Zeng Shen-yan, Hu Jun, Huang Gui-xiu, He Chao-zu
National Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Wei Sheng Wu Xue Bao. 2007 Jun;47(3):402-6.
Xanthomonas oryzae pv. oryzae (Xoo), a Gram-negative bacterium, is the causal agent of rice bacterial blight disease, which can cause severe yield loss of rice worldwide. To identify genes contributing to virulence and explore the possible mechanism of pathogenicity, transposon mutagenesis was used to isolate nonpathogenic mutants. By screening of a high-quality Tn5-like transposon (EZ: :TN) insertional mutant library of Xoo PXO99 against a host plant (rice cultivar IR24), one virulence-deficient mutant, XOG11, was identified. Genomic fragment flanking the insertion site of the mutant was amplified by thermal asymmetric interlaced polymerase chian reaction ( TAIL-PCR) and sequenced. The result of NCBI blast homologue searching of the fragment shows that the transposon was inserted into a hrp associated gene, hpaB. Xoo hpaB gene is one of the hrp gene cluster members that encode a type [I secretion system (TTSS) and locates at the downstream of hrpE. The product of hpaB in Xoo is a small (Molecular Weight, 17.6kDa), acidic (PI, 4.28) and Leucine-rich (14.4%) protein and shares high homology with corresponding proteins in other Xanthomonas. It suggests that HpaB may play as a TTSS chaperone. Mutant XOGl1 was confirmed both by PCR and Southern blotting: The PCR result by using primers upstream and downstream of hpaB respectively verified Tn5 insertion in hpaB and excluded the rare case of second transfer of the transposon associated with flanking sequence; Southern blot of digested genomic DNA with the probe of Km resistance gene aph proved that XOG11 was inserted by a single-copy transposon, indicating that the loss of pathogenicity in XOG11 was due to the Tn5 insertion in hpaB gene. Genetic complementation by cloning hpaB in the wide host range plasmid pHMI and transferring the recombinant plasmid into XOG11 restored its pathogenicity in IR24. These results suggest that the pathogenicity deficiency of XOG11 is due to the mutation of hpaB gene.
水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae，Xoo）是一种革兰氏阴性细菌，是水稻白叶枯病的病原菌，可在全球范围内导致水稻严重减产。为了鉴定与毒力相关的基因并探索可能的致病机制，利用转座子诱变分离无毒突变体。通过针对宿主植物（水稻品种IR24）筛选高质量的Xoo PXO99类Tn5转座子（EZ::TN）插入突变体文库，鉴定出一个毒力缺陷突变体XOG11。通过热不对称交错聚合酶链反应（TAIL-PCR）扩增突变体插入位点侧翼的基因组片段并进行测序。该片段在NCBI上进行同源性搜索的结果表明，转座子插入到一个与hrp相关的基因hpaB中。Xoo hpaB基因是hrp基因簇成员之一，编码I型分泌系统（TTSS），位于hrpE下游。Xoo中hpaB的产物是一种小蛋白（分子量17.6kDa），呈酸性（PI为4.28），富含亮氨酸（14.4%），与其他黄单胞菌中的相应蛋白具有高度同源性。这表明HpaB可能作为TTSS的分子伴侣发挥作用。通过PCR和Southern杂交对突变体XOGl1进行了验证：分别使用hpaB上下游引物进行PCR，结果证实了Tn5插入hpaB，并排除了转座子与侧翼序列相关的二次转移这种罕见情况；用卡那霉素抗性基因aph探针消化基因组DNA后的Southern杂交证明XOG11是由单拷贝转座子插入的，表明XOG11致病性的丧失是由于hpaB基因中的Tn5插入。通过在广宿主范围质粒pHMI中克隆hpaB并将重组质粒转入XOG11进行遗传互补，恢复了其在IR24中的致病性。这些结果表明，XOG11的致病性缺陷是由于hpaB基因的突变。
