水稻白叶枯病菌TonB-Dep-Rec蛋白家族成员Tdrxoo的功能鉴定

Identification and functional analysis of Tdrxoo, the member of TonB-dependent-receptor family proteins in Xanthomonas oryzae pv. Oryzae

摘要:

摘要：【目的】旨在揭示水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae，Xoo)致病性和运动性及其基因表达的调控途径。【方法】本研究通过基因克隆、序列分析和缺失突变方法，对与应答调节子GacAxoo互作的Tdrxoo的分子特征和功能进行了鉴定。【结果】利用序列特异性引物进行基因扩增，成功地从野生型菌株PXO99A中克隆了tdrxoo基因。Tdrxoo与其它病原黄单胞菌的同源序列高度保守，具有TonB-Dependent-Receptor (TDR)结构域，推测其是位于细菌外膜、可能接收来自细菌体外环境信号的蛋白。用基因标记交换法，构建了△tdrxoo基因缺失突变体。与PXO99A相比，Δtdrxoo在人工培养条件下的生长受到影响，致病性完全丧失，胞外纤维素酶和木聚糖酶活性和运动能力显著减弱，基因互补可以使之恢复；Δtdrxoo嗜铁素产生无明显改变。【结论】Tdrxoo作为一种细胞外膜蛋白，可能参与调控了病菌的生长、致病性、胞外酶活性和运动性等表型。

Abstract:

Abstract: [Objective] To demonstrate the novel regulatory pathways mediated in bacterial pathogenicity and motility in Xanthomonas oryzae pv. oryzae (Xoo), the casual agent of bacterial blight in rice. [Methods] Molecular identification and functional characterization of Tdrxoo, which interacts with GacAxoo of the two-component regulatory system (GacSxoo/GacAxoo) in Xoo, were performed through gene cloning, sequencing and disrupt analysis. [Results] tdrxoo was successfully cloned from the genomic DNA of wild-type PXO99A by using polymerase chain reactions with the degenerated primers tdrxooF/R. The tdrxoo gene was found to be highly conserved in the plant-pathogenic Xanthomonas spp. Sequence analysis showed that Tdrxoo was homological to a protein with the TonB-Dependent-Receptor (TDR) domain. Tdrxoo is probably localized in the outer membrane of bacterial cells, recognizing the signals from extracellular environment, and inducing the intracellular signal transduction. △tdrxoo, the disrupted mutant, was obtained after a single cross-over recombination event between tdrxoo and the plasmid pK-tdr with the tdrxoo segment. The mutant lost the ability of causing the disease, and was affected in growth in vitro compared to PXO99A. In addition, the motility and the extracellular enzymes production of △tdrxoo were reduced, which can be restored through complementation of the △tdrxoo mutant by introduction of tdrxoo. tdrxoo deficiency didn’t affect siderophore production. [Conclusion] According to the existence of tdrxoo in Xoo genome and phenotype of △tdrxoo, Tdrxoo, as the outer membrance protein, is proposed to be involved in regulation of pathogenicity, extracellular enzyme production, the growth and motility of Xoo.