比较基因组、转录组和基因功能研究揭示核盘菌致病和发育的分子机理
发布时间：2018-08-18 19:20
【摘要】：核盘菌(Sclerotinia sclerotiorum)是一种寄主范围广泛、世界性分布的典型死体营养型植物病原真菌,能够在多种经济作物(例如油菜、向日葵、大豆和扁豆等)上引起菌核病,给农作物生产造成巨大的经济损失。为了从基因组水平上研究核盘菌的发育特性,对核盘菌及已测序真菌的基因组进行了功能注释和功能基因组比较分析。发现核盘菌基因组中与转座元件相关的类似CENP-B的DDE超家族核酸内切酶和反转录酶要远远多于其它真菌,这种显著的差异暗示了转座事件在核盘菌基因组的进化中起到了重要作用。进一步详细分析了核盘菌中的转座元件的种类和它们与类似CENP-B的DDE超家族核酸内切酶基因和反转录酶基因在基因组中的相对位置关系,表明这些酶家族基因的周围确实存在典型的转座元件,且绝大多数类似于CENP-B的DDE超家族核酸内切酶基因都与DNA转座元件相关,其中TcMar-Fot1类群最多;所有反转录酶基因都与反转录转座元件相关,其中LINE/Tad1,LTR/Gypsy和LTR/Copia三个类群占比最多。对核盘菌及其它真菌中转座元件的种类和数量的比较发现,DNA和RNA转座元件均在核盘菌基因组的进化过程中起着重要作用。系统发育分析揭示类似CENP-B的DDE超家族核酸内切酶和反转录酶基因家族的扩张可能是核盘菌中相应转座元件在进化过程中短时间内大量复制扩增的结果。转录分析表明尽管这两大类转座子中的大部分已经“失活”,但少部分仍然具有“活性”,插入宿主功能基因的下游是其保持活性的方式之一。利用数字表达谱检测了核盘菌在6个关键发育阶段(包括菌丝营养生长、侵染、菌核形成、菌核菌丝萌发、菌核子囊柄萌发和子囊柄形成阶段)的转录组。为了从整体上阐述核盘菌不同功能模块的活性在各个发育阶段的动态变化,建立一种“功能谱”的分析方法来衡量各种功能模块(例如,GO、Interpro功能模块和KEGG代谢途径)的相对活性。功能谱分析可以将基因表达水平和基因功能注释结合起来描绘相应功能模块的相对活性。通过比较“功能谱”,明确了各个功能模块在核盘菌不同发育阶段相对活性的动态变化。此外,基因功能富集分析显示了核盘菌在不同发育阶段相对菌丝营养生长阶段所富集到的多个功能模块。功能谱分析结果显示一条不完整的碳固定相关通路在核盘菌侵染、菌核菌丝萌发和菌核子囊柄萌发过程中被显著地激活。光合作用过程中植物的碳固定途径是将太阳能转化为生物质、生物产品和生物燃料的过程。对这条通路进行深入研究发现大量异养型真菌也拥有和植物碳固定途径相关的各种酶类,其中的许多酶在真菌中都是保守的。在异养型的核盘菌中存在17个植物碳固定途径相关酶类的同源蛋白,在calvin-benson-basham(cbb)还原型磷酸戊糖途径中有10个,在c4-二羧酸循环中有7个。尤其是在cbb循环中,只有5-二磷酸羧化加氧酶(rubisco)和磷酸核酮糖激酶(prk)在核盘菌中找不到同源蛋白。rnai沉默试验证明许多和这条途径相关的酶类都在核盘菌的侵染和菌核形成过程中发挥重要作用。遗传发育分析表明许多碳固定相关酶类在进化中都经历了基因复制、基因丢失或获得和基因功能多样化事件。这些发现在碳固定层面展示了自养型生物和异养型生物之间的联系,表明在进化过程中碳固定相关基因的功能在不同物种中是动态变化的。在核盘菌发育过程中,碳水化合物活性酶类相关的功能模块也受到了显著地诱导。比较基因组分析显示死体营养型和半活体营养型病原真菌的植物细胞壁降解酶和真菌细胞壁降解酶的数量都要比大多数活体营养型病原真菌中的多。然而,对核盘菌、禾谷镰刀菌、青杨叶锈菌和麦类杆锈菌中的碳水化合物活性酶类的转录分析表明在死体营养型和活体营养型真菌的侵染过程中,许多编码植物细胞壁降解酶类的基因和真菌细胞壁降解酶类的基因都显著上调表达,表明植物病原真菌中存在一种植物细胞壁的降解和真菌细胞壁的重组或修饰相伴随的普遍机制,该机制可能与病原真菌的侵染密切相关。此外,本研究结果表明植物病原真菌细胞壁的重组和修饰还与其自身发育相关。小分泌蛋白在活体、半活体营养型真菌和其寄主植物的互作中发挥了重要作用,然而对它们在寄主范围广泛的死体营养型真菌中的作用还知之甚少。转录组分析显示许多分泌蛋白编码基因在核盘菌菌核形成和侵染过程中显著上调表达。选取两个在核盘菌侵染过程中显著上调表达的富含半胱氨酸的小分泌蛋白,即sscvnh和ssssvp1为例进行深入研究,发现它们均在核盘菌的致病过程中发挥重要作用。对ssssvp1的进一步研究表明,ssssvp1从菌丝中被分泌出来后可被植物细胞内化并且可在细胞间自主转运,这种转运不依赖于核盘菌本身。ssssvp1主要定位于寄主的细胞质并可诱导植物细胞坏死。酵母双杂交、免疫共沉淀和荧光双分子互补试验均证实ssssvp1与植物中蛋白qcr8互作,qcr8是植物线粒体呼吸链上细胞色素b-c1复合体中的一个亚基。双定点突变结果表明两个半胱氨酸残基(c38和c44)同时突变使SsSSVP1不能形成同源二聚体,不能和QCR8互作并失去了诱导植物细胞坏死的能力,说明部分半胱氨酸残基在维持SsSSVP1的结构和功能过程中发挥了重要作用。荧光共定位试验和荧光双分子互补试验显示SsSSVP1可以在QCR8进入线粒体之前将其“劫持”到细胞质中从而扰乱了QCR8的亚细胞定位。在烟草中进行的病毒介导的基因沉默试验表明沉默QCR8导致植株发育异常且引起植物细胞的坏死反应,提示SsSSVP1诱导的植物细胞坏死与SsSSVP1-QCR8之间的互作导致QCR8亚细胞定位的改变相关,QCR8亚细胞定位的改变可能使其丧失了生物学功能。这些结果表明小分泌蛋白在寄主非特异性死体营养型真菌中也作为潜在的效应因子发挥着重要作用。本研究揭示了小分泌蛋白在寄主范围广泛的死体营养型真菌和其寄主植物互作过程中的新功能,阐明这种互作类型的致病机制具有重要意义。本文从基因组、转录组和单个小分泌蛋白生物学功能等多个水平研究了核盘菌致病和发育的分子机制。本研究的结果表明核盘菌所有发育阶段都是多个功能上密切联系的基因组成一个网络协同起作用的结果,但是这个网络中每个基因所发挥的具体作用和所处的地位并不相同,存在某些关键的“节点基因”对特定的生物学过程起着决定性作用。本文结合核盘菌的数字表达谱和生物信息学方法一方面全面分析了核盘菌发育过程中各种功能模块的整体表现,另一方面深入研究了某些关键“节点”基因的生物学功能,分别从宏观和微观的角度研究了核盘菌各种生物学过程的分子基础。通过以上两种思路和策略,本研究全方位综合展示了核盘菌致病和发育的分子机理,为菌核病的防治提供了新的视野和理论支撑。
[Abstract]:Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) is a typical dead-body vegetative pathogenic fungus with wide host range and worldwide distribution. Sclerotinia sclerotiorum can cause Sclerotinia in many economic crops (such as rape, sunflower, soybean and lentil) and cause great economic losses to crop production. The genomes of Sclerotinia sclerotiorum and sequenced fungi were annotated and compared. It was found that there were much more endonucleases and retrotransferases in the DDE superfamily of Sclerotinia sclerotiorum than in other fungi, suggesting that transposal events occurred in the nucleic disk. The species of transposable elements in Sclerotinia sclerotiorum and their relative position with the DDE superfamily endonuclease gene and reverse transcriptase gene similar to CENP-B were analyzed in detail, which indicated that there were typical transposable elements around the genes of these enzymes family. Most of the DDE superfamily endonuclease genes similar to CENP-B are related to DNA transposable elements, among which TcMar-Fot1 group is the most; all the retrotranspose genes are related to retrotransposon elements, of which LINE/Tad1, LTR/Gypsy and LTR/Copia are the most common. Phylogenetic analysis revealed that the expansion of the DDE superfamily endonuclease and reverse transcriptase gene family similar to CENP-B may be the result of a large number of replication and amplification of the corresponding transposable elements in Sclerotinia within a short period of time. Transcription analysis showed that although most of the two types of transposons were inactivated, a few of them were still "active" and insertion into the downstream of the host functional gene was one of the ways to maintain their activity. Transcriptions of nuclear hyphae germination, sclerotium ascospores germination and ascospores formation. In order to explain the dynamic changes of activities of different functional modules of Sclerotinia in different developmental stages, a "functional spectrum" analysis method was established to measure the phase of various functional modules (e.g., GO, Interpro functional modules and KEGG metabolic pathways). Functional spectrum analysis can combine the level of gene expression with the annotation of gene function to describe the relative activity of the corresponding functional modules. By comparing the "functional spectrum", the dynamic changes of the relative activity of each functional module in different developmental stages of Sclerotinia sclerotiorum were clarified. Functional spectrum analysis showed that an incomplete carbon fixation pathway was significantly activated during the infection of Sclerotinia sclerotiorum, the germination of Sclerotinia mycelium and the germination of Sclerotinia cyst stalk. Biomass, biological products, and biofuel processes. In-depth studies of this pathway have revealed that a large number of heterotrophic fungi also possess enzymes associated with plant carbon fixation pathways, many of which are conserved in fungi. 10 of the alvin-benson-basham (cbb) reductive pentose phosphate pathways and 7 of the C4-dicarboxylic acid cycles, especially in the CBB cycle, only 5-diphosphate carboxylation oxygenase (rubisco) and ribonuclease phosphate kinase (prk) can not find homologous proteins in Sclerotinia. RNAi silencing tests have shown that many enzymes associated with this pathway are located in the nuclear disk. Genetic development analysis shows that many carbon fixation-related enzymes undergo events of gene replication, gene loss or acquisition, and gene functional diversity in evolution. These findings demonstrate the relationship between autotrophic and heterotrophic organisms on the carbon fixation level, indicating evolution. During the development of Sclerotinia sclerotiorum, functional modules related to carbohydrate-activated enzymes were also significantly induced. Comparative genomic analysis showed that cell wall degrading enzymes and fungal cells of dead and semi-living trophic pathogenic fungi were also significantly induced. However, transcriptional analysis of carbohydrate-degrading enzymes in Sclerotinia sclerotiorum, Fusarium graminearum, Populus Populus leaf rust and Stem rust of wheat showed that many of the enzymes encoding plant cell wall degrading enzymes were present during the infection of dead and living vegetative fungi. Both genes and genes of fungal cell wall degrading enzymes were significantly up-regulated, suggesting that there is a common mechanism associated with the degradation of plant cell wall and the recombination or modification of fungal cell wall in plant pathogenic fungi, which may be closely related to the infection of pathogenic fungi. The recombination and modification of the wall are also related to its own development. Small secretory proteins play an important role in the interactions between living, semi-living and host plants, but their roles in a wide range of dead-body trophic fungi are poorly understood. Transcription analysis shows that many secretory protein-coding genes are located in the nuclear disk. Two small cysteine-rich secretory proteins, sscvnh and ssssssvp1, which were significantly up-regulated during Sclerotinia formation and infection, were selected as examples for further study. Both of them played an important role in the pathogenesis of sclerotinia. Further studies on ssssssvp1 showed that ssssssvp1 might play an important role in the pathogenesis of sclerotinia. Ssssvp1 is mainly localized in the host cytoplasm and can induce plant cell necrosis. Yeast two-hybrid, immunoprecipitation and fluorescent bimolecular complementarity assay confirmed that ssssvp1 and plant protein qcr8 Interaction, qcr8 is a subunit of the cytochrome b-c1 complex in the plant mitochondrial respiratory chain. The results of two site-directed mutagenesis showed that the two cysteine residues (c38 and c44) could not form homologous dimers with SsSSVP1, could not interact with QCR8 and lost the ability to induce plant cell necrosis, suggesting that some cysteine residues were in the vitamin B. Fluorescence co-localization test and fluorescence bimolecular complementarity test showed that SsSSVP1 could "hijack" QCR8 into the cytoplasm before it entered the mitochondria, thereby disrupting the subcellular localization of QCR8. Virus-mediated gene silencing test in tobacco showed that QCR8 was silenced. These results suggest that SsSSVP1-induced plant cell necrosis is associated with the alteration of subcellular localization of QCR8, and the alteration of subcellular localization of QCR8 may deprive it of its biological function. This study reveals the new function of small secretory proteins in the interaction between dead vegetative fungi and their host plants, and elucidates the pathogenic mechanism of this type of interaction. Molecular mechanisms of pathogenesis and development of Sclerotinia sclerotiorum have been studied at various levels, such as protein biological functions. The results of this study show that all developmental stages of Sclerotinia sclerotiorum are the result of a network of closely related genes, but each gene in this network plays a specific role and is in a position to play. Different from each other, there are some key "node genes" that play a decisive role in specific biological processes. In this paper, the digital expression profiles of Sclerotinia and bioinformatics methods are combined to analyze the overall performance of various functional modules in the development process of Sclerotinia on the one hand, on the other hand, some key "node" bases are studied in depth. The molecular basis of various biological processes of Sclerotinia sclerotiorum has been studied from macroscopic and microscopic perspectives due to its biological functions. Through the above two ideas and strategies, the molecular mechanism of pathogenesis and development of Sclerotinia sclerotiorum has been demonstrated comprehensively, which provides a new perspective and theoretical support for the prevention and treatment of Sclerotinia sclerotiorum.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S432.44
，
本文编号：2190433
[Abstract]:Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) is a typical dead-body vegetative pathogenic fungus with wide host range and worldwide distribution. Sclerotinia sclerotiorum can cause Sclerotinia in many economic crops (such as rape, sunflower, soybean and lentil) and cause great economic losses to crop production. The genomes of Sclerotinia sclerotiorum and sequenced fungi were annotated and compared. It was found that there were much more endonucleases and retrotransferases in the DDE superfamily of Sclerotinia sclerotiorum than in other fungi, suggesting that transposal events occurred in the nucleic disk. The species of transposable elements in Sclerotinia sclerotiorum and their relative position with the DDE superfamily endonuclease gene and reverse transcriptase gene similar to CENP-B were analyzed in detail, which indicated that there were typical transposable elements around the genes of these enzymes family. Most of the DDE superfamily endonuclease genes similar to CENP-B are related to DNA transposable elements, among which TcMar-Fot1 group is the most; all the retrotranspose genes are related to retrotransposon elements, of which LINE/Tad1, LTR/Gypsy and LTR/Copia are the most common. Phylogenetic analysis revealed that the expansion of the DDE superfamily endonuclease and reverse transcriptase gene family similar to CENP-B may be the result of a large number of replication and amplification of the corresponding transposable elements in Sclerotinia within a short period of time. Transcription analysis showed that although most of the two types of transposons were inactivated, a few of them were still "active" and insertion into the downstream of the host functional gene was one of the ways to maintain their activity. Transcriptions of nuclear hyphae germination, sclerotium ascospores germination and ascospores formation. In order to explain the dynamic changes of activities of different functional modules of Sclerotinia in different developmental stages, a "functional spectrum" analysis method was established to measure the phase of various functional modules (e.g., GO, Interpro functional modules and KEGG metabolic pathways). Functional spectrum analysis can combine the level of gene expression with the annotation of gene function to describe the relative activity of the corresponding functional modules. By comparing the "functional spectrum", the dynamic changes of the relative activity of each functional module in different developmental stages of Sclerotinia sclerotiorum were clarified. Functional spectrum analysis showed that an incomplete carbon fixation pathway was significantly activated during the infection of Sclerotinia sclerotiorum, the germination of Sclerotinia mycelium and the germination of Sclerotinia cyst stalk. Biomass, biological products, and biofuel processes. In-depth studies of this pathway have revealed that a large number of heterotrophic fungi also possess enzymes associated with plant carbon fixation pathways, many of which are conserved in fungi. 10 of the alvin-benson-basham (cbb) reductive pentose phosphate pathways and 7 of the C4-dicarboxylic acid cycles, especially in the CBB cycle, only 5-diphosphate carboxylation oxygenase (rubisco) and ribonuclease phosphate kinase (prk) can not find homologous proteins in Sclerotinia. RNAi silencing tests have shown that many enzymes associated with this pathway are located in the nuclear disk. Genetic development analysis shows that many carbon fixation-related enzymes undergo events of gene replication, gene loss or acquisition, and gene functional diversity in evolution. These findings demonstrate the relationship between autotrophic and heterotrophic organisms on the carbon fixation level, indicating evolution. During the development of Sclerotinia sclerotiorum, functional modules related to carbohydrate-activated enzymes were also significantly induced. Comparative genomic analysis showed that cell wall degrading enzymes and fungal cells of dead and semi-living trophic pathogenic fungi were also significantly induced. However, transcriptional analysis of carbohydrate-degrading enzymes in Sclerotinia sclerotiorum, Fusarium graminearum, Populus Populus leaf rust and Stem rust of wheat showed that many of the enzymes encoding plant cell wall degrading enzymes were present during the infection of dead and living vegetative fungi. Both genes and genes of fungal cell wall degrading enzymes were significantly up-regulated, suggesting that there is a common mechanism associated with the degradation of plant cell wall and the recombination or modification of fungal cell wall in plant pathogenic fungi, which may be closely related to the infection of pathogenic fungi. The recombination and modification of the wall are also related to its own development. Small secretory proteins play an important role in the interactions between living, semi-living and host plants, but their roles in a wide range of dead-body trophic fungi are poorly understood. Transcription analysis shows that many secretory protein-coding genes are located in the nuclear disk. Two small cysteine-rich secretory proteins, sscvnh and ssssssvp1, which were significantly up-regulated during Sclerotinia formation and infection, were selected as examples for further study. Both of them played an important role in the pathogenesis of sclerotinia. Further studies on ssssssvp1 showed that ssssssvp1 might play an important role in the pathogenesis of sclerotinia. Ssssvp1 is mainly localized in the host cytoplasm and can induce plant cell necrosis. Yeast two-hybrid, immunoprecipitation and fluorescent bimolecular complementarity assay confirmed that ssssvp1 and plant protein qcr8 Interaction, qcr8 is a subunit of the cytochrome b-c1 complex in the plant mitochondrial respiratory chain. The results of two site-directed mutagenesis showed that the two cysteine residues (c38 and c44) could not form homologous dimers with SsSSVP1, could not interact with QCR8 and lost the ability to induce plant cell necrosis, suggesting that some cysteine residues were in the vitamin B. Fluorescence co-localization test and fluorescence bimolecular complementarity test showed that SsSSVP1 could "hijack" QCR8 into the cytoplasm before it entered the mitochondria, thereby disrupting the subcellular localization of QCR8. Virus-mediated gene silencing test in tobacco showed that QCR8 was silenced. These results suggest that SsSSVP1-induced plant cell necrosis is associated with the alteration of subcellular localization of QCR8, and the alteration of subcellular localization of QCR8 may deprive it of its biological function. This study reveals the new function of small secretory proteins in the interaction between dead vegetative fungi and their host plants, and elucidates the pathogenic mechanism of this type of interaction. Molecular mechanisms of pathogenesis and development of Sclerotinia sclerotiorum have been studied at various levels, such as protein biological functions. The results of this study show that all developmental stages of Sclerotinia sclerotiorum are the result of a network of closely related genes, but each gene in this network plays a specific role and is in a position to play. Different from each other, there are some key "node genes" that play a decisive role in specific biological processes. In this paper, the digital expression profiles of Sclerotinia and bioinformatics methods are combined to analyze the overall performance of various functional modules in the development process of Sclerotinia on the one hand, on the other hand, some key "node" bases are studied in depth. The molecular basis of various biological processes of Sclerotinia sclerotiorum has been studied from macroscopic and microscopic perspectives due to its biological functions. Through the above two ideas and strategies, the molecular mechanism of pathogenesis and development of Sclerotinia sclerotiorum has been demonstrated comprehensively, which provides a new perspective and theoretical support for the prevention and treatment of Sclerotinia sclerotiorum.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S432.44
，
本文编号：2190433
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