水稻条斑病细菌hrp基因诱导表达系统的建立

Establishment of the hrp-inducing systems for the expression of the hrp genes of Xanthomonas oryzae pv.oryzicola

水稻条斑病细菌(Xanthomonas oryzae pv.oryzicola,Xooc)决定在非寄主植物上激发过敏反应(hypersensitive response)和在寄主水稻上具致病性(pathogenicity)的hrp基因簇是诱导表达的。为研究hrp基因的功能,利用hpa1和hrpX基因的启动子与gfp基因进行融合,构建了hrp基因诱导表达系统。绿色荧光蛋白表达揭示,Xooc的hrp基因在营养丰富的NB培养基上不能有效表达,在hrp诱导培养基XOM3上可有效表达。以hrpX和hrpG突变体为参照,RT-PCR研究结果提示,Xooc野生型菌株hpa1基因在NB上不能有效表达,在XOM3培养基上可有效表达。相应地,hrpX突变体中hpa1基因不能被诱导表达,而在hrpG突变体中hpa1基因转录表达水平低于野生菌。研究结果还证实,水稻悬浮细胞能高效诱导Xooc的hrp基因表达。Xooc hrp基因诱导表达系统的建立为研究hrp基因功能、发掘T3SS效应分子以及开展Xooc致病性研究奠定了基础。

The hrp genes of Xanthomonas oryzae pv. oryzicola （Xooc）, which is the causal agent of bacterial leaf streak in rice, possesses the ability to elicit hypersensitive response on nonhost plants and the pathogenicity in host rice. In order to analyze the function of the hrp genes, we developed hrp-inducing systems using transcriptional hrp ： ： gfp fusions with the promoters of hrpX and hpal of Xooc. The levels of GFP protein expression indicated that the hrp gene expression in Xooc was not efficiently induced in NB medium, but efficiently in XOM3 medium. Using the hrpG and hrpX mutants of Xooc as the controls, the results by RT-PCR demonstrated that in wild type strain the expression of the hpal gene was suppressed in NB medium, but was increased in XOM3 medium. When incubated in XOM3, the expression of the hpa 1 gene was abolished in hrpX mutant, while the level of the hpa 1 gene expression was lower in the hrpG mutant than that in wild-type strain. More importantly, it was found that the induction of the hrp gene expression was strongly increased in response to rice suspension cells and callus in this study. This suggests that the hrp-inducing systems, XOM3 or rice suspension cells or rice callus, for the induction of the hrp genes expression be useful for functionally analyzing the hrp genes, mining effectors secreted by the type Ⅲ secretion apparatus and understanding pathogenicity determinats of Xooc.