In planta analysis of protein–protein interactions related to light signaling by bimolecular fluorescence complementation

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Abstract
The determination of protein–protein interactions is becoming more and more important in the molecular analysis of signal transduction chains. To this purpose the application of a manageable and simple assay in an appropriate biological system is of major concern. Bimolecular fluorescence complementation (BiFC) is a novel method to analyze protein–protein interactions in vivo. The assay is based on the observation that N- and C-terminal subfragments of the yellow-fluorescent protein (YFP) can only reconstitute a functional fluorophore when they are brought into tight contact. Thus, proteins can be fused to the YFP subfragments and the interaction of the fusion proteins can be monitored by epifluorescence microscopy. Pairs of interacting proteins were tested after transient cotransfection in etiolated mustard seedlings, which is a well characterized plant model system for light signal transduction. BiFC could be demonstrated with the F-box protein EID1 (empfindlicher im dunkelroten Licht 1) and the Arabidopsis S-phase kinase-related protein 1 (ASK1). The interaction of both proteins was specific and strictly dependent on the presence of an intact F-box domain. Our studies also demonstrate that etiolated mustard seedlings provide a versatile transient assay system to study light-induced subcellular localization events.