The Use of FRET/FLIM to Study Proteins Interacting with Plant Receptor Kinases

The investigation of protein interactions in living plant tissue has become of increasing importance in recent years. A high spatial and temporal resolution for the observation of in vivo protein interaction is needed, e.g., in order to follow changes of plant receptor kinase interactions and complex formation over time. In vivo fluorescence or Förster resonance energy transfer (FRET) measurements allow for detailed analyses of interacting proteins in their natural environment at a subcellular level. Especially FRET-FLIM (fluorescence lifetime imaging microscopy) measurements provide an extremely powerful and reliable tool meeting the demands for investigating in vivo protein interaction quantitatively and with high precision. Here, we will describe in detail how to practically perform in vivo FRET measurements of receptor kinases in plants and discuss potential pitfalls and points of consideration.