Poster: Emerging technologies

Abs # 904: High-throughput fluorescent tagging of full-length Arabidopsis gene products of unknown function

Presenter:	Tian, Guo-Wei , gutian@notes.cc.sunysb.edu
Authors	Tian, Guo-Wei  (A)   Mohanty, Amitabh  (B)   Chary, Narasimha  (C)   Li, Shijun  (D)   Paap, Brigitte  (A)   Drakakaki, Georgia  (C)   Ehrhardt, David  (E)   Jackson, David  (B)   Rhee, Seung Y  (D)   Raikhel, Natasha  (C)   Citovsky, Vitaly  (A)
Affiliations:	(A): Department of Biochemistry & Cell Biology, State University of New York, Stony Brook, NY 11794 (B): Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 (C): Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, CA 92521 (D): The Arabidopsis Information Resource (TAIR), Carnegie Institution of Washington, Department of Plant Biology, Stanford, CA 94305 (E): Carnegie Institution of Washington, Department of Plant Biology, Stanford, CA 94305

Arabidopsis is a model system for identifying plant genes and characterizing their function. Currently, about one third of all predicted proteins in Arabidopsis cannot be assigned to any functional category. One way to shed light on the function of an unknown protein is to determine its subcellular locations and supracellular patterns of expression. Toward this end, we developed a high-throughput method, termed Fluorescent Tagging of Full-Length Proteins (FTFLP), which includes following three major steps: (i) selection of Arabidopsis genes with unknown function and fluorescent tagging of these genes with YFP or CFP that is introduced into the selected site by triple template PCR, (ii) generation of transgenic Arabidopsis plants that stably express each tagged gene, and (iii) analysis of subcellular localization and expression pattern of each tagged protein using laser-scanning confocal microscopy. In addition to being streamlined for high-throughputness, our approach offers two significant advantages: first, it produces internally-tagged full length proteins that are likely to exhibit native subcellular localization, and second, it yields information about the tissue specificity of gene expression by the use of native promoters. Using this method, we tagged several known proteins with diverse subcellular targeting patterns to prove the concept of the approach as well as several proteins with unknown function and unassigned subcellular localization. The results indicated that this approach could be used for characterization of the Arabidopsis proteome in general. More information about the progress and results can be found from our website at: http://aztec.stanford.edu/gfp/.