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水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)引起的白叶枯病(bacterial leaf blight,BLB)是水稻上最严重的细菌病害之一。Xoo与寄主水稻的互作依赖由hrp基因编码的Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS),将效应蛋白(T3SS effectors,T3SEs)注射入水稻细胞,引起BLB症状的扩展。HrpG是hrp基因转录表达的主要调控因子。为了鉴定未知的hrpG调控子,本研究在以hrpG∷gusA为报道基因构建的转座子突变体库中,筛选获得4个候选的hrpG负调控子突变体G24-46、G48-22、G19-14和G57-41。GUS活性测定、荧光定量PCR以及烟草组织的GUS染色试验均显示,在这些突变体中hrpG的表达显著增加。Southern杂交结果显示,突变体中转座子均为单一位点的插入。插入位点分析结果显示,在G24-46、G48-22、G57-41和G19-14中转座子分别插入在minD、pilA、metB和wxoB基因中。毒性测定结果显示,这4个突变体在水稻上的毒性显著降低。这些hrpG调控子基因的鉴定为进一步解析稻黄单胞菌hrpG上游调控网络提供了新的科学线索。
Bacterial leaf blight (BLB) caused by Xoohomonas oryzae pv. Oryzae (Xoo) is one of the most serious bacterial diseases in rice. The interaction between Xoo and host rice depends on the type Ⅲ secretion system (T3SS) encoded by the hrp gene and the T3SS effectors (T3SEs) are injected into rice cells to cause the expansion of the symptoms of BLB. HrpG is the main transcription factor of hrp gene. In order to identify unknown hrpG regulators, we selected four candidate hrpG negative regulator G24-46, G48-22, G19- 14 and G57-41. GUS activity assay, fluorescence quantitative PCR, and GUS staining of tobacco tissues all showed a significant increase in hrpG expression in these mutants. The result of Southern hybridization showed that the transposon of the mutant all inserted into a single site. Insert site analysis showed that the transposons were inserted in the minD, pilA, metB and wxoB genes at G24-46, G48-22, G57-41 and G19-14, respectively. Toxicity assay results showed that the four mutants had significantly lower toxicity on rice. The identification of these hrpG regulatory genes provides new scientific clues for further analysis of the upstream regulatory network of hrpG in rice.