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.
