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和交流，所有细胞都被细胞外基质所包围，细胞外基质提供了这些能力。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。几种细胞表面蛋白作为细胞壁状态的传感器或受体，监测其完整性和组成。细胞壁相关激酶（Wall Associated kinase, WAKs）是细胞表面受体，与细胞壁上的果胶结合，调节细胞的扩大。WAKs也被病原体、机械应力和正常发育变化产生的碎片果胶激活。在这个项目中进行的实验将开始帮助理解WAKs如何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。这项工作将在一所小型文理学院进行，重点是本科教育和研究。通过本项目的工作，学生们在实验科学方面得到了全面的训练，并且很大一部分学生将继续进入研究生和专业学校学习。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所在机构的核心细胞生物学和遗传学实验室课程，并参与了为准备不足的学生开设的科学课程。被子植物的细胞壁由纤维素、半纤维素和果胶的复杂排列组成。果胶可以被选择性地和局部地修饰成交联的结构网络，这对细胞的扩大有显著的影响。植物分泌的聚半乳糖醛酸酶的局部消化也可以通过放松细胞壁来改变果胶网络，从而使细胞定向膨胀。许多病原体和机械破坏破坏果胶，往往导致植物的应激反应。该项目涉及壁相关激酶（WAKs），它结合到交联果胶的长聚合物和病原体和损伤诱导的果胶片段，或低聚半乳糖醛酸酯（OGs）。与天然聚合物结合的WAKs是正常细胞扩增所必需的，但与新生成的OGs结合的亲和力更高，随后激活一个独特的应激反应途径。本项目将对拟南芥OG处理后磷酸化的激酶、细胞质激酶和潜在支架蛋白等受体进行表征。利用一种新的基于CRISPR/Cas9的WAK缺失突变体，研究拟南芥中聚集在一个30 kb位点上的5个单独的WAK亚型的作用。令人困惑的是，在发育过程和对环境干扰的反应中，一种受体如何区分不同类型的果胶，并以不同的方式做出反应。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，并与不同的共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.