Investigating the dynamics of protein–protein interactions in plants

SUMMARY Both stable and transient protein interactions play an important role in the complex assemblies required for the proper functioning of living cells. Several methods have been developed to monitor protein–protein interactions in plants. However, the detection of dynamic protein complexes is very challenging, with few technologies available for this purpose. Here, we developed a new platform using the plant UBIQUITIN promoter to drive transgene expression and thereby to detect protein interactions in planta . Typically, to decide which side of the protein to link the tags, the subcellular localization of the protein fused either N‐terminal or C‐terminal mCitrine was firstly confirmed by using eight different specific mCherry markers. Following stable or transient protein expression in plants, the protein interaction network was detected by affinity purification mass spectrometry. These interactions were subsequently confirmed by bimolecular fluorescence complementation (BiFC), bioluminescence resonance energy transfer and co‐immunoprecipitation assays. The dynamics of these interactions were monitored by Förster resonance energy transfer (FRET) and split‐nano luciferase, whilst the ternary protein complex association was monitored by BiFC‐FRET. Using the canonical glycolytic metabolon as an example, the interaction between these enzymes was characterized under conditions that mimic physiologically relevant energy statuses.