INVESTIGATORS: Beers, E., Brunner, A., Dickerman, A., Helm, R.
INSTITUTION: Virginia Tech
NON-TECHNICAL SUMMARY: The final characteristics of any wood are the result of the coordinated actions of enzymes and structural proteins. The resulting three-dimensional composite that we see as wood is ultimately dependent on the proteins active in the cells of secondary xylem, the wood-forming tissue. The proteins that do the work in cells typically interact with other proteins to perform their roles, and the sum total of these interactions is known as the protein-protein interactome. Presently only a small portion of the protein-protein interactome has been identified and characterized. The overall goal of the proposed research is to identify key interactions between proteins that are associated with wood formation in poplar, a woody biomass crop. Several of these identified members will subsequently be the subjects of in-depth functional analyses using transgenic poplar engineered for simultaneous increases or decreases in expression of interacting proteins. This functional interactomics approach will allow evaluation of the potential of components of the biomass protein-protein interactome to contribute to the development of poplar trees with superior biomass feedstock potential.
OBJECTIVES: 1. Perform a high throughput screen (yeast two-hybrid; Y2H) of a library of biomass-associated prey proteins to identify proteins that interact with 200 biomass bait proteins. 2. Perform independent tests in vitro and in vivo of protein-protein interactions involving proteins selected for functional analysis. 3. Perform functional analysis of selected pairs of interactors using transgenic poplar. 4. Maintain the project website for reporting new protein-protein interaction and functional interactomics data.
APPROACH: To expand the list of interacting proteins relevant to woody biomass, Y2H analysis will be used to screen the majority of the biomass proteins cloned during a previous project (http://xylome.vbi.vt.edu/) against a library of prey proteins. To determine whether interactors identified by Y2H can interact in plant cells or in vitro, independent protein-protein interaction tests such as bimolecular fluorescence complementation, immunoprecipitation or pull-down assays will be performed. For functional analysis, transgenic poplar will be prepared for gene silencing and/or ectopic expression of selected interactors. Transgenics will be characterized with regard to plant development, morphology and biomass and several aspects of wood quality: density, anatomy, soluble carbohydrates, starch and cellulose and hemicellulose and lignin content and linkage. The internal, password protected, side of the project website will enable project participants to upload data. The public external face of the website will present the project in a slightly reduced version of the full detailed data available internally.
Name: Eric Beers