RUI: Cell Wall Associated Kinases As Pectin Receptors

所有复杂的生物体都是由细胞组成的，这些细胞必须相互结合并相互通信，所有细胞都被提供其中一些能力的细胞外基质所包围。在植物中，细胞外基质被称为细胞壁，部分由碳水化合物纤维素和果胶组成。几种细胞表面蛋白充当细胞壁状态的传感器或受体，监测细胞壁的完整性和组成。壁相关蛋白(WAKs)是一种细胞表面受体，与细胞壁中的果胶结合，调节细胞的扩大。WAKS还被病原体、机械压力以及正常发育变化产生的碎裂果胶激活。将在该项目期间进行的实验将有助于理解WAKS可以区分和响应细胞壁中不同类型的果胶的机制，从而了解植物如何感知它们细胞壁的状态，这对它们的生长和发育至关重要。这项工作将在一所专注于本科教育和研究的小型文理学院进行。通过这个项目的工作，学生们接受了全面的实验科学培训，其中很大一部分学生继续读研究生和专业学校。PI还将他实验室的植物细胞生物学和遗传学的许多方面纳入了科学家研究所的核心细胞生物学和遗传学实验室课程，并参与了为科学知识准备不足的学生开设的课程。被子植物的细胞壁由纤维素、半纤维素和果胶组成的复杂排列。果胶可以被选择性地局部修饰，使其交联成一个结构网络结构，对细胞扩大有显著影响。植物分泌的聚半乳糖醛酸酶的局部消化也可以通过松动细胞壁来改变果胶网络，从而允许细胞的定向扩张。许多病原体和机械破坏使果胶碎裂，常常导致植物的逆境反应。该项目涉及壁相关激酶(WAKS)，它既能与交联果胶的长聚合物结合，又能与病原体和损伤诱导的果胶片段或寡聚半乳糖醛酸脂(OGs)结合。与天然聚合物结合的WAK是正常细胞扩张所必需的，但它以更高的亲和力结合新生成的OG，随后激活不同的应激反应途径。这个项目将表征在OG处理拟南芥时被磷酸化的类似受体的蛋白、细胞质蛋白和潜在的支架蛋白。在拟南芥中，5个单独的WAK亚型中的每一个都聚集在一个30kb的基因座上，其作用将通过一个新的基于CRISPR/Cas9的WAK缺失突变体进行遗传剖析。令人困惑的是，在发育过程中，一种受体类型如何以不同的方式区分和响应不同类型的果胶，以及对环境干扰的反应。我们的目标是了解这是否通过不同的WAK来实现，每个WAK结合不同的果胶，并与不同的辅助受体和配体结合来激活替代途径。

All complex organisms are composed of cells that must bind to and communicate with each other, and all cells are surrounded by an extra-cellular matrix that provides some of these abilities. In plants, the extracellular matrix is termed the cell wall, and is partially composed of the carbohydrates cellulose and pectin. Several cell surface proteins act as sensors or receptors for the state of the cell wall, monitoring its integrity and composition. The Wall Associated Kinases (WAKs) are cell surface receptors that bind to the pectin in the cell wall, and regulate cell enlargement. WAKs are also activated by fragmented pectins generated by pathogens, mechanical stress and perhaps by normal developmental changes. The experiments that will be conducted during this project will begin to help in understanding the mechanism by which WAKs can distinguish and respond to different types of pectin in the cell wall, and thereby how plants sense the state of their cell wall, essential for their growth and development. This work will be carried out at a small liberal arts college where the focus is on undergraduate education and research. Students receive a thorough training in experimental science through work in this project, and a significant portion go on to graduate and professional school. The PI also incorporates many aspects of plant cell biology and genetics from his lab into the core cell biology and genetics lab course at the scientist's institution, and is involved in courses for underprepared students in the sciences.The cell walls of angiosperms are composed of a complex arrangement of cellulose, hemicellulose and pectin. The pectins can be selectively and locally modified to be cross-linked into a structural network that can have dramatic effects on cell enlargement. Localized digestion by plant secreted polygalacturonases can also modify the pectin network by loosening the cell wall to permit directional expansion of cells. Numerous pathogens and mechanical disruptions fragment pectin, often leading to a plant stress response. This project involves the Wall Associated Kinases (WAKs) that bind to both long polymers of cross-linked pectin and to pathogen and damage induced pectin fragments, or oligo-galacturonides (OGs). WAKs bound to native polymers are required for normal cell expansion, but bind newly generated OGs with higher affinity, and subsequently activate a distinct stress response pathway. This project will characterize receptor like kinases, cytoplasmic kinases, and potential scaffolding proteins that are phosphorylated upon OG treatment of Arabidopsis. The role of each of the 5 individual WAK isoforms, clustered in one 30 kb locus in Arabidopsis will be genetically dissected using a new CRISPR/Cas9 based WAK deletion mutant. The puzzle is how one receptor type can distinguish and respond in different ways to different types of pectin, during development and the response to environmental disturbance. The goal is to understand if this is achieved through different WAKs each binding different pectins, and in combination with different co-receptors and ligands to activate alternate pathways.