【作者】 Sami Mohammed Adam Mohammed；

【导师】 戴小枫；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2014， 博士

【摘要】 大丽轮枝菌属子囊真菌可导致600多种双子叶植物引发黄萎病,对农业生产连年造成重大经济损失,其中包括多种重要经济作物如棉花,橄榄,番茄,草莓等。目前人们对大丽轮枝菌的致病机理了解甚少,而筛选和鉴定大丽轮枝菌关键致病因子是揭示大丽轮枝菌致病分子机理的重要前提。随着基因组和蛋白组研究新技术的快速发展,为高效系统的筛选和鉴定大丽轮枝菌致病相关基因提供了有利条件。本研究基于大丽轮枝菌基因组数据,选择了坏死及乙烯诱导蛋白（NEP）家族为大丽轮枝菌候选致病相关的基因进行深入的功能研究。从VdG1菌株基因组中获得NEP家族的8个基因VdNEP1至VdNEP8.其中VdNEP6基因编码256个氨基酸,包含一个保守基序GHRHDWE,研究认为含有该保守序列的基因可影响植物细胞质膜的通透性,并能触发植物细胞的防卫反应从而诱导细胞坏死反应。VdNEP6基因在20株不同棉产区收集到的大丽轮枝菌菌株中具有较多的非同义SNP突变,该基因在不同菌株中较高的变异性表明其可能与大丽轮枝菌致病毒力相关。同时信号肽活性检测实验表明VdNEP6为胞外分泌蛋白。但是VdNEp6基因敲除突变株的致病力及生长表型与野生型菌株VdG1均无明显差异。利用PVX载体PGR107通过农杆菌介导的VdNEP6在烟草中的瞬时转化实验结果表明该基因不能够诱导植物细胞的坏死反应。实验结果表明VdNEP6基因不能够引发植物细胞的坏死反应且与大丽轮枝菌致病力没有直接相关性。基于前期大丽轮枝菌比较分泌蛋白组数据,获得了大量高效应答棉花组织的糖基水解酶家族蛋白,表明该类蛋白与大丽轮枝菌致病力具有潜在的相关性。本研究挑选了10个在棉花组织培养条件下高效诱导表达的CAZy基因,并对其毒力功能进行了初步的鉴定。对10个CAZy基因进行了全长克隆,并利用PVX介导的烟草瞬时表达系统检测了10个基因的细胞诱导坏死及抑制坏死效应。结果显示这些基因均不能够诱导烟草叶片细胞的坏死反应,然而其中的两个基因（VDG1₀.3897和VDG1₀.3197）,分别预测为角质酶和羟肽酶Y,能够有效的抑制细胞凋亡调节蛋白BAX及VdNEP1激发子蛋白所诱导的细胞死亡。该实验结果表明两个候选基因可能通过调节寄主的免疫反应从而增强病原菌的致病力。基因表达分析显示两个候选基因在棉花组织诱导培养基中能够被强烈的诱导表达,说明了其在参与大丽轮枝菌侵染过程中可能发挥的作用,值得进一步深入研究。此外,为了揭示大丽轮枝菌特异定殖于植物维管束组织机理,通过比较基因组学对14种已经测序的植物病原真菌（维管束和非维管束病害真菌）基因组数据进行了比较分析。参试的真菌分为三组,分别为粪壳菌（Fusarium graminearum, F. oxysporum, Verticilium dahliae, V. albo-atrum, Neurospora crassa, Magnaporthe grisea and Chaetomium globosum）,散囊菌（Aspergillus nidulans and A. terreus）和榷舌菌（Botrytis cinerea）。大规模序列比对分析结果显示有57个基因特异存在于大丽轮枝菌中。将57个基因在已报道的135个真菌基因组中进行深入的比对分析发现,有5个基因高度特异的存在于典型植物维管束病害真菌中,如大丽轮枝菌（V. dahliae）,尖孢镰刀菌（Fusarium oxysporum）及黑白轮枝菌（V. albo-atrum）。这5个基因被分别命名为VdVSl, VdVS2, VdVS3, VdVS4, VdVS5。基因表达分析表明,在大丽轮枝菌侵染棉花过程中,5个维管束相关基因能够被显著地诱导表达。其中将野生型菌株VDGh1.991中VdVS3和VdVS5敲除后并不影响菌落的生长状况及孢子的产生,但与野生型相比这两个基因的突变株的致病力显著下降,表明VdVS3和VdVS5在大丽轮枝菌侵染寄主过程中发挥重要作用。因此对VdVS3和VdVSS基因功能的进一步研究对于揭示大丽轮枝菌及维管束病害真菌的致病机理具有重要的意义。综上所述,该研究发现了一些与大丽轮枝菌致病相关的靶标基因,进一步的基因功能鉴定将会大大增进大丽轮枝菌和维管束真菌病害致病机理的研究。更多还原

【Abstract】 The Ascomycete fungus Verticillium dahliae Klebahn causes vascular wilt disease across over600plant species, including many high-value crops, such as cotton, olive, tomato, strawberry, etc. Due to less understanding of pathogenesis mechanism, Verticillium wilt is still a chronic economic problem in crop production, causing great losses in annual crop yields. To address this issue, screening and identification of pathogenicity-related factors is a key step to further decipher the mechanism of pathogenicity in V. dahliae. Accompanied by rapid development of omics techniques, including genomics and proteomics, which enable us to obtain a big data and screen the critical and useful information fastly and systematically.In this study, based on V. dahliae genome data, the gene family of Necrosis and ethylene-inducing protein （NEP） that may contribute to virulence was selected for in-depth functional characterization. More specifically, we identified eight VdNEP genes in the genome of VdGl, a V. dahliae strain isolated from cotton, designated as VdNEP1to VdNEP8. Of which, VdNEP6gene, encoding a secreted protein of256amino acids, contains a conserved heptapeptide motif GHRHD WE that is important in plasma membrane permeability and accordingly assisting triggering of plant defense response through damaging plant cells causing leaf necrosis. Especially, VdNEP6showed a high ratio of non-synonymous SNPs among twenty V. dahliae isolates which were collected from different cotton production fields, revealing their potential roles in pathogenesis in cotton V. dahliae. Functional validation of the signal peptide indicated that VdNEP6exactly located in extracellular. However, gene disruption of VdNEP6using homologous recombination and Agrobacterium tumefaciens mediated transformation （ATMT） techniques can not affect the pathogenicity, growth and conidiation of the wild type strain VdGl. Transient expression assay carried by Potato virus X （PVX） vector pGR107revealed that VdNEP6was neither involved in cell death induction nor suppression activity. Thus, we suggested that VdNEP6has no direct effect in cotton V. dahliae strain, since it failed to affect reducing the virulence of the wild type and no clear host cell death pattern was accomplished.Based on the quantitative analysis between the V. dahliae secretomes that were induced or non-induced by cotton tissue extract, numerous CAZy proteins showed a high sensitivity to cotton tissue, revealing their potential roles contributing to virulence in V. dahliae. Ten of CAZy genes that showed high expression level in the cotton tissue induced secretome were selected for functional characterization on virulence. To test cell death induction or suppression activities, ten of CAZy genes were cloned and transiently expressed in tobacco leaves via PVX （Potato virus X）-mediated expression system. The results indicated that none of genes can induce cell death in tobacco, whereas two genes including a putative cutinase and a Carboxypeptidase Y genes （VDG1₀.3897and VDG1₀.3197） can effectively suppress cell death triggered both by apoptosis regulator BAX and VdNEP1elicitor that known to trigger necrosis on cotton leaves. The suppression ability of the candidates suggested that the two genes may play potential roles in V. dahliae pathogenesis and possibly enhance the virulence through the regulation of host immunity. In addition, gene expression analysis indicated that the two candidate genes showed a strong induction in V. dahliae when cultivated in the medium with cotton tissue extract. Through homologous recombination, the gene knock-out mutants without the cutinase VDG1₀.3897candidate gene was successfully obtained. The disease assay of the mutant on cotton is in progress.In addition, to disclose why the V. dahliae specifically colonize in plant vascular niches, we conducted a comparative genomics among14sequenced plant pathogenic fungi including vascular and non-vascular pathogens. The tested fungi are ascomycetes and belong to three classes, including Sordariomycetes （Fusarium graminearum, F. oxysporum, Verticilium dahliae, V. albo-atrum, Neurospora crassa, Magnaporthe grisea and Chaetomium globosum）, Eurotiomycetes （Aspergillus nidulans and A. terreus） and Leotiomycetes （Botrytis cinerea）. The data revealed57genes that are specific to vascular wilt fungus Verticilium dahliae （VDGhl.991）. In-depth comparative analysis of these57genes among the reported135fungal genomes further revealed five genes, namely, VdVS1to VdVS5that showed high presence ratio in vascular related fungal genomes such as V. dahliae, Fusarium oxysporum and V. albo-atrum but not in non-vascular related pathogens. Further gene expression analysis indicated that five vascular-related genes of V. dahliae can be strongly induced by cotton tissue, an in vitro interaction system between V. dahliae and cotton. Gene disruption of VdVS3and VdVS5in V. dahliae wild-type strain VDGhl.991did not affect the growth and sporulation but caused virulence reduction on cotton compared with the wild type strain, indicating that VdVS3and VdVS5may play important roles in V. dahliae pathogenicity. Further functional characterization of these vascular-related candidate genes may facilitate our understanding on the pathogenicity mechanism of vascular infected fungal pathogens.In summary, this study provided the target genes related to pathogenicity in V. dahliae, further functional investigation may greatly enhance the exploration of pathogenesis mechanism of this vascular-parasitic fungus.更多还原