A Monoclonal Antibody Toolkit for Functional Genomics of Plant Cell Walls

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A Monoclonal Antibody Toolkit for Functional Genomics of Plant Cell WallsProject Summary List of Senior PersonnelPI: Michael G. Hahn, CCRC and Department of Plant Biology, University of Georgia (UGA)Co-PI: Geert-Jan Boons, CCRC and Department of Chemistry, UGACo-PI: Christopher S. King, College of Veterinary Medicine, UGACo-PI: Malcolm A. O'Neill, CCRC, UGACo-PI: William S. York, CCRC and Department of Biochemistry and Molecular Biology, UGAIntroductionPlant 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 polysaccharide structures as tools for the functional identification of genes that encode cell wall biosynthetic and modifying enzymes; genes for which there are limited alternative assays. The availability of diverse antibodies against plant cell wall polysaccharide structures will provide additional tools that are complementary to existing genetic and biochemical experimental approaches for studies of plant cell walls. Project Description and Expected OutcomesThe specific goals are: 1) To isolate monoclonal antibodies that recognize plant cell wall carbohydrate structures either from hybridoma cell lines or from phage display libraries. Mice will be immunized with diverse neoglycoproteins prepared using cell wall extracts or purified poly/oligosaccharides isolated from diverse plants, and hybridoma cell lines will be generated from splenic lymphocytes of the immunized animals. Resulting hybridoma cell lines, or alternatively, antibody phage display libraries, will be screened for production of antibodies reactive with plant carbohydrate structures. 2) To characterize in detail the carbohydrate structure (epitope) recognized by the cell wall-reactive antibodies. The antibodies obtained will be grouped by their abilities to bind to a set of purified wall polysaccharides from diverse plants. The epitope structure(s) recognized by selected antibodies in each reactivity group will be characterized in detail. The ~100 unique antibodies generated during the course of this project will be instrumental to those interested in the functional characterization of genes involved in the biosynthesis and modification of plant cell walls, and will also serve as valuable tools for chemical and biological investigations of plant cell wall structure and function. Outreach and TrainingAn explicit effort will be made to involve undergraduate students in the research carried out under this grant. The participation of historically underrepresented groups in the research will be fostered by recruiting undergraduate students through the UGA Summer Undergraduate Research Program (SURP) (www.gradsch.uga.edu/rr/) and through a newly established interaction between the Department of Plant Biology, the CCRC and Morehouse College in Atlanta that brings Morehouse undergraduates to the Department/Center for summer research projects. Access to Expected Outcomes Information about all antibodies generated through this project, including details about the polysaccharide structures recognized by each antibody, will be made available immediately to the scientific community via posting to an antibody database on the NSF-funded Plant Cell Wall Biosynthesis Research Network web site (http://xyloglucan.prl.msu.edu/). A link to this database will also be posted on the website of the Complex Carbohydrate Research Center (CCRC) (http://www.ccrc.uga.edu/). The antibodies will be made available to the research community without restriction through antibody stock centers established at the CCRC at UGA (Athens, GA) and the Centre for Plant Sciences at the University of Leeds (UK). Antibodies may be ordered from and will be distributed by CarboSource (http://www.ccrc.uga.edu/web/services/carbosource/CSS_home.html) and Plant Probes (http://www.plantprobes.co.uk/). The distribution of the antibodies will be self-supported by modest fees charged to those ordering the antibodies. The fee structure will be posted to the CarboSource and Plant Probes web sites. The hybridoma cell lines themselves will be replicated and stored in three physically separated facilities: 1) the CCRC and 2) the Monoclonal Antibody Facility of the College of Veterinary Medicine, both at UGA; and 3) with Dr. Paul Knox at the University of Leeds..