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8th Annual Plant Genome Awardees Meeting A Monoclonal Antibody Toolkit for Functional Genomics of Plant Cell Walls Michael G. Hahn, PI University of Georgia, Athens [email protected], (706) 542-4457 Plants to be Studied: Not applicable. Project Objectives: 1. To generate a library of monoclonal antibodies against a diversity of plant cell wall carbohydrate structures that can be used for functional characterization of genes involved in cell wall biosynthesis, modification, and metabolism. 2. To characterize in detail the carbohydrate structures (epitopes) recognized by both newly generated and existing uncharacterized antibodies. Experimental Approaches: 1. The principal approach to be used for the generation of new antibodies utilizes hybridoma technology. Mice will be immunized with either cell wall preparations or with polysaccharide/ protein complexes (either covalent or non-covalent) using polysaccharides from diverse plants. Hybridoma lines generated from the immunized mice will be screened for production of antibodies against purified plant cell wall polysaccharides. Available phage display antibody libraries will also be screened for such antibodies. 2. All antibodies generated will be screened for their abilities to bind to their cognate epitopes in plant tissues by immunofluorescence and immunogold microscopy. 3. The specificities of antibodies against plant cell wall carbohydrate structures will be determined by a combination of chemical, biochemical, and immunological approaches. Polysaccharide structures will be selectively altered using highly purified glycosylhydrolases and the effects on antibody binding will be determined. In addition, homogeneous oligosaccharides will be prepared either by enzymatic/chemical cleavage of cell wall polysaccharides or by de novo chemical synthesis. These purified oligosaccharides will be added to existing glycoarrays for rapid, high-throughput epitope characterization and will be used in competitive immunoassays to delineate epitope structures. Information/Materials to be Generated: 1. Monoclonal antibodies (~100 different) against plant cell wall carbohydrate structures whose binding specificities (epitopes) have been characterized. 2. Homogeneous, structurally characterized oligosaccharides (~100) derived from plant cell wall polysaccharides. 3. Enlarged glycoarray for characterization of carbohydrate-binding proteins (e.g., antibodies). Contact Information for Outcomes and Deliverables: 1. 2. 3. 4. CarboSource http://www.ccrc.uga.edu/~carbosource/CSS_home.html Plant Probes http://www.plantprobes.co.uk/ WallBioNet http://xyloglucan.prl.msu.edu/antibodies.html Consortium for Functional Glycomics http://www.functionalglycomics.org/static/consortium/resources/resourcecoreh.shtml 5. Michael G. Hahn [email protected] 68 8th Annual Plant Genome Awardees Meeting Internet Addresses (Web or Email) for Project Information: 1. [email protected] 2. http://cell.ccrc.uga.edu/~mao/wallmab/Home/Home.phphttp://xyloglucan.prl.msu.edu/antibod ies.html Informatics Contact Person(s): 1. Malcolm O’Neill 2. Michael G. Hahn [email protected] [email protected] Practical Applications of Research: All plant cells are surrounded by cell walls that give shape and structure to the cells, tissues, and ultimately organs of the plant. Furthermore, cell wall components have been demonstrated to play active roles in plant growth and development, and plant responses to environmental stimuli. Plant cell walls are composed primarily of polysaccharides, many of whose structures are complex due to multiple sugar components and branching between the sugars. Available evidence suggests that over 2000 genes in a given plant may encode enzymes involved in cell wall synthesis and modification. To date, only a handful of these genes have been functionally identified and characterized. This project will provide a large, diverse, well-characterized library of monoclonal antibodies against plant cell wall carbohydrate (polysaccharide) structures as tools for the functional identification of genes that encode cell wall biosynthetic and modifying enzymes and genes for which there are limited alternative assays. The availability of diverse antibodies against plant cell wall carbohydrate structures will provide additional tools that are complementary to existing genetic and biochemical experimental approaches for studies of plant cell walls. The antibodies generated under this project will also be valuable to the broader community of plant scientists interested in the role(s) of cell walls and their components in plant growth, development, and responses to other organisms and environmental stimuli. Project Participants: Last Name Hahn First Name Michael Boons Geert-Jan King Christoher O’Neill Malcolm York William Institution University of Georgia University of Georgia Unversity of Georgia University of Georgia University of Georgia 69 Role PI E-Mail [email protected] Co-PI [email protected] Co-PI [email protected] Co-PI [email protected] Co-PI [email protected]