Minisymposium 21: Emerging technologies

Abs # 42003: 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, S. 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 and 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 sub-cellular location and pattern of expression. To this end, we developed a high-throughput method, termed Fluorescent Tagging of Full-Length Proteins (FTFLP), which includes the following three major steps. (1) Selection and fluorescent tagging of Arabidopsis genes with unknown function. Each selected gene is amplified in two fragments, one from up to 3 kb upstream of the transcription start of the gene to the tag insertion site within the coding sequence, and second from the tag insertion site to 0.5-1 kb downstream of the gene. Our default position for insertion of the YFP/CFP tags is 10 amino acids upstream of the stop codon. The YFP/CFP coding sequences were also amplified. A second round of PCR, termed triple-template PCR, was then performed using all three amplified fragments as templates to introduce the YFP tag into the selected site and create an internally-tagged full-length gene. (2) Generation of transgenic Arabidopsis lines that stably express the tagged genes. Each tagged gene was transferred by Gateway recombination cloning into a donor vector and then into binary destination vectors developed in our labs. The resulting constructs were introduced into Agrobacterium, and then transformed into Arabidopsis plants by the floral dip method. (3) Analysis of sub-cellular localization and expression pattern of each tagged protein using laser-scanning confocal microscopy. In addition to being streamlined for high-throughput, our approach offers two significant advantages: first, it produces internally-tagged full length proteins that are likely to exhibit native sub-cellular localization, and second, it yields information about the tissue specificity of gene expression by the use of native promoters. Using FTFLP, we tagged numerous known proteins with diverse sub-cellular targeting patterns to prove the concept of the approach as well as proteins with unknown function and unassigned sub-cellular localization. Our data indicate that FTFLP can be used both for studies of individual proteins of interest and for functional characterization of the Arabidopsis proteome. Additional information about the project can be found on our website http://aztec.stanford.edu/gfp/.