Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry.
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.
Universität Tübingen, ZMBP, Plant Physiology, Auf der Morgenstelle 1, D-72076, Tübingen, Germany. klaus.harter@zmbp.uni-tuebingen.de.
Kenneth Wayne
KW
Berendzen
Maik
M
Böhmer
Niklas
N
Wallmeroth
Sébastien
S
Peter
Marko
M
Vesić
Ying
Y
Zhou
Franziska Katharina Elisabeth
FK
Tiesler
Frank
F
Schleifenbaum
Klaus
K
Harter