运用高通量组分分析技术深入探索植物细胞壁在葡萄与霜霉菌互作机制中的作用

Downy mildew is recognized as one of the most serious diseases of grapevine (Vitis Vinifera), and can severely disrupt the entire industry chain of grape and wine. The elevation of the plant natural resistance or breeding of the new cultivar with higher resistant level may reduce the use of farm chemicals effectively. Plant cell wall, as the primary barrier of plant to the outside environment, has been thought to be one of the most important factors which contributes to the resistant level. However, it is always a challenge for in-depth analysis of plant cell wall due to its complex nature and various crosslink between the polymers. This study intends to employ the recently established and validated cell wall profiling technique (Comprehensive Microarray Polymer Profiling) which combined with other classical cell wall analytical tools, to directly observe the differences on polymer level (polysaccharides, glycoprotein, etc.) between the grape cultivars with different resistance levels (newly established level system). The aims of this profiling study include the mapping of the molecular differences based on the cell wall polymers, as well as identifying the specific polymers which determine significantly the resistance level. These datasets will aid the future molecular breeding and plant defense study. This proposed study will also analyse the differences of response to the infection (downy mildew) on the cell wall level between cultivars, and use the datasets acquired as the guideline to investigate the cell wall related genes from the RNA-seq datasets, for more in-depth understanding of how cell wall get involved in grapevine-downy mildew interaction. The analysis of cell wall oligosaccharides in this infection study can also help us to validate the potential value of grape-based oligosaccharides as an elicitor for elevation of grape basic defense level.

葡萄霜霉病作为最严重的葡萄病害之一，会对葡萄的生产及之后的各类深加工产业造成严重影响，提高植物自然抗性或培育高抗性的新品种可有效降低化学农药的使用。细胞壁作为植物与外界初始屏障，被认为是影响植物抗性的重要因素之一，但其复杂的结构及分子间链接方式一直给分析带来很大的挑战。本项目将运用最新建立并验证的细胞壁多糖抗体微矩阵技术，结合其他经典的细胞壁分析技术，首次对新建立的葡萄霜霉菌抗性等级中特定葡萄品种进行直接的大分子（多糖，糖蛋白）层面的细胞壁检测，旨在建立葡萄抗霜霉菌的细胞壁分子图谱，并发现细胞壁中决定葡萄抗性的重要成分，为今后的抗病分子育种打好基础。本研究同时会对葡萄应对霜霉菌侵染的细胞壁调控进行深入探索，并以此为导向挖掘葡萄转录组中相关基因，揭示结构层面上葡萄与霜霉菌的互作机制，而以此为基础的细胞壁降解寡糖分析，也会帮助验证葡萄组织来源的寡聚糖作为应激子提高葡萄基础抗性的潜在价值。

葡萄霜霉病是葡萄生长发育中常见的病害之一，其造成的减产会给葡萄产业带来不可忽视的影响。传统的化学防治手段除了造成额外的人力物力支出，对环境与人体健康也有着潜在的影响。研究发现一些野生葡萄资源在进化过程中获得了对病害天然的抗性或免疫能力，明晰其抗病机制，对培育高抗性高品质的新品种有着重要的意义。植物细胞壁作为植物细胞防御外界胁迫的第一道屏障，被认为在植物抗病中扮演着基本支撑功能之外更多的角色，然而其组分与结构的复杂性一直是前期研究的瓶颈。本课题运用最前沿的高通量细胞壁多糖抗体技术，首次从细胞壁多糖层面探索葡萄的抗病机制。实验首先建立了葡萄各组织的细胞壁多糖图谱，明晰了各组织间细胞壁成分的差异，并着重分析了病菌主要侵入区域的葡萄果肉与叶片组织的深层结构。之后实验比较分析了双红、感病品种赤霞珠及其杂交种（中等抗性）叶片细胞壁的病菌侵染前状态，发现抗性品种中高甲酯化半乳糖醛酸聚糖、阿拉伯聚糖、鼠李糖半乳糖醛酸聚糖的含量与抗病性呈正相关。随后的实验发现霜霉菌侵染24小时后，感病品种赤霞珠的果胶结构失去其原有的紧密型而变得疏松，对其叶片的寡聚糖检测也证实了果胶的裂解，而抗病性强的双红叶片细胞壁则基本未受病菌的影响，其细胞壁组分与结构在侵染48小时后表现出更好的完整度。综上所述，实验发现霜霉菌在早期的侵染主要以降解果胶为主要手段，叶片细胞壁中果胶甲酯链、阿拉伯聚糖等支链在一定程度上提高了果胶结构、乃至细胞壁结构的紧致性，从而提高了霜霉菌降解果胶的难度。