75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

89 Acquisition and Distribution of Stocks for Genomics and Phenomics by ABRC
Randy Scholl, Luz Rivero, Deborah Crist, Emma Knee, Natalie Case, Heather Joesting, Daniel Johnson, Kate
Ludwig, James Mann, Cori Phillips, Garret Posey, Pamela Vivian, Zhen Zhang, Ling Zhou
Arabidopsis Biological Resource Center, Department of Plant Cellular and Molecular Biology and Plant
Biotechnology Center, The Ohio State University
The Arabidopsis Biological Resource Center (ABRC) maintains many stocks relevant to genome exploration and
functional genomics. Several flank-tagged insertion collections are distributed by ABRC, specifically: the T-DNA lines
from a) SALK Institute, J. Ecker lab, b) Syngenta Biotechnology, including the ca. 9,000 lines, originally associated with
MTAs, c) the Wisconsin Ds-Lox population, d) GABI-Kat lines, and e) RNAi lines from AGRIKOLA.
Developing initiatives related to insertion lines and their application to phenomics include: a) receipt of 1,900
confirmed, purified insertion lines from J. Ecker; b) efforts to make these lines available for large phenotypic studies;
and c) regrowth of the entire ABRC collection of natural accessions to reestablish single seed sources which will be
genetically fingerprinted by J. Borevitz and collaborators.
Full length open reading frame (ORF) cDNA clones are still being received. The total number of full length clones
is approximately 15,000 including donations from SSP and Salk (J. Ecker), TIGR (C. Town), Peking/Yale and J. Callis.
In addition, we are receiving a large collection of ORF clones cloned into Gateway Expression constructs – 1,100 of
these (from S. P. Dinesh Kumar) are currently in house. Versatile Destination vectors (for Gateway and other systems)
for various expression applications in plants, bacteria and yeast are available. We have formatted 11,000 of the new
cDNA clones into plates. We plan to distribute other similar large collections in this way.
ABRC is supported by the National Science Foundation.
90 BRET: A Method to Detect in vivo Protein Interactions in Plant Cells
Chitra Subramanian, Jongchan Woo, Albrecht von Arnim
University of Tennessee, Knoxville
Sensing extracellular signals and transmitting them into specific responses involves protein-protein interactions within
a cell. In order to detect protein-protein interactions in vivo we have used Bioluminescence Resonance Energy transfer
(BRET). BRET involves radiation-less energy transfer between Renilla luciferase (RLUC) and YFP if the candidate
fusion protein partners interact with each other. We have generated a suite of BRET expression vectors for both restriction
cloning and gateway cloning. A codon optimized version of RLUC enhanced the stable expression of fusion proteins in
transgenic Arabidopsis. BRET has been demonstrated between a B-box protein, STH, and an E3 ubiquitin ligase, COP1
and also between two b zip transcription factors HY5 and HYH in stable transgenic Arabidopsis seedlings. RLUC can
be targeted to a variety of subcellular organelles like chloroplasts, mitochondria, golgi and peroxisomes. Finally, we
have also demonstrated the use of Renilla luciferase as a reporter of protein stability in a cycloheximide chase assay.
Results will be presented for a survey of pair wise protein-interactions thought to play a role in the Arabidopsis light
signaling network.