应答调节蛋白基因flgRRxoo对水稻白叶枯病菌毒性表达的调控作用鉴定

Molecular identification and functional analysis of flgRRxoo,the gene encoding a putative response regulator in regulation of virulence expression of Xanthomonas oryzae pv. oryzae

为了阐明水稻白叶枯病菌(Xanthomonas oryzaepv.oryzae,简称Xoo)应答调节蛋白FlgRRxoo的生物学功能,本研究通过基因克隆和序列分析、用标记交换法构建突变体以及表型测定,对flgRRxoo基因进行了分子鉴定。通过设计引物进行特异性扩增,成功地从Xoo野生型菌株PXO99A中克隆了flgRRxoo。FlgRRxoo为N端具有REC结构域的单一结构应答调节蛋白,其基因序列与其他黄单胞病菌中的同源序列高度保守。与PXO99A相比,△flgRRxoo胞外多糖(EPS)合成能力以及对水稻的毒性显著降低,基因互补可以使之恢复;△flgRRxooEPS合成基因gumBDGKM表达均有所下降;但其运动性、胞外纤维素酶和木聚糖酶活性无明显改变。表明FlgRRxoo可能主要通过调控EPS合成能力,影响了病菌在水稻上的毒性。

To be better understand the structure and biological function of flgRRxoo in Xanthomonas oryzae pv.oryzae（Xoo）,the casual pathogen of bacterial blight in rice,molecular identification of the flgRRxoo gene was performed through gene cloning,sequencing and mutant construction.flgRRxoo cloned from geno-mic DNA of the wild-type strain PXO99^A was found to be highly conserved in plant-pathogenic Xanthomonas spp.,and FlgRRxoo was structurally featured with REC domain.The mutant was constructed after a double crossover recombination event between gentamycin resistance gene（Gm^R） and flgRRxoo through the marker exchange and validated by PCR assay.The △flgRRxoo mutant demonstrated great deficiency in exopolysaccharide（EPS） production and obviously low virulence compared with PXO99A,which could be restored through complementation of the mutant by introducing flgRRxoo gene;expression of EPS biosynthesis-related genes gumBDGKM was down-regulated in △flgRRxoo.Whereas,no significant changes in flagellar motility and production of extracellular cellulase and xylanase in vitro were observed in △flgRRxoo compared to PXO99^A.Therefore,FlgRRxoo might act as a regulator involved in regulation of EPS production and virulence of Xoo.