水稻白叶枯病菌核苷酸信号受体蛋白Clpxoo的分子鉴定及其功能

Molecular identification and functional analysis of Clpxoo,a homologue to the nucleotide receptor protein in Xanthomonas oryzae pv. oryzae

【目的】本文的目的为阐明水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae,简称Xoo)核苷酸信号途径及其作用机理。【方法】本研究对推导的信号受体蛋白Clpxoo进行了基因克隆、序列分析、缺失突变和互补及其相关表型的鉴定。【结果】克隆的clpxoo基因序列与大肠杆菌crp和铜绿假单胞菌vfr的同源性较高,与其它几种病原黄单胞菌clp高度保守。Clpxoo序列N端具有环化核苷酸cNMP结合结构域(CAP-ED),C端具有保守的DNA结合结构域(HTH-CRP)。用双交换法构建了基因缺失突变体(△clpxoo)。与野生型菌株PXO99A相比,△clpxoo的运动性、胞外多糖产生能力和对H2O2的抗性均显著降低,基因互补可使之部分恢复;△clpxoo胞外酶产生和对烟草致敏性无显著改变。【结论】Clpxoo可能作为全局性的保守调控因子之一,调控了Xoo的鞭毛运动性、胞外多糖产生和对H2O2的抗性。

[Objective] To better understand the mechanisms of cyclic di-GMP signaling in Xanthomonas oryzae pv. oryzae （Xoo）, the casual pathogen of bacterial blight of rice, molecular identification of Clpxoo. [ Methods] A putative signal receptor protein was performed through gene cloning, sequencing and deletion analysis. [ Results] Sequence analysis showed Clpxoo was a homologue of Crp and Vfr, the cAMP receptor proteins in Escherichia coli and Pseudomonas aeruginosa respectively, which had the cNMP-binding domains （CAP ED） at N terminal and the DNA-binding domains （HTH CRP） at C terminal and is highly conserved in the plant-pathogenic Xanthomonas spp. We constructed △ clpxoo through a double crossover recombination and validated by PCR assay, △ clpxoo displayed the reduced motility and extracellular polysaccharide （EPS） production, and increased sensitive to H2O2 toxicity compared with PXO99^A. All these phenotype changes could be partly restored through complementation of mutants by introducing clpxoo. Moreover, we observed no significant changes in production of extracellular cellulase and xylanase in vitro, biofilm formation and induction of hypersensitive response （HR） on non-host tobacco in △clpxoo compared to PXO99^A. [Conclusionl Therefore, Clpxoo played a role as one of the global regulator in regulation of flagellar motility, EPS production and H2O2 resistance.