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Summary Integration of signalling on the cellular level is essential for the survival of organisms. Protein-protein interaction studies provide valuable insights in these signalling events. One of the best understood signalling pathways in plants is the brassinosteroid (BR) hormone signalling pathway, which is mediated by the receptor brassinosteroid insensitive 1 (BRI1) with its co-receptor bri 1- associated kinase (BAK1). Both BRI1 and BAK1 have been shown to interact with receptor like protein 44 (RLP44), which was implicated in cell wall integrity sensing by modulation of BL signalling. Here we provide evidence by quantitative in vivo three-fluorophore FRET-FLIM measurements, that RLP44, BRI1 and BAK1 form a trimeric complex in the plasma membrane of N. benthamiana leaf cells, with an estimated distance between them below 15 nm. The immune receptor flagellin sensing 2 (FLS2), which is also a receptor-like kinase like BRI1, is not integrated in a similar complex with RLP44 and BAK1. Our study supports that BRI1 and FLS2 are localized in distinct nanodomains in the PM. As the fluorescence lifetime of the donor is monitored, our method circumvents the extensive calculations necessitated by intensity-based FRET interaction assays and thus provides a feasible base for studying the sub-compartmentalization in the plasma membrane of living plant cells with a nanoscale resolution. Graphical abstract Principle of three fluorophore FRET-FLIM Förster resonance energy transfer (FRET) describes the energy transfer of a donor fluorophore (D) to an acceptor fluorophore (A1) if they are in close proximity, typically below 10 nm. Three fluorophore FRET-FLIM combines two FRET pairs in such a fashion, that the acceptor (A1) of the first pair, is at the same time donor to a second acceptor (A2). Thus, energy of D can either sequentially move from D via A1 to A2 and directly between D-A1 and D-A2. Previous studies have quantified the fluorescence intensities to monitor the distances between the three fluorophores. Here we establish the use of fluorescence lifetime imaging microscopy (FLIM), which measures the donor fluorescence lifetime (τ) to assess complex formation of three proteins of interest in the plasma membrane. Specifically, if both A1 and A2 drain energy from D, (or sequentially from D via A1 to A2), then the fluorescence lifetime decreases. Both the distance below 15 nm between D and A2 and the trimeric complex formation will result in an additional decrease of τ 3 compared to τ 2 and τ 2’ . |
