 that belongs to the GntR family regulates many cellular processes such as the movement and synthesis of extracellular polysaccharides and extracellular enzymes, and is associated with hypersensitive response (HR) and pathogenicity. On the other hand, the global transcriptional regulator Clp regulates the secretion and synthesis of extracellular enzymes and extracellular polysaccharides, and is associated with the pathogenicity of Xanthomonas. Previous studies have shown that both HpaR1 and Clp bind to the promoter region of the glycoside hydrolase encoding gene (named ghy gene). This study investigates the molecular mechanism of the co-regulation of HpaR1 and Clp on the expression of ghy gene. Through electrophoresis mobility shift assay (EMSA), we found that both HpaR1 and Clp bind to the promoter regions of gene ghy in vitro. Both HpaR1 and Clp also bind to the promoter regions of gene ghy in vivo by chromatin immunoprecipitation (ChIP) assays. DNase I footprinting and 5'-RACE assays showed that both HpaR1 and Clp bind to the -35 region upstream of the ghy promoter. The HpaR1 binding site was located upstream of the Clp binding site. RT-qPCR and in vitro transcription assays showed that HpaR1 negatively while Clp positively regulates the transcription of gene ghy. Furthermore, HpaR1 inhibits the activation of Clp on the transcription of gene ghy in vitro. Our findings indicate that HpaR1 and Clp exhibit opposite effect on the transcription of gene ghy. It is speculated that HpaR1 may regulate the expression of gene ghy by inhibiting the activity of RNA polymerase.
十字花科黑腐病菌(Xanthomonas campestris pv. campestris, Xcc)是一种维管束致病菌,能够引起寄主的黑腐病,是研究植物病原细菌与植物互作的一种重要模式菌株。在Xcc中,GntR家族的全局性转录调控因子HpaR1参与调控Xcc的运动、胞外多糖和胞外酶的合成等许多细胞过程,并与Xcc的过敏反应(hypersensitive response, HR)和致病相关;全局性转录调控因子Clp则参与调控胞外酶和胞外多糖的分泌与合成,并与黄单胞菌的致病相关。前期研究发现,Xcc中的转录调控因子HpaR1和Clp均能与糖苷水解酶(glycoside hydrolase)编码基因(命名为ghy基因)的启动子区结合。为探究转录调控因子HpaR1和Clp共同调控ghy基因表达的分子机理,本研究首先通过凝胶阻滞分析(electrophoresis mobility shift assay, EMSA)发现HpaR1和Clp在体外能够结合在ghy基因的启动子区;利用染色质免疫共沉淀(chromatin immunoprecipitation, ChIP)方法,进一步证实HpaR1和Clp在细胞内能够结合在ghy基因启动子区。通过5ʹ-cDNA末端快速扩增(rapid amplication of 5ʹ-cDNA ends, 5ʹ- RACE)和DNase I保护实验(DNase I footprinting)确定HpaR1和Clp均结合在ghy基因启动子的-35区上游,并且HpaR1的结合位点位于Clp结合位点的上游。通过实时荧光定量PCR(real time fluorescence quantitative PCR, RT-qPCR)和体外转录的方法,发现HpaR1抑制ghy基因的转录,而Clp激活ghy基因的转录。当二者共同存在时,HpaR1能够抑制Clp对ghy基因的转录激活作用。HpaR1和Clp单独存在时,分别负调控和正调控ghy基因的转录,推测HpaR1尽管位于ghy基因启动子-35区上游,但可能通过抑制RNA聚合酶的活性来调控ghy基因的表达。.
Keywords: Clp; HpaR1; Xanthomonas campestris pv. campestris; gene ghy; transcriptional regulation.