Cloning and functional analysis of Acidovorax avenae subsp. citrulli partial hypersensitive response and pathogenicity(hrp) gene cluster | 瓜类细菌性果斑病菌过敏性反应和致病性(hrp)基因簇部分基因的克隆及功能分析

Chinese. 过敏性反应和致病性(hrp)基因存在于革兰氏阴性植物病原细菌中，决定病原细菌对寄主植物致病性和诱导非寄主及抗病植物过敏性反应(hypersensitive response, HR)。本研究从果斑菌(Acidovorax avenae subsp. citrulli)的hrp基因簇中克隆了hpaA、hrcT、hrcC和hrpG基因，通过同源重组的方法，分别构建了它们的突变体。电镜观察发现，hpaA和hrpG基因突变体的鞭毛缺失且细胞形态发生显著变化，而hrcT和hrcC基因突变体的鞭毛和细胞形态未发生明显变化。在烟草(Nicotiana tabacam)和哈密瓜(Hami cantaloupe)叶片上的测定结果显示，hpaA、hrcT和hrcC的突变体均失去在烟草上的HR激发能力和在哈密瓜叶片上的致病性；hrpG在烟草上的HR激发能力和在哈密瓜上的致病性则显著减弱；进一步的生长曲线测定结果表明，hpaA、hrcT、hrcC和hrpG的突变体的定殖能力均显著下降。相应地，功能互补后突变体基本恢复至野生表型。证明瓜类细菌性果斑病菌hrp基因作为Ⅲ型分泌系统关键组份影响病原细菌对寄主植物的致病性和对非寄主及抗病植物的过敏性反应。

English. The hypersensitive response and pathogenicity(hrp) genes exist in Gram-negative plant pathogenic bacteria and are responsible for the pathogenicity of bacteria．They can induce hypersensitive response (HR) on non-host and resistant plants. hpaA, hrcT, hrcC and hrpG genes were cloned from the hrp gene cluster of Acidovorax avenae subsp. citrulli. Disruption mutants of strain xjl12 were successfully generated by a single cross-over event. Electron microscopy observation showed that the cells of hpaA and hrpG mutants lacked detectable flagella and obviously changed in cell morphology, while hrcT and hrcC mutants’ flagella and cell morphology did not change. Surprisingly, hpaA, hrcT and hrcC mutants lost HR-induced capacity in tobacco (Nicotiana tabacam) and the pathogenicity in melon(Hami cantaloupe) leaves; while the hrpG mutant was significantly reduced. Furthermore, growth analysis results showed that colonization of hpaA, hrcT, hrcC and hrpG mutants were significantly decreased, respectively. Accordingly, the mutant phenotypes nearly recovered after complement. Overall, these results indicate that the hrp genes of bacterial fruit blotch of melon as a key component of type Ⅲ secretion system affect pathogenicity on host plants and disease resistance on non-host and resistant plants.