Three-fluorophore FRET enables the analysis of ternary protein association in living plant cells

Abstract

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 BRI1-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.

Computational modeling and quantitative physiology reveal central parameters for brassinosteroid-regulated early cell physiological processes linked to elongation growth of theArabidopsisroot

“…For example, the BRI1-dependent regulation of the vascular cell fate in the MZ of the root or the BRI1-mediated cross-tissue control of the cell wall homeostasis require nano-organized BRI1 complexes that contain at least additionally RLP44 ( Wolf et al, 2014 ; Holzwart et al, 2018 ). Moreover, RLP44-containing BRI1/BAK1 nanoclusters are spatially distinct from for instance FLS2/BAK1 nanoclusters ( Glöckner et al, 2020 ).…”

Visualizing protein-protein interactions in plants by rapamycin-dependent delocalization

“…Trifluorophore FRET-FLIM and trimolecular fluorescence complementation (Glöckner et al, 2019;Offenborn et al, 2015) have been shown to work in plants. The spatial tagging limitations required to have a positive readout using the above tools however are likely limiting to apply this for any random complex of choice.…”

“…Recently FRET-FLIM has been developed into a three-fluorophore system which enabled to visualize triple PPIs (Glöckner et al, 2019). Nevertheless, FRET-FLIM is quite-labor intense, requires careful interpretation and multiple control assays.…”

Deviating from the Beaten Track: New Twists in Brassinosteroid Receptor Function

“…However, the cell wall also feeds back on BR signalling [153]. The state of pectin, an important component of the plant cell wall and a dynamic regulator of its properties [154][155][156][157], is relayed to the BR receptor complex by RECEPTOR-LIKE PROTEIN44 (RLP44), which directly interacts with both BRI1 and BAK1, likely acting as a scaffold to promote their association [103,158,159]. In line with this notion, RLP44 is sufficient to promote BR signalling, at least partially independent of BRs [103].…”