Identification of virulence genes in the crucifer anthracnose fungus Colletotrichum higginsianum by insertional mutagenesis
Identification of virulence genes in the crucifer anthracnose fungus Colletotrichum higginsianum by insertional mutagenesis
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通过插入诱变鉴定十字花科炭疽真菌Colletotrichum higginsianum 毒力基因
DOI：
10.1016/j.micpath.2013.06.001
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发表时间：
2013-11-01
影响因子：
3.8
通讯作者：
Huang, Junbin
中科院分区：
文献类型：
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作者：
Liu, Liping;Zhao, Dian;Huang, Junbin
为研究黄瓜炭疽病菌对拟南芥的致病性分子遗传机制，利用拟南芥炭疽病菌T-DNA插入突变体库，构建了拟南芥炭疽病菌T-DNA插入突变体库。利用根癌农杆菌介导法建立了十字花科植物炭疽病病原菌higginsianum的遗传转化体系。在875个转化子中，获得了6个毒性突变体，包括一个附着胞黑色素缺陷型突变体T734，两个穿透缺陷型突变体T45和B30，以及3个引起拟南芥过敏反应的突变体T679、T732和T801。Southern杂交分析表明，突变体T732和T734含有单位点的T-DNA整合，而B30含有两个T-DNA插入。通过反向聚合酶链反应（PCR）和热不对称交错PCR从这些突变体中回收T-DNA的边界侧翼序列。序列分析表明，在突变体T734中的单一T-DNA插入靶向一个未知功能的基因的编码区，并在突变体T732中靶向编码铜胺氧化酶的基因。突变体B30中的两个T-DNA插入位点被发现在编码外泌体组分的基因的编码区和DUF 221结构域基因的上游区中。这些基因中没有一个先前与植物病原真菌的毒力有关。在这些无毒突变体中，T734在菌落生长中表现出改变的颜色，并产生黑色素缺陷的白化病附着胞。T734中的T-DNA插入片段位于一个名为C. higginsianum melanin-deficiency gene（Ch-MEL 1），其与编码胶孢炭疽菌（Colletotrichum gloeosporioides）中的假定蛋白质的基因（GenBank ELA 33048）高度相似。为了验证Ch-MEL 1基因是否与突变体1734的毒力相关，使用靶向基因破坏和互补方法。Delta Ch-mel 1无效突变体的附着胞在黑化中有缺陷，并且未能穿透宿主表皮细胞。当接种到受伤的叶组织上时，Delta Ch-mel 1突变体在宿主组织上生长，但未能引起伤口部位以外的病变。与此相反，无论是补体C三角洲Ch-mel 1 -2和野生型产生黑化附着胞，并导致拟南芥叶片坏死。Ch-MEL 1在C. higginsianum和宿主组织中的侵入后生长。结合其他无毒突变体及其相关基因的鉴定，本研究为半生养型梭菌致病性的分子机制提供了新的见解。Higginsianum。(c)2013爱思唯尔有限公司保留所有权利。
To investigate the molecular and genetic mechanisms underlying virulence of Colletotrichum higginsianum on Arabidopsis thaliana, a T-DNA insertion mutant library of C. higginsianum, the causal agent of crucifer anthracnose, was established using Agrobacterium tumefaciens-mediated transformation. Among 875 transformants tested for virulence on Arabidopsis, six mutants with altered virulence, including an appressorial melanin-deficient mutant T734, two mutants defective in penetration, T45 and B30, and three mutants, T679, T732 and T801, that cause hypersensitive reactions on host Arabidopsis, were obtained. Southern blot analysis indicated that the mutants T732 and T734 harbored single-site T-DNA integrations, while B30 harbored two T-DNA insertions. Border flanking sequences of T-DNAs from these mutants were recovered by inverse polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR. Sequence analyses revealed that single T-DNA insertions in mutant T734 targeted the coding region of a gene with unknown function, and in mutant T732 targeted a gene encoding a copper amine oxidase. The two T-DNA insertion sites in mutant B30 were found in the coding region of a gene encoding an exosome component and in the upstream region of a DUF221-domain gene. None of these genes have previously been implicated in virulence of the phytopathogenic fungi. Among these avirulent mutants, T734 showed altered color in colony growth and produced melanin-deficient, albino appressoria. The T-DNA insert in T734 was detected in the coding region of a gene named C. higginsianum melanin-deficiency gene (Ch-MEL1), which is highly similar to a gene encoding a hypothetical protein in Colletotrichum gloeosporioides (GenBank ELA33048). To validate whether the Ch-MEL1 gene was associated with virulence of the mutant 1734, a targeted gene disruption and complementation approach was used. The appressoria of Delta Ch-mel1 null mutants were defective in melanization and failed to penetrate the host epidermal cells. When inoculated onto the wounded leaf tissues, the Delta Ch-mel1 mutants grew on host tissues but failed to cause lesions beyond the wound site. In contrast, both the complement C Delta Ch-mel1-2 and the wild type produced melanized appressoria and caused necrosis on leaves of Arabidopsis. Ch-MEL1 is required for both appressorial melanin production in C. higginsianum and post-invasive growth in host tissues. Together with identification of other avirulent mutants and their associated genes, this study provides novel insights into molecular mechanisms underlying virulence of the hemibiotroph, C. higginsianum. (c) 2013 Elsevier Ltd. All rights reserved.