Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry

被引：42

作者：

Berendzen, Kenneth Wayne ^{[1
]}

Boehmer, Maik ^{[2
,3
]}

Wallmeroth, Niklas ^{[1
]}

Peter, Sebastien ^{[1
]}

Vesic, Marko ^{[1
]}

Zhou, Ying ^{[1
]}

Tiesler, Franziska Katharina Elisabeth ^{[1
]}

Schleifenbaum, Frank ^{[1
]}

Harter, Klaus ^{[1
]}

机构：

[1] Univ Tubingen, ZMBP, D-72076 Tubingen, Germany

[2] Univ Calif San Diego, Div Biol Sci, Cell & Dev Biol Sect, La Jolla, CA 92093 USA

[3] Ctr Mol Genet 0116, La Jolla, CA 92093 USA

来源：

PLANT METHODS | 2012年 / 8卷

关键词：

FACS; BiFC; In planta; In vivo; Protein-protein interaction screen; CPK3; KINASE CPK3; ARABIDOPSIS; SYSTEM; GENE; ENCODES; DNA; IDENTIFICATION; VISUALIZATION; COMPONENTS; RECEPTOR;

D O I：

10.1186/1746-4811-8-25

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Understanding protein and gene function requires identifying interaction partners using biochemical, molecular or genetic tools. In plants, searching for novel protein-protein interactions is limited to protein purification assays, heterologous in vivo systems such as the yeast-two-hybrid or mutant screens. Ideally one would be able to search for novel protein partners in living plant cells. We demonstrate that it is possible to screen for novel protein-protein interactions from a random library in protoplasted Arabidopsis plant cells and recover some of the interacting partners. Our screen is based on capturing the bi-molecular complementation of mYFP between an YN-bait fusion partner and a completely random prey YC-cDNA library with FACS. The candidate interactions were confirmed using in planta BiFC assays and in planta FRET-FLIM assays. From this work, we show that the well characterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3, HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This is one of the first random in planta screens to be successfully employed.

引用

页数：17

共 63 条

[21] Identification of LOV KELCH PROTEIN2 (LKP2)-interacting factors that can recruit LKP2 to nuclear bodies [J].