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

ABSTRACT: Understanding protein and gene function requires identifying interaction partners usingbiochemical, molecular or genetic tools. In plants, searching for novel protein-proteininteractions 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 proteinpartners in living plant cells. We demonstrate that it is possible to screen for novel proteinproteininteractions from a random library in protoplasted Arabidopsis plant cells and recoversome of the interacting partners. Our screen is based on capturing the bi-molecularcomplementation of mYFP between an YN-bait fusion partner and a completely random preyYC-cDNA library with FACS. The candidate interactions were confirmed using in plantaBiFC assays and in planta FRET-FLIM assays. From this work, we show that the wellcharacterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3,HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This isone of the first random in planta screens to be successfully employed.