Three-Fluorophore FRET Enables the Analysis of Ternary Protein Association in Living Plant Cells

Abstract

Protein-protein interaction studies provide valuable insights into cellular signaling. Brassinosteroid (BR) signaling is initiated by the hormone-binding receptor Brassinosteroid Insensitive 1 (BRI1) and its co-receptor BRI1 Associated Kinase 1 (BAK1). BRI1 and BAK1 were shown to interact independently with the Receptor-Like Protein 44 (RLP44), which is implicated in BRI1/BAK1-dependent cell wall integrity perception. To demonstrate the proposed complex formation of BRI1, BAK1 and RLP44, we established three-fluorophore intensity-based spectral Förster resonance energy transfer (FRET) and FRET-fluorescence lifetime imaging microscopy (FLIM) for living plant cells. Our evidence indicates that RLP44, BRI1 and BAK1 form a ternary complex in a distinct plasma membrane nanodomain. In contrast, although the immune receptor Flagellin Sensing 2 (FLS2) also forms a heteromer with BAK1, the FLS2/BAK1 complexes are localized to other nanodomains. In conclusion, both three-fluorophore FRET approaches provide a feasible basis for studying the in vivo interaction and sub-compartmentalization of proteins in great detail.

One Pattern Analysis (OPA) for the quantitative determination of protein interactions in plant cells

“…to the loss of valuable information (Immink et al 2009;Denay et al 2019;Tonaco et al 2006;Immink et al 2002;Glockner et al 2022).…”

“…Compared to BiFC, FRET assays have the advantage of having higher spatial resolution and, more importantly, are more specific, as complementing FP fragments show a tendency for self-assembly resulting in false positive interactions (Horstman et al 2014;Shyu and Hu 2008). FLIM in contrast to intensity-or spectral-based FRET methods, is more gentle on cells and tissues due to lower required laser intensity and is generally considered the more accurate method to detect FRET (Strotmann and Stahl 2022;Long et al 2017;Long et al 2018;Stahl et al 2013;Russinova et al 2004;Glockner et al 2022).…”

“…Intensity-based FRET usually requires strict controls and correction for spectral bleed-through, whereas lifetime acquisition by FLIM is more robust (Strotmann and Stahl 2022;Godet and Mely 2019;Bucherl et al 2014;Spatola Rossi et al 2022;Piston and Kremers 2007). Additionally, FLIM-FRET is independent from local Donor and Acceptor concentrations and requires only relatively low irradiation of cells, and is thus considered to be superior compared to intensity-based techniques (Long et al 2017;Glockner et al 2022;Long et al 2018;Stahl et al 2013;Russinova et al 2004;Denay et al 2019). In time-domain FLIM experiments, arrival times of single photons after excitation with a pulsed laser are recorded and binned into a histogram, resulting in a characteristic fluorescence decay for a specific fluorophore.…”

The WAK-like protein RFO1 acts as a sensor of the pectin methylation status in Arabidopsis cell walls to modulate root growth and defense

“…In the context of plant growth, the Arabidopsis PM-localized receptor kinase FERONIA has recently been shown to regulate pavement cell morphogenesis upon binding to highly dmPectin ( Lin et al., 2022 ). In Arabidopsis, the PM receptors BRASSINOSTEROID INSENSITIVE 1 (BRI1) and RECEPTOR-LIKE PROTEIN 44 (RLP44), together with the BRI1 co-receptor BRI1-Associated Kinase 1 (BAK1), have been reported to coordinately mediate plant responses to abnormally high levels of mPectin present at the cell wall ( Wolf et al., 2012 , 2014 ; Glöckner et al., 2022 ). Similarly, the WALL-ASSOCIATED KINASEs (WAKs) and WAK-likes (WAKLs) participate in controlling cell expansion ( Lally et al., 2001 ; Wagner and Kohorn, 2001 ; Wu et al., 2020 ; Li et al., 2021 ), presumably by sensing pectin perturbations.…”

Controlling Three-Color Förster Resonance Energy Transfer in an Optical Fabry–Pérot Microcavity at Low Mode Order

“…In addition, there can also be an energy transfer from the donor to the lowest-energy acceptor via the intermediate acceptor. Several recent studies have successfully demonstrated the development of single-molecule triple FRET techniques. − However, the extension of FRET to triple FRET is not as straightforward as it appears, and there are many challenges that limit the quantitative analysis of triple FRET data, e.g., severe photobleaching, small absorption/emission spectral overlap, and large distances between multicolor fluorescent dyes.…”