Journal
PLANT METHODS
Volume 8, Issue -, Pages -Publisher
BMC
DOI: 10.1186/1746-4811-8-25
Keywords
FACS; BiFC; In planta; In vivo; Protein-protein interaction screen; CPK3
Categories
Funding
- DFG [SPP1212, HA 2146 / 9-1]
- Julian Schroeder's laboratory [NIH R01GM060396]
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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.
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