Over the rainbow: A practical guide for fluorescent protein selection in plant FRET experiments

Receptor‐like kinases (RLK) and receptor‐like proteins (RLP) often interact in a combinatorial manner depending on tissue identity, membrane domains, or endo‐ and exogenous cues, and the same RLKs or RLPs can generate different signaling outputs depending on the composition of the receptor complexes they are involved in. Investigation of their interaction partners in a spatial and dynamic way is therefore of prime interest to understand their functions. This is, however, limited by the technical complexity of assessing it in endogenous conditions. A solution to close this gap is to determine protein interaction directly in the relevant tissues at endogenous expression levels using Förster resonance energy transfer (FRET). The ideal fluorophore pair for FRET must, however, fulfil specific requirements: (a) The emission and excitation spectra of the donor and acceptor, respectively, must overlap; (b) they should not interfere with proper folding, activity, or localization of the fusion proteins; (c) they should be sufficiently photostable in plant cells. Furthermore, the donor must yield sufficient photon counts at near‐endogenous protein expression levels. Although many fluorescent proteins were reported to be suitable for FRET experiments, only a handful were already described for applications in plants. Herein, we compare a range of fluorophores, assess their usability to study RLK interactions by FRET‐based fluorescence lifetime imaging (FLIM) and explore their differences in FRET efficiency. Our analysis will help to select the optimal fluorophore pair for diverse FRET applications.