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

智力上的优点。美国国家科学基金会资助的伯科维茨博士最近开展的工作确定了第一个编码功能性质膜钙传导离子通道的植物基因。该基因产物是环核苷酸(CNMP)门控的非选择性阳离子通道(CNGC)，在植物天然免疫/病原菌响应信号转导通路中促进下游一氧化氮(NO)的产生。本项目利用与CNGC功能和调控相关的分子结构的理解来阐明该信号系统中的进一步步骤。Berkowitz博士将研究该CNGC从向内钙传导的上游的信号步骤(即将病原体的感知与通道激活联系起来)，(B)将钙传导到胞浆中与NO的产生联系起来，(C)导致胞质二级信使对该通道的调节，以及(D)涉及钙通道与植物对无毒病原体的超敏反应(HR)。研究方法包括用从野生型(WT)和CNGC功能缺失突变植物中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转导过程中调节CNGC功能的可能调控系统，如cNMP浓度、钙调蛋白(CaM)、腺苷和鸟苷环化酶、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，还将确定CNGC激活剂/调节剂对植物钙吸收和NO生成的影响，以阐明植物先天免疫/病原体响应信号级联的步骤。在经历HR的植物中，二级信号分子cAMP和cGMP的水平将受到监测。将在整个工厂层面上进行工作，以检验几个相关的假设。Berkowitz博士先前的工作表明，通过CNGC的内流钙流量通过一氧化氮合酶的CaM/Ca激活下游NO产生，从而介导植物对无毒病原菌紫丁香假单胞菌的HR反应。这一假设也将得到检验。更广泛的影响。作为该项目的一部分，伯科维茨博士将继续发展分子遗传学领域的创新高中教师培训计划。该计划旨在为高中生物教师提供教案、实验室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充实他们目前的生物遗传学课程。这门高中分子遗传学课程的体验方面是，学生参与一系列研究，将基因序列与蛋白质结构、蛋白质结构与细胞水平的功能以及蛋白质在整个植物表型、生长和发育中的作用联系起来。该计划包括一套(由伯科维茨博士开发的)PowerPoint演示文稿、蛋白质建模软件、高中实验室教案、教师指南、一本80页的分子遗传学实验手册，以及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国际学生联合会进一步开发并发布在URL(www.biologyteacher.uConn.edu)上，以便向广大高中生物教师提供所有这些教学资源。

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.