【作者】 张明哲；

【导师】 秦庆明；

【作者基本信息】 吉林大学 ， 植物学， 2020， 博士

【摘要】 灰葡萄孢菌（Botrytis cinerea）引起的植物灰霉病属于世界性病害,是露地和保护地作物常见且较难防治的一种真菌性病害。灰葡萄孢菌是典型的死体营养型病原真菌,可侵染1400多种不同的寄主植物,包括几乎所有的蔬菜及果树。寄主植株从苗期到挂果期、运输、储存期均可发病,造成巨大的经济损失,特别是在现代设施农业的快速发展阶段,为灰霉病的发生提供了适宜的环境条件,灰霉病的危害呈逐年提高的趋势。正因为它寄主广泛、危害巨大,灰葡萄孢菌名列分子植物病理学中十大重要真菌病原物的第二位。目前对灰葡萄孢菌的防治还没有十分有效的方法,传统的解决方式是使用大量的农药,不仅涉及食品安全,而且还造成环境污染,严重危害人们的身体健康。因此,亟需深入研究灰葡萄孢菌的致病机制,以便寻找到有效和环境友好的灰霉病防治方法。本研究同时对寄主植物番茄（Solanum lycopersicum）和灰葡萄孢菌进行转录组（RNA-seq）分析,并对部分差异表达基因（DEG）进行功能验证,便于我们能够更好地了解其相互作用的机制。分析灰葡萄孢菌侵染番茄叶片24小时后的混合转录组,此时是病原菌在叶表皮中渗透和发育的关键时间点,灰葡萄孢菌已经侵入但尚未出现大的坏死病斑。分析结果确定了涉及番茄和灰葡萄孢菌的复杂基因网络。编码效应子、次级代谢产物生物合成酶、激素和活性氧（ROS）产生以及自噬相关蛋白的真菌转录物的表达水平上调,表明这些基因可能参与了灰葡萄孢菌最初的侵染过程。与此同时,参与植物防御反的基因,如抗毒素、胁迫反应的基因、ATP结合盒转运蛋白[ATP-binding cassette（ABC）transporter proteins]、病程相关蛋白[pathogenesis‐related（PR）proteins]和WRKY DNA结合转录因子均被上调。作者通过基因敲除和致病性分析功能研究了几种灰葡萄孢菌DEG,对部分表达量上升基因进行了致病力、分生孢子形态发育和萌发、侵染相关发育、对逆境的响应、活性氧（ROS）等相关实验。证明了BcCGF1是一种新型的致病力相关因子,可通过调节分生孢子萌发、菌丝发育、侵染结构形成、宿主穿透和胁迫适应来介导真菌的侵染相关发育和致病力。研究表明BcCGF1基因通过控制灰葡萄孢菌ROS的产生调控包括分生孢子萌发、侵染结构形成等与侵染相关的发育,外源性cAMP可恢复突变体侵染相关发育的不足。作者的发现为阐明病原物攻击和寄主防御的策略提供了新的见解。对BcCGF1在介导真菌致病机理中的系统研究可能为真菌病害的控制提供新的靶标。根据数据分析结果,还分别选择了两个自噬的相关基因BcSNF1和BcATG1、两个调控次生代谢产物相关基因BcLAE1和BcVEL1、6个细胞壁降解酶相关基因β葡萄糖苷酶（β-glucosidases,BcBGS）,编码葡萄糖-甲醇-胆碱（GMC）氧化还原酶基因BCIN₀3g01540作为研究对象。分别敲除了BcCGF1、BcSNF1、BcATG1、BcLAE1、BcVEL1、6个β-glucosidases（BcBGS1-6）、BCIN₀3g01540,获得了敲除突变体并进行了相关功能研究。6个β-glucosidases（BcBGS1-6）缺失突变体的致病力与野生型菌株无显著性差异;BcSNF1、BcATG1、BcLAE1、BcVEL1、BCIN₀3g01540的缺失突变体致病力显著低于野生型菌株。此外,作者优化了农杆菌介导灰葡萄孢菌分生孢子的遗传转化体系,显著提高了ATMT（Agrobacterium tumefaciens-mediated transformation）效率。基于该优化方法,本研究团队构建了约含5万个T-DNA随机插入的突变体库,利用功能基因组学方法从全基因组范围筛选及鉴定灰葡萄孢菌功能性致病相关因子,并阐明其中多个新发现的致病相关因子在灰葡萄孢菌侵染过程中的作用及其分子机理。在本研究中,作者通过TAIL-PCR（Thermal asymmetric interlaced-PCR）的方法获得其T-DNA所插入基因的上下游侧翼序列,鉴定被插入失活的致病相关基因,并根据PCR的方法获取该基因全长的核苷酸序列。作者找到了一个乙醇脱氢酶基因,该基因在转录组分析中表达量是显著上调的。概言之,本项工作为我们进一步系统分析灰葡萄孢菌-寄主相互作用的分子机制及深入阐析灰葡萄孢菌新的致病机理奠定了基础。更多还原

【Abstract】 The necrotrophic phytopathogenic fungus Botrytis cinerea causes gray mold on over 1400 plant species.Gray mold is a worldwide fungal disease that is difficult to control.The pathogen infects almost all vegetables and fruit crops including numerous economically important crops such as grapevine,strawberry,raspberry,ornamental flowers and annually causes enormous economic losses worldwide.The pathogen attacks many plant organs,including stems,fruits,leaves and flowers,at both pre-and post-harvest stages.The huge economic losses,especially in the rapid development stage of modern facility agriculture that provides suitable environmental conditions for the occurrence of gray mold,are increasing year by year.Due to economic and scientific importance,B.cinerea is considered as an important pathogen for studying pathogenesis of necrotrophic fungal pathogens,which ranks second in the top ten important fungal pathogens in molecular plant pathology.At present,there is no effective method for the control of gray mold.The traditional solution is to use a large amount of pesticides,which causes not only a big concern for food safety,but also environmental pollution.Therefore,an in-depth study of the molecular pathogenesis of B.cinerea is helpful to find an effective and environmentally friendly strategy for controlling the disease.Transcriptome analyses of both host plants and pathogens,and functional validation of the identified differentially expressed genes（DEGs）allow us to better understand the mechanisms underlying their interactions.Here,we analyse the mixed transcriptome derived from B cinerea infected tomato（Solanum lycopersicum）leaves at 24 hr post inoculation,a critical time point at which the pathogen has penetrated and developed in leaf epidermis,whereas necrotic symptoms have not yet appeared.Our analyses identified a complex network of genes involved in tomato–B.cinerea interaction.The expression of fungal transcripts encoding candidate effectors,enzymes for secondary metabolite biosynthesis,hormone and reactive oxygen species（ROS）production,and autophagy-related proteins were up-regulated,suggesting that these genes may be involved in the initial infection processes.Moreover,tomato genes involved in phytoalexin production,stress responses,ATP-binding cassette（ABC）transporters,pathogenesis-related proteins,and WRKY DNA-binding transcription factors were up-regulated.We functionally investigated several B.cinerea DEGs via gene replacement and pathogenicity assays.Our results demonstrated that BcCGF1 was a novel virulence-associated factor that mediates fungal development and virulence via regulation of conidial germination,conidiation,infection structure formation,host penetration,and stress adaptation.The fungal infection-related development was controlled by BcCGF-mediated ROS production and exogenous cAMP restored the mutant infection-related development.Our findings provide new insights into the elucidation of the simultaneous tactics of pathogen attack and host defence.Our systematic elucidation of BcCGF1 in mediating fungal pathogenesis may open up new targets for fungal disease control.In addition,genes regulating autophagy,secondary metabolites,cell wall degrading enzymes,and hypoxia that may affect the pathogenicity of B.cinerea were chosen for further functional verification.These genes included two autophagy-related genes（BcATG1 and BcSNF1）,two genes that regulate secondary metabolites（BcLAE1,BcVEL1）,and six cell wall-degrading enzyme-related genes β-glucosidases（BcBGS1 to BcBGS6）,BCIN₀3g01540,a gene encoding a glucose-methanol-choline（GMC）oxidoreductase that may be involved in B.cinerea response to hypoxic environment.We generated the gene knockedout mutants of BcSNF1,BcATG1,BcLAE1,BcVEL1,BcBGS1 to BcBGS6,and BCIN₀3g01540 and performed related functional studies.Our findings indicated that BcSNF1,BcATG1,BcLAE1,BcVEL,BcCGF1 regulate the pathogenicity of B.cinereaWe also optimized the Agrobacterium tumefaciens-mediated transformation（ATMT）in B.cinerea.Compared to the previus ATMT in B.cinerea,the efficiency of our optimized transformation improves about 8-10 folds.We generated a B.cinerea T-DNA insertion mutant library containing about 50,000 transformants and systematically dissected molecular mechanisms of pathogeneses of some novel B.cinerea factors identified from the library.We obtained the upstream and downstream flanking sequences of a T-DNA inserted gene that encodes an alcohol dehydrogenase from a pathogenicity-attenuated mutant strain identified from our library by using the approach of thermal asymmetric interlaced polymerase chain reaction（TAIL-PCR）.Interestingly,the gene was also identified from our list of DEGs and displayed a significant up-regulation during interaction between B cinerea and tomato.Taken together,this work has laid a foundation for our further systematic dissection of molecular mechanisms underlying B.cinerea-host interactions.更多还原