Plant Calcium Conducting Channels: Linking Molecular Architecture to Roles in Innate Immunity Signal Transduction

知识价值。由美国国家科学基金会资助的伯科维茨博士最近进行的一项研究发现了第一个编码功能性质膜钙传导离子通道的植物基因。该基因产物是一个环核苷酸（cNMP）门控的非选择性阳离子通道（CNGC），在植物先天免疫/病原体反应信号级联反应中促进下游一氧化氮（NO）的产生。本项目利用对CNGC功能和调控相关分子结构的理解来阐明该信号系统的进一步步骤。Berkowitz博士将研究以下信号传导步骤：(a)通过CNGC向内传导Ca的上游（即将病原体感知与通道激活联系起来），(b)将Ca传导到细胞质与NO生成联系起来，(c)导致细胞质次级信使对通道的调节，以及(d)涉及Ca通道与植物对无毒病原体的超敏反应（HR）。研究方法包括利用野生型（WT）和CNGC功能缺失突变体分离的原生质体进行膜片钳实验。这项工作旨在描述在病原体信号级联过程中调节CNGC功能的可能调控系统，如cNMP浓度、钙调素（CaM）、腺苷酸和鸟苷酸环化酶、病原体相关分子模式（PAMP）激发子，如脂多糖（LPS）、G蛋白和cNMP磷酸二酯酶。此外，CNGC激活剂/调节剂对植物Ca摄取和NO生成的影响将被确定，以阐明植物先天免疫/病原体反应信号级联的步骤。次生信号分子cAMP和cGMP的水平将在植物发生HR时被监测。工作将在整个工厂层面进行，以测试几个相关的假设。Berkowitz博士之前的工作表明，通过CNGCs向内的Ca通量介导了植物对无毒病原体丁香假单胞菌的HR反应，这是由于下游通过CaM/Ca激活一氧化氮合酶产生NO。这一假设也将得到验证。更广泛的影响。作为该项目的一部分，Berkowitz博士将继续在分子遗传学领域开发一项创新的高中教师培训计划。该计划旨在为高中生物教师提供课程计划、实验练习以及所有必要的信息和支持，以创新的体验式学习计划来增加他们目前的生物学课程。这门高中分子遗传学课程的体验方面是学生参与一系列的研究，将基因序列与蛋白质结构、蛋白质结构与细胞水平上的功能、蛋白质在整个植物表型、生长和发育中的作用联系起来。该项目包括一套（由Berkowitz博士开发的）ppt演示、蛋白质建模软件、高中实验课程计划、教师指南、80页的分子遗传学实验手册和相关网络资源的注释目录。教师培训计划和高中实验课程将由PI进一步开发并发布在URL （www.biologyteacher.uconn.edu）上，以便所有这些教学资源将提供给广泛的高中生物教师。

Intellectual merit. NSF-funded work recently undertaken by Dr. Berkowitz identified the first plant gene encoding a functional plasma membrane calcium-conducting ion channel. This gene product, a cyclic nucleotide (cNMP) gated nonselective cation channel (CNGC), facilitates downstream nitric oxide (NO) generation in plant innate immunity/pathogen response signaling cascades. This project takes advantage of understanding of the molecular architecture related to CNGC function and regulation to elucidate further steps in this signaling system. Dr. Berkowitz will investigate the signaling steps (a) upstream from inward Ca conduction by this CNGC (i.e. linking pathogen perception to channel activation), (b) linking Ca conduction into the cytosol to NO generation, (c) resulting in regulation of the channel by cytosolic secondary messengers, and (d) that involve the Ca channel with the hypersensitive response (HR) of plants to avirulent pathogens. The research approach includes patch clamp experiments with protoplasts isolated from wild type (WT) and CNGC loss-of-function mutant plants. The work aims to characterize possible regulatory systems that modulate CNGC function during pathogen signaling cascades such as cNMP concentration, calmodulin (CaM), adenylate and guanylate cyclase, pathogen associated molecular pattern (PAMP) elicitors such as lipopolysaccharide (LPS), G proteins, and cNMP phosphodiesterases. In addition, effect of CNGC activators/modulators on Ca uptake and NO generation in plants will be ascertained to elucidate steps in the plant innate immunity/pathogen response signaling cascades. The level of the secondary signaling molecules cAMP and cGMP will be monitored in plants undergoing HR. Work will be undertaken at the whole plant level to test several related hypotheses. Dr. Berkowitz's prior work suggests that inward Ca flux through CNGCs mediates HR response of plants to the avirulent pathogen Pseudomonas syringae due to downstream NO generation via CaM/Ca activation of nitric oxide synthase. This hypothesis will also be tested. Broader impacts. As part of this project, Dr. Berkowitz will continue development of an innovative high school teacher-training program in the area of molecular genetics. The program aims to provide high school biology teachers with lesson plans, laboratory exercises, and all the information and support necessary to augment their current biology course curricula in genetics with an innovative experiential learning program. The experiential aspect of this high school curriculum in molecular genetics is that students participate in a series of investigations that link gene sequence to protein structure, protein structure to function at the cell level, and role of the protein in whole plant phenotype, growth and development. This program includes a package (developed by Dr. Berkowitz) of PowerPoint presentations, protein modeling software, high school lab lesson plans, teacher's guides, an 80-page molecular genetics lab manual, and an annotated catalog of relevant web resources. The teacher-training program and high school lab curriculum will be further developed and posted on a URL (www.biologyteacher.uconn.edu) by the PI so that all of these teaching resources will be made available to a wide audience of high school biology teachers.