Download 68 A Monoclonal Antibody Toolkit for Functional Genomics of Plant

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]
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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
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Role
PI
E-Mail
[email protected]
Co-PI
[email protected]
Co-PI
[email protected]
Co-PI
[email protected]
Co-PI
[email protected]