Stephenson S A, Hatfield J, Rusu A G, Maclean D J, Manners J M
Cooperative Research Centre for Tropical Plant Pathology, The University of Queensland, Brisbane, Australia.
Mol Plant Microbe Interact. 2000 Sep;13(9):929-41. doi: 10.1094/MPMI.2000.13.9.929.
A gene of Colletotrichum gloeosporioides that is induced by nitrogen starvation in axenic culture and is expressed at the early stages of infection of the host Stylosanthes guianensis has been identified and its role in pathogenicity tested. The sequence of this gene, named CgDN3, indicated that it encodes a protein of 74 amino acids that contains a predicted 18 amino acid signal sequence for secretion of a basic 54 amino acid mature protein with weak homology to an internal region of plant wall-associated receptor kinases. Mutants of C. gloeosporioides were produced by homologous recombination in which part of the coding sequence and promoter region of the CgDN3 gene was replaced with a hygromycin-resistance gene cassette. Mutations in the CgDN3 gene were confirmed in two independent transformants and Northern (RNA) analysis demonstrated the disrupted CgDN3 gene was not expressed. The mutants had faster mycelial growth rates in vitro but produced spores that germinated to form appressoria normally on the leaf surface. However, the CgDN3 mutants were unable to infect and reproduce on intact host leaves. Microscopic analysis revealed small clusters of necrotic host cells at inoculation sites on leaves, suggesting that these mutants elicited a localized, host hypersensitive-like response. The mutants were able to grow necrotrophically and reproduce on leaves when conidia were inoculated directly onto wound sites. The putative promoter region of the CgDN3 gene was fused to a gene encoding a modified jellyfish green fluorescent protein and introduced into the fungus. Following inoculation, strong expression of green fluorescent protein was observed in primary infection vesicles in infected epidermal cells with weaker expression evident in hyphae growing within infected leaf tissue. These findings indicate that CgDN3 encodes a novel pathogenicity determinant associated with the biotrophic phase of primary infection and required to avert a hypersensitive-like response by a compatible host.
已鉴定出一种来自胶孢炭疽菌的基因，该基因在无菌培养中受氮饥饿诱导，并在侵染宿主圭亚那柱花草的早期阶段表达，且已对其在致病性方面的作用进行了测试。这个名为CgDN3的基因序列表明，它编码一种由74个氨基酸组成的蛋白质，该蛋白质含有一个预测的18个氨基酸的信号序列，用于分泌一种由54个氨基酸组成的碱性成熟蛋白质，该成熟蛋白质与植物细胞壁相关受体激酶的一个内部区域具有微弱的同源性。通过同源重组产生了胶孢炭疽菌突变体，其中CgDN3基因的部分编码序列和启动子区域被潮霉素抗性基因盒取代。在两个独立的转化体中证实了CgDN3基因的突变，Northern（RNA）分析表明，被破坏的CgDN3基因不表达。这些突变体在体外菌丝生长速度更快，但产生的孢子在叶表面能正常萌发形成附着胞。然而，CgDN3突变体无法在完整的宿主叶片上侵染和繁殖。显微镜分析显示，叶片接种部位有小簇坏死的宿主细胞，这表明这些突变体引发了局部的、类似宿主过敏反应。当分生孢子直接接种到伤口部位时，这些突变体能够在叶片上进行坏死营养生长并繁殖。将CgDN3基因的推定启动子区域与编码一种修饰的水母绿色荧光蛋白的基因融合，并导入该真菌。接种后，在受感染表皮细胞的初次感染泡囊中观察到绿色荧光蛋白的强烈表达，而在受感染叶组织内生长的菌丝中表达较弱。这些发现表明，CgDN3编码一种与初次感染的活体营养阶段相关的新型致病性决定因子，是避免相容宿主产生类似过敏反应所必需的。
