A fundamental aspect of organism fitness involves the detection of a "non-self" presence in the extracellular environment. In the case of a pathogenic microbe invader, perception of non-self presence needs to be translated through a cellular signal transduction cascade so that the cells of the organism under attack can mount a defense or immune response. The aim of this project is to provide new insights into the early events of pathogen perception in plant cells. Prior research has shown that calcium (Ca) flow into the cytoplasm of cells through a cyclic nucleotide gated cation channel (CNGC) protein, and downstream nitric oxide (NO) generation were critical to the plant immune response signaling cascades. The research focus of this project will take advantage of our understanding of the molecular architecture related to CNGC function and regulation to elucidate further steps in this signaling cascade. Hypotheses will be tested regarding the signaling steps upstream from inward Ca conduction by this CNGC; i.e. linking pathogen perception to channel activation. Work will determine if and how cyclic nucleotide elevation occurs in plant cells responding to pathogen recognition. One component of the project aims to examine some plant cell membrane receptor proteins for possible ability to generate cyclic nucleotide, the signaling molecule that activates Ca conduction through CNGCs. Other studies will focus on identifying regulatory molecules that may impact activation of the CNGC channels, and therefore modulate plant immune response to pathogens. Broader impacts. Project activity includes use of an innovative molecular genetics teacher-training program for outreach and education efforts. This two-week full day program trains participants in molecular genetics experimental methods and underlying principles in an integrated fashion. Participants are trained, and provided with a package of tools and resources (informational and biological) to allow them to present a molecular biology experiential lab educational program in high school biology classes. This program includes a package (developed for use as part of this project) of PowerPoint presentations, protein modeling software, lab lesson plans, teacher's guides, an 80-page molecular genetics lab manual, an annotated catalog of web resources to assist high school teachers in obtaining biological and informational resources, and more. As part of the proposed broader impacts of the project, the two-week training program based on this material will be delivered to five undergraduate School of Education students as well as five current high school biology teachers each summer during the funding period. Assistance will be provided to high school teachers as they implement the program in schools throughout the State. All of the information in the teacher-training program and high school lab curriculum will be posted on a URL and made available to a wide audience of high school biology teachers through this and other web sites. The educational component of the project includes continued training of a current female African-American graduate student who will undertake research objectives of the project as part of her Ph.D. degree.
Animals such as humans have an innate immune system that provides the organism with the ability to evoke defense responses against pathogenic microbes that can proilferate upon infection. The innate immune response of animals to infectious microbes is facilitated by a vascular system in which mobile sentry cells (neutrofils, a type of white blood cell) circulate, guarding against pathogen attack. Plants on the other hand do not have such a defense system with mobile sentries that perceive pathogen perception and then evoke defense responses. Alternatively, each cell in a plant has receptors that can monitor the presence of an invading pathogen. Pathogen perception by these cell membrane ‘pattern recognition receptors’ (PRRs) can lead to altered cell function, the generation of antimicrobial compounds, increased expression of proteins encoded by defense genes, and other immune responses that provide a defense reponse to pathogen invasion. One mystery concerning the ‘biological warfare’ between invading pathogen and the plant cells under assault, is how the intial perception of pathogens by plant cells is translated into an evocation of defense responses that facilitate immunity. The signal tranduction system that facilitates plant cell immune responses was the focus of the ressearch undertaken as a component of this project. One way in which all cells- animal, plant and procaryote, translate perception of external cues into altered cell function and adaptation is through a secondary messenger system involving the transient increase in intracellular (cytosolic) Ca2+. This Ca2+ is then decoded by a signal transduction system to intiate specific cell responses that facilitate adaptation and cell fitness in the face of environmental challenges such as pathogen assault. We have linked this Ca2+ signaling system with the binding of an endogenous plant peptide to it’s receptor. Theses signaling peptides are one way in which plants ‘amplify’ the defense response to pathogen perception. The peptides are either generated by plant cells under assault, or released from plant cells as they die from pathogen infection. These endogenous plant peptides act as secondary signals to evoke immune responses in cells neighboring those under assault by a microbe. The immune response intiated by the peptide signaling system leads to an immune reaction that limits the spread of infection within the plant. This Peptide-evoked defense response involves a number of steps identified by the work undertaken for this project. Binding of the plant peptide to a PRR is associated with the generation of cyclic nucleotide (cAMP and/or cGMP). In turn, the cAMP or cGMP activates cyclic nucleotide gated Ca2+ conducting ion channels which generate the ‘warning alarm’ of a temporary cytosolic Ca2+ elevation. The Ca2+ signal is ‘decoded’ by a series of proteins. This signal transduction system includes Ca2+-dependent protein kinases (CDPKs), and Ca2+-binding calmodulin (CAM) (or calmodulin-like) proteins. CDPKs and CAMs activate other proteins downstream in the signaling cascade. This immune response leads to generation of the antimicrobial compounts hydrogen peroxide (and other reactive oxygen species), and nitric oxide, a diffusible volatile signal that can pass from cell to cell. CDPKs and possibly CAMs activate other proteins such as mitogen activated protein kinases (MAPKs) and transcription factors. This leads to signals that lead to altered gene expression; enhanced expression of other immune defense-related proteins. The plant peptide signals were shown to be important signals that facilitate the immune signaling cascade. Broader impacts. A two-week (8 hr/day), summer molecular biology training practicum was developed and presented to undergraduates in the Teacher Education Program (TEP) training to begin careers as high school science teachers. The goal of this practiccum was provide training to education majors so that they can more effectively develop lab-based pedagogy for molecular biology teaching in high schools. A ~80 page lab manual and associated teaching resources were developed for the future teachers. Participating students received electronic and hard copies of the lab manual developed for the practicum, as well as other teaching tools to support their future use of the information. The program included protocols and underlying principles for plant DNA extraction, gel electrophoresis, bacterial cultures, PCR, plasmid isolation, DNA manipulation and purification, cloning with plasmid vectors, bacterial and yeast transformation, growing and working with Arabidopsis, use of DeepView Swiss Pdb and RAS-MOL for protein modeling, web-based tutorials, and an annotated list of web sites useful for teaching high school biology (e.g.movies and tutorials in molecular biology subjects). Information from this program is posted on a web site (see www.biologyteacher.uconn.edu). At this url, teachers are provided with everything from lesson plans to lab outlines, lab write-up formats, grading sheets, protocols, physical set up of classroom, etc. The information is freely available for any teacher to download with no restrictions (NSF support is noted). This should have an impact on teachers beyond those trained by the PI. in the aforementioned training practicuum.
