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

智力上的优点。最近由Berkowitz博士进行的NSF资助的工作确定了第一个编码功能性质膜钙离子传导通道的植物基因。这种基因产物，环核苷酸（cNMP）门控非选择性阳离子通道（CNGC），促进下游一氧化氮（NO）的产生在植物先天免疫/病原体反应信号级联。该项目利用对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步骤：（a）通过CNGC向内Ca传导的上游（即将病原体感知与通道激活联系起来），（B）将Ca传导到细胞质中与NO生成联系起来，（c）通过细胞质二级信使调节通道，以及（d）涉及Ca通道与植物对无毒病原体的过敏反应（HR）。研究方法包括用从野生型（WT）和CNGC功能丧失突变体植物中分离的原生质体进行膜片钳实验。这项工作的目的是表征可能的调节系统，在病原体信号级联过程中调节CNGC功能，如cNMP浓度，钙调蛋白（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/调节剂对植物中Ca吸收和NO产生的影响将被确定，以阐明植物先天免疫/病原体应答信号级联中的步骤。二级信号分子cAMP和cGMP的水平将在进行HR的植物中进行监测。工作将在整个植物水平上进行，以测试几个相关的假设。Berkowitz博士先前的工作表明，通过CNGCs向内的Ca通量介导植物对无毒病原体假单胞菌的HR反应，这是由于下游NO生成通过CaM/Ca激活一氧化氮合酶。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，Berkowitz博士将继续开发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学教师提供教案，实验室练习以及所有必要的信息和支持，以通过创新的体验式学习计划来增强他们目前在遗传学方面的生物学课程。在分子遗传学这个高中课程的经验方面是学生参与一系列的调查，链接基因序列蛋白质结构，蛋白质结构在细胞水平上的功能，以及蛋白质在整个植物表型，生长和发育中的作用。该计划包括一个PowerPoint演示文稿包（由Berkowitz博士开发），蛋白质建模软件，高中实验室教案，教师指南，80页的分子遗传学实验室手册，以及相关网络资源的注释目录。教师培训计划和高中实验室课程将由PI进一步开发并发布在URL（www.biologyteacher.uconn.edu）上，以便将所有这些教学资源提供给广大高中生物教师。

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.