微管蛋白介导的对座腔菌属顶枯病的葡萄内源免疫早期信号转导研究

Plant innate immunity is comprised of two layers: Pathogen associated molecular pattern triggered immunity (PTI) and Effecter triggered immunity (ETI). Based on our previous research on grapevine cytoskeleton mediated innate immunity, microtubule, as one of the early factors responding in early plant cell defence, is highly involved. In addition to their well-known roles as the executor of down-stream signaling and rigid scaffold, the microtubules can sense the very early signals during pathogen infection as a ‘detector’. Grapevine Trunk Disease is a fungal disease which leads to serious disaster for vine and wine. In this study, V. rupestris expressing GFP-AtTUB6 suspension cells will be investigated in vivo and be observed for their array in Nano-level. Upon pathogen infection, the grapevine RNA-sequencing and comparison will be performed to mine the candidate microtubule associated genes. The mutant cell lines will be constructed. This will facilitate the studies of microtubule associated genes involved in microtubule signaling and array in PTI, as well as cell cycle regulation during programmed cell death in ETI. To elucidate the molecular and cell regulation mechanisms during Botryosphaeriaceae infection will shed light to functional analysis of cytoskeleton signalling in the early innate immunity in grapevine-fungus interactions, Plant innate immunity is comprised of two layers: Pathogen associated molecular pattern triggered immunity (PTI) and Effecter triggered immunity (ETI). Based on our previous research on grapevine cytoskeleton mediated innate immunity, microtubule, as one of the early factors responding in early plant cell defence, is highly involved. In addition to their well-known roles as the executor of down-stream signaling and rigid scaffold, the microtubules can sense the very early signals during pathogen infection as a ‘detector’. Grapevine Trunk Disease is a fungal disease which leads to serious disaster for vine and wine. In this study, V. rupestris expressing GFP-AtTUB6 suspension cells will be investigated in vivo and be observed for their array in Nano-level. Upon pathogen infection, the grapevine RNA-sequencing and comparison will be performed to mine the candidate microtubule associated genes. The mutant cell lines will be constructed. This will facilitate the studies of microtubule associated genes involved in microtubule signaling and array in PTI, as well as cell cycle regulation during programmed cell death in ETI. To elucidate the molecular and cell regulation mechanisms during Botryosphaeriaceae infection will shed light to functional analysis of cytoskeleton signalling in the early innate immunity in grapevine-fungus interactions, to enhance the disease resistance in vine and wine industries.

植物内源免疫由两个层面组成：病原相关分子模式诱发的免疫（PTI）和效应因子诱发的免疫（ETI）。基于我们在葡萄细胞骨架介导的内源免疫的前期工作发现，作为植物抗性的早期细胞因子，微管蛋白有高度的相关性，在内源免疫两个层面除了执行信号分子的下游响应和骨架支持作用，还可以作为感知元件在病原入侵初期做出反应。葡萄树干病害是对葡萄生产造成严重影响的真菌为主的病害。本研究将对葡萄微管蛋白绿色荧光蛋白标记细胞系进行活体实时示踪、在纳米级水平观察细胞骨架的精细结构。通过对病原侵染前后葡萄转录组测序和比对，确定目的基因、构建突变体细胞系，研究葡萄座腔菌科真菌胁迫初期微管蛋白相关基因在PTI层面介导的信号传递、自身结构重排及ETI层面引起细胞程序化死亡过程中对细胞周期的调控作用。对阐释葡萄树干真菌病害提供细胞骨架介导的早期内源免疫领域的葡萄属植物与真菌互作的理论依据，增强生产中葡萄抗病功能。

植物内源免疫由两个层面组成：病原相关分子模式诱发的免疫（PTI）和效应因子诱发的免疫（ETI）。基于我们在葡萄细胞骨架介导的内源免疫的前期工作发现，作为植物抗性的早期细胞因子，微管蛋白（MT）有高度的相关性，在内源免疫两个层面除了执行信号分子的下游响应和骨架支持作用，还可以作为感知元件在病原入侵初期做出反应。葡萄树干病害是对葡萄生产造成严重影响的真菌为主的病害。本研究通过将对葡萄MT微管蛋白绿色荧光蛋白标记细胞系进行活体实时示踪、在纳米级水平观察细胞骨架的精细结构。通过对病原侵染前后葡萄转录组测序和比对，确定目的基因、构建突变体细胞系，研究葡萄座腔菌科真菌胁迫初期微管蛋白MT相关基因在PTI层面介导的信号传递、自身结构重排及ETI层面引起细胞程序化死亡过程中对细胞周期的调控作用。.从感染座腔菌属顶枯病的葡萄枝条中，采用生物学特性观察以及rDNA-ITS测序，分离鉴定出6种隶属5个属的病原真菌。获得感病欧洲葡萄蛇龙珠MT标记系。对抗病美洲沙地葡萄MT标记系使用N. parvum、D. seriata及B. dothidea胞外分泌物分别处理24 h和3 d，进行转录组测序分析，分别得到1365-5605个差异表达基因（其中，转录因子50余个，分布于20个家族）。它们参与类黄酮生物合成、植物-病原互作和激素信号转导等通路。非靶代谢组学结果显示有121-142种差异代谢物，其中苯丙烷和聚酮类、脂质和类脂分子在病原菌胁迫下上调。对病原真菌诱导的抗病细胞系MT进行显微示踪，结合细胞形态学和微管排布稳定性指标，将三种病原真菌受MT调控的致病性的响应力划分为N.parvum>B. dothidea>D. seriata。MT相关基因VrKIN-5C，在生物与非生物胁迫下表现出相反的结果，暗示其在生物/非生物胁迫中将信号进行分配、传递到不同下游信号通路。该基因定位于细胞核中，与纺锤体关键成员MDA2相互作用。在绿色荧光蛋白MT标记系中，插入带有红色荧光标记的VrKIN-5C构建过表达细胞系，观察到明显抗MT解聚剂效应。在N. parvum Bt-67侵染下，丙二醛含量在VrKIN-5C过表达系中升高，暗示其在脂膜过氧化过程中的正向调控，使其解聚延迟起始时刻推迟2倍以上。本研究结果对阐释葡萄树干真菌病害提供细胞骨架介导的早期内源免疫领域的葡萄属植物与真菌互作的理论依据。

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