title: Screening a cDNA library for protein-protein interactions directly in planta.
abstract: Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein- or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension (CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ~2 × 10(5) cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species.
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392, USA.
authors.forename: Lan-Ying
authors.initials: LY
authors.lastname: Lee
authors.forename: Fu-Hui
authors.initials: FH
authors.lastname: Wu
authors.forename: Chen-Tran
authors.initials: CT
authors.lastname: Hsu
authors.forename: Shu-Chen
authors.initials: SC
authors.lastname: Shen
authors.forename: Hsuan-Yu
authors.initials: HY
authors.lastname: Yeh
authors.forename: De-Chih
authors.initials: DC
authors.lastname: Liao
authors.forename: Mei-Jane
authors.initials: MJ
authors.lastname: Fang
authors.forename: Nien-Tze
authors.initials: NT
authors.lastname: Liu
authors.forename: Yu-Chen
authors.initials: YC
authors.lastname: Yen
authors.forename: Ladislav
authors.initials: L
authors.lastname: Dokládal
authors.forename: Eva
authors.initials: E
authors.lastname: Sykorová
authors.forename: Stanton B
authors.initials: SB
authors.lastname: Gelvin
authors.forename: Choun-Sea
authors.initials: CS
authors.lastname: Lin
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D015723:Gene Library; D011485:Protein Binding
chemical_list: D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; C036483:T-DNA