Identification of pathogenicity genes regulated by two kinds of transcription regulators of the fungal plant pathogen Fusarium oxysporum

我们之前鉴定了两个基因FOW2和FOW3，它们分别编码Zn(II) 2cys6型和his2cys2型转录调控因子，这两个基因是甜瓜枯萎病病原菌尖孢镰刀菌（Fusarium oxysporum f. sp. melonis）侵染植物所必需的。在本研究中，我们试图分离fow2或fow3调控基因。我们构建了cDNA减法文库，其中包含由FOW2和FOW3突变引起的转录水平变化的基因。通过cDNA斑点杂交和RNA凝胶杂交对这些文库进行筛选，鉴定出6个受Fow2正调控的基因。我们制作了这些基因的突变体，并鉴定出一个由ren1调控的致病基因VRF2-1。vrf2-1突变体对甜瓜的毒力降低，而fow2突变体完全丧失致病性。这一结果表明，Fow2调控了包括VRF2-1在内的多个致病基因的转录。我们对一个编码FOW2的BAC克隆进行了测序，发现VRF2-1也被这个克隆编码。该克隆包含另外29个基因，其中10个基因被发现可能受Ren1调控，这表明FOW2调控基因与FOW2密切相关。数据库同源性检索和PCR分析表明，丝状子囊菌种具有FOW2同源物。这表明FOW2同源物也控制其他真菌的植物感染。我们还发现了两个基因，REN1和FoSTUA，它们编码不同类型的转录调节因子，对尖孢镰刀菌的分生至关重要。在植物病原真菌中，分生孢子作为播散繁殖体和初、次生接种体在疾病循环中起着重要作用。我们在营养生长和分生过程中进行了表达序列标记分析，鉴定出了分生cDNA文库中特异性检测到的496个基因。比较野生型菌株与ren1突变体和fostuA突变体之间496个基因的转录水平，分别鉴定出35个和3个受ren1和fostuA调控的基因。少

We previously identified two genes, FOW2 and FOW3, which encode Zn(II)2Cys6-type and His2Cys2-type transcription regulators, respectively, essential for plant infection in the melon wilt pathogen Fusarium oxysporum f. sp. melonis. In this research, we attempted to isolate Fow2-or Fow3-regulated genes.We constructed cDNA subtraction libraries, which contained genes with changes in transcription levels caused by mutations in FOW2 and FOW3. Screening of these libraries by cDNA dot-blot differential hybridization and RNA gel blot analyses identified six genes positively regulated by Fow2. We made mutants of these genes and identified a Ren1-regulated pathogenicity gene, named VRF2-1. The vrf2-1 mutant showed reduced virulence on melon plants, although the fow2 mutant completely lost pathogenicity. This result indicates that Fow2 regulates the transcription of multiple pathogenicity genes, including VRF2-1. We sequenced a BAC clone encoding FOW2 and found that VRF2-1 is also encoded by this … More clone. This clone contains additional 29 genes, of which 10 were found to be possibly regulated by Ren1, suggesting that the Fow2-regulated genes are closely linked with FOW2. Database homology search and PCR analysis revealed that the filamentous ascomycetes species have the FOW2 homologues. This suggests that the FOW2 homologues also control plant infection in other fungi.We also identified two genes, REN1 and FoSTUA, which encode different types of transcription regulators essential for conidiation in F. oxysporum. In plant pathogenic fungi, conidia play important roles in the disease cycle as disseminated propagules and primary and secondary inocula. We performed expressed sequence tag analysis during vegetative growth and conidiation and identified 496 genes specifically detected in the conidiation cDNA library. Comparison of the transcription levels of the 496 genes between the wild-type strain and the ren1 mutant or the fostuA mutant identified 35 and three genes regulated by Ren1 and FoStuA, respectively. Less