Acquisition of a Laser Scanning Confocal Microscope for Plant Science Research

We seek to acquire a laser scanning ,onfocal microscope with !pectral Imaging (CSI) for visualization of biological molecules in plant science research. This instrument will open new frontiers for at least 10 research programs in the College of Agriculture. Several projects in the college involve functional genomics of plants and their pathogens. This has spawned a need for simultaneous visualization of multiple proteins and or nucleic acids in biological samples. Current confocal microscopes on the UK campus are unable to resolve the emission spectra of certain fluors, limiting the types of experiments that can be performed. A CSI does not have this limitation and, therefore, would greatly expand the scope of plant science research on the UK campus. This proposal outlines several experiments that require resolution of multiple fluors. Nagy has developed yeast as a model host system for investigating host factors involved in replication/recombination of a plant virus. Screening of a library of single-gene knock-out mutants, resulted in identification of 112 yeast genes with putative roles in viral replication and 7 with predicted functions in viral recombination. Many of the former genes are involved in protein localization/trafficking, suggesting that they deliver the viral replication machinery, and possibly the viral RNA, to cellular replication sites (peroxisomal membranes). Nagy plans to test this hypothesis by using the CSI and autofluorescent reporter-protein fusions to investigate (co)localization of the viral replicase protein with the host factors, together with a peroxisomal marker. Also planned are confocal experiments to investigate in vivo interactions between host factors, the viral replicase and viral RNA using Fluorescence resonance energy transfer (FRET). These studies will provide unprecedented insights into host factors involved in viral replication and recombination. Farman studies the organization, evolution and instability of fungal genomes. He has discovered a novel mechanism of genetic instability in the fungus Magnaporthe grisea that causes a specific gene (BUF1) to be deleted in up to 40% of meioses. Based on their genetic behavior, Farman hypothesizes that the deletions occur pre-meiotically and are caused by mis-pairing between homologous chromosomes. He will test this hypothesis by using CSI and fluorescent probes to visualize the two BUF1 alleles in pre-meiotic and meiotic tissues. It is anticipated that these studies will establish a new paradigm for meiotic chromosome instability. Perry seeks to elucidate mechanisms by which the Arabidopsis MADS-box protein AGL 15 regulates embryonic development. Current studies are focused on identification and functional characterization of genes regulated by AGL 15 and several candidates have been identified by chromatin immunoprecipitation. The CSI will be used to determine the cellular locations of proteins encoded by AGL 15-regulated genes, as a means to confirm/assign function. Gene regulation by AGL 15 appears to be modulated by other proteins. Several candidates that interact with AGL 15 have been identified using a yeast 2-hybrid screen. Perry will utilize FRET to confirm these interactions in vivo. Goodin investigates interactions between rhabdoviruses and plants. Using cell biology and microarrays, he has identified plant genes that are differentially expressed in response to rhabdovirus infection as well characterizing changes in plant cell endomembranes that occur in virus-infected cells. The CSI will be used for in situ hybridization and live cell imaging studies in order to determine where and when particular host genes are expressed in relation to the cells that are actually virus-infected. This study will greatly enhance understanding of plant responses to viral infection. The intellectual merits of the above research activities are many. Novel and fundamental insights will be gained in biological systems as diverse as viral replication/recombination, plant responses to viral infection, chromosome instability, regulation of development in plants; and other areas of investigation pursued by additional plant science faculty who plan to use fluorescence microscopy methods in their research. The broader impact of acquiring a CSI will be an enhancement of research and training experiences for postdoc, graduate students and, especially, the many undergraduates who perform research projects in the Plant Science Building, to satisfy the independent study requirement of the Agricultural Biotechnology major. In addition, the knowledge gained may lead to: methods for control of viral diseases in humans, animals and plants; development of new viral-based gene expression vectors; ways to prevent genetic diseases; and improved crop productivity.