A Toolkit for in Vivo Visualization/Modulation of Plant Cell Wall Polysaccharides

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PI: Michael G. Hahn (University of Georgia)CoPIs: Geert-Jan Boons, Harry Gilbert, Christopher S. King, Malcolm A. O'Neill, and William S. York (University of Georgia)There are currently no tools that can be used for in vivo imaging plant cell wall polysaccharides and glycoproteins. This project will provide a set of molecular probes for cell wall polysaccharides in the form of single-chain antibody fragments (ScFvs) and carbohydrate-binding modules (CBMs) as tools for in vivo imaging of plant cell walls using microscopy. The specific goals are: 1) to generate, either from hybridoma cell lines or from carbohydrate binding domains, a comprehensive series of tagged molecular probes that can be expressed in plant cells and will bind to cell wall glycans in vivo; and 2) to demonstrate that the probes generated will permit the localization of diverse carbohydrate structures within living plant cells or alternatively alter cell wall structure by selectively disrupting polysaccharide incorporation into walls in transgenic plants. It is anticipated that the molecular probes generated during the course of this project will be instrumental to those interested in the dynamics of plant cell wall polysaccharides and that some of these probes will disrupt cell wall assembly and structure through their interaction with the polysaccharides/glycoproteins and serve as valuable tools to discover the functions of cell wall polysaccharides in the biology of plant cells. With respect to outreach and training, project personnel will teach a one week course to be held during each summer at the Complex Carbohydrate Research Center during the tenure of the grant to teach polysaccharide localization methods to interested scientists. An 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 University of Georgia Summer Undergraduate Research Program (SURP) (http://www.grad.uga.edu/outreach&diversity/surp.html). All students and post-doctoral associates will be closely mentored to encourage and foster their development as research scientists. Information about all antibodies, ScFvs, and CBMs generated through this project, will be made available immediately to the scientific community via posting to a web-based antibody database (http://www.wallmabdb.net). All molecular probes will be made available to the research community without restriction through CarboSource (http://www.carbosource.net). Plasmids and transgenic plant lines generated in the molecular probe function validation studies will be available through the Arabidopsis Biological Resource Center (http://abrc.osu.edu).