Open Access Methodology

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

Kenneth Wayne Berendzen1, Maik Böhmer2, Niklas Wallmeroth1, Sébastien Peter3, Marko Vesić1, Ying Zhou1, Franziska Katharina Elisabeth Tiesler1, Frank Schleifenbaum3 and Klaus Harter1*

Author Affiliations

1 Universität Tübingen, ZMBP, Plant Physiology, Auf der Morgenstelle 1, D-72076, Tübingen, Germany

2 University of California, San Diego, Division of Biological Sciences, Cell and Developmental Biology Section & Ctr for Mol. Genetics 0116, 9500 Gilman Drive #0116, La Jolla, CA, 92093-0116, USA

3 Universität Tübingen, ZMBP, Biophysical Chemistry, Auf der Morgenstelle 18, D-72076, Tübingen, Germany

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Plant Methods 2012, 8:25  doi:10.1186/1746-4811-8-25

Published: 12 July 2012

Abstract

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 randomin planta screens to be successfully employed.

Keywords:
FACS; BiFC; In planta; In vivo; Protein-protein interaction screen; CPK3