比較基因組、轉錄組和基因功能研究揭示核盤菌致病和發(fā)育的分子機理
發(fā)布時間：2018-08-18 19:20
【摘要】：核盤菌(Sclerotinia sclerotiorum)是一種寄主范圍廣泛、世界性分布的典型死體營養(yǎng)型植物病原真菌,能夠在多種經濟作物(例如油菜、向日葵、大豆和扁豆等)上引起菌核病,給農作物生產造成巨大的經濟損失。為了從基因組水平上研究核盤菌的發(fā)育特性,對核盤菌及已測序真菌的基因組進行了功能注釋和功能基因組比較分析。發(fā)現(xiàn)核盤菌基因組中與轉座元件相關的類似CENP-B的DDE超家族核酸內切酶和反轉錄酶要遠遠多于其它真菌,這種顯著的差異暗示了轉座事件在核盤菌基因組的進化中起到了重要作用。進一步詳細分析了核盤菌中的轉座元件的種類和它們與類似CENP-B的DDE超家族核酸內切酶基因和反轉錄酶基因在基因組中的相對位置關系,表明這些酶家族基因的周圍確實存在典型的轉座元件,且絕大多數(shù)類似于CENP-B的DDE超家族核酸內切酶基因都與DNA轉座元件相關,其中TcMar-Fot1類群最多;所有反轉錄酶基因都與反轉錄轉座元件相關,其中LINE/Tad1,LTR/Gypsy和LTR/Copia三個類群占比最多。對核盤菌及其它真菌中轉座元件的種類和數(shù)量的比較發(fā)現(xiàn),DNA和RNA轉座元件均在核盤菌基因組的進化過程中起著重要作用。系統(tǒng)發(fā)育分析揭示類似CENP-B的DDE超家族核酸內切酶和反轉錄酶基因家族的擴張可能是核盤菌中相應轉座元件在進化過程中短時間內大量復制擴增的結果。轉錄分析表明盡管這兩大類轉座子中的大部分已經“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。利用數(shù)字表達譜檢測了核盤菌在6個關鍵發(fā)育階段(包括菌絲營養(yǎng)生長、侵染、菌核形成、菌核菌絲萌發(fā)、菌核子囊柄萌發(fā)和子囊柄形成階段)的轉錄組。為了從整體上闡述核盤菌不同功能模塊的活性在各個發(fā)育階段的動態(tài)變化,建立一種“功能譜”的分析方法來衡量各種功能模塊(例如,GO、Interpro功能模塊和KEGG代謝途徑)的相對活性。功能譜分析可以將基因表達水平和基因功能注釋結合起來描繪相應功能模塊的相對活性。通過比較“功能譜”,明確了各個功能模塊在核盤菌不同發(fā)育階段相對活性的動態(tài)變化。此外,基因功能富集分析顯示了核盤菌在不同發(fā)育階段相對菌絲營養(yǎng)生長階段所富集到的多個功能模塊。功能譜分析結果顯示一條不完整的碳固定相關通路在核盤菌侵染、菌核菌絲萌發(fā)和菌核子囊柄萌發(fā)過程中被顯著地激活。光合作用過程中植物的碳固定途徑是將太陽能轉化為生物質、生物產品和生物燃料的過程。對這條通路進行深入研究發(fā)現(xiàn)大量異養(yǎng)型真菌也擁有和植物碳固定途徑相關的各種酶類,其中的許多酶在真菌中都是保守的。在異養(yǎng)型的核盤菌中存在17個植物碳固定途徑相關酶類的同源蛋白,在calvin-benson-basham(cbb)還原型磷酸戊糖途徑中有10個,在c4-二羧酸循環(huán)中有7個。尤其是在cbb循環(huán)中,只有5-二磷酸羧化加氧酶(rubisco)和磷酸核酮糖激酶(prk)在核盤菌中找不到同源蛋白。rnai沉默試驗證明許多和這條途徑相關的酶類都在核盤菌的侵染和菌核形成過程中發(fā)揮重要作用。遺傳發(fā)育分析表明許多碳固定相關酶類在進化中都經歷了基因復制、基因丟失或獲得和基因功能多樣化事件。這些發(fā)現(xiàn)在碳固定層面展示了自養(yǎng)型生物和異養(yǎng)型生物之間的聯(lián)系,表明在進化過程中碳固定相關基因的功能在不同物種中是動態(tài)變化的。在核盤菌發(fā)育過程中,碳水化合物活性酶類相關的功能模塊也受到了顯著地誘導。比較基因組分析顯示死體營養(yǎng)型和半活體營養(yǎng)型病原真菌的植物細胞壁降解酶和真菌細胞壁降解酶的數(shù)量都要比大多數(shù)活體營養(yǎng)型病原真菌中的多。然而,對核盤菌、禾谷鐮刀菌、青楊葉銹菌和麥類桿銹菌中的碳水化合物活性酶類的轉錄分析表明在死體營養(yǎng)型和活體營養(yǎng)型真菌的侵染過程中,許多編碼植物細胞壁降解酶類的基因和真菌細胞壁降解酶類的基因都顯著上調表達,表明植物病原真菌中存在一種植物細胞壁的降解和真菌細胞壁的重組或修飾相伴隨的普遍機制,該機制可能與病原真菌的侵染密切相關。此外,本研究結果表明植物病原真菌細胞壁的重組和修飾還與其自身發(fā)育相關。小分泌蛋白在活體、半活體營養(yǎng)型真菌和其寄主植物的互作中發(fā)揮了重要作用,然而對它們在寄主范圍廣泛的死體營養(yǎng)型真菌中的作用還知之甚少。轉錄組分析顯示許多分泌蛋白編碼基因在核盤菌菌核形成和侵染過程中顯著上調表達。選取兩個在核盤菌侵染過程中顯著上調表達的富含半胱氨酸的小分泌蛋白,即sscvnh和ssssvp1為例進行深入研究,發(fā)現(xiàn)它們均在核盤菌的致病過程中發(fā)揮重要作用。對ssssvp1的進一步研究表明,ssssvp1從菌絲中被分泌出來后可被植物細胞內化并且可在細胞間自主轉運,這種轉運不依賴于核盤菌本身。ssssvp1主要定位于寄主的細胞質并可誘導植物細胞壞死。酵母雙雜交、免疫共沉淀和熒光雙分子互補試驗均證實ssssvp1與植物中蛋白qcr8互作,qcr8是植物線粒體呼吸鏈上細胞色素b-c1復合體中的一個亞基。雙定點突變結果表明兩個半胱氨酸殘基(c38和c44)同時突變使SsSSVP1不能形成同源二聚體,不能和QCR8互作并失去了誘導植物細胞壞死的能力,說明部分半胱氨酸殘基在維持SsSSVP1的結構和功能過程中發(fā)揮了重要作用。熒光共定位試驗和熒光雙分子互補試驗顯示SsSSVP1可以在QCR8進入線粒體之前將其“劫持”到細胞質中從而擾亂了QCR8的亞細胞定位。在煙草中進行的病毒介導的基因沉默試驗表明沉默QCR8導致植株發(fā)育異常且引起植物細胞的壞死反應,提示SsSSVP1誘導的植物細胞壞死與SsSSVP1-QCR8之間的互作導致QCR8亞細胞定位的改變相關,QCR8亞細胞定位的改變可能使其喪失了生物學功能。這些結果表明小分泌蛋白在寄主非特異性死體營養(yǎng)型真菌中也作為潛在的效應因子發(fā)揮著重要作用。本研究揭示了小分泌蛋白在寄主范圍廣泛的死體營養(yǎng)型真菌和其寄主植物互作過程中的新功能,闡明這種互作類型的致病機制具有重要意義。本文從基因組、轉錄組和單個小分泌蛋白生物學功能等多個水平研究了核盤菌致病和發(fā)育的分子機制。本研究的結果表明核盤菌所有發(fā)育階段都是多個功能上密切聯(lián)系的基因組成一個網絡協(xié)同起作用的結果,但是這個網絡中每個基因所發(fā)揮的具體作用和所處的地位并不相同,存在某些關鍵的“節(jié)點基因”對特定的生物學過程起著決定性作用。本文結合核盤菌的數(shù)字表達譜和生物信息學方法一方面全面分析了核盤菌發(fā)育過程中各種功能模塊的整體表現(xiàn),另一方面深入研究了某些關鍵“節(jié)點”基因的生物學功能,分別從宏觀和微觀的角度研究了核盤菌各種生物學過程的分子基礎。通過以上兩種思路和策略,本研究全方位綜合展示了核盤菌致病和發(fā)育的分子機理,為菌核病的防治提供了新的視野和理論支撐。
[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.
【學位授予單位】：華中農業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S432.44
，
本文編號：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.
【學位授予單位】：華中農業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S432.44
，
本文編號：2190433
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