SignificanceCell-fate determination and cellular behavior in plants rely mainly on positional information and intercellular communication. A plethora of cues are perceived by surface receptors and integrated into an adequate cellular output. Here, we show that the small receptor-like protein RLP44 acts as an intermediary to connect the receptors for two well-known signaling molecules, brassinosteroid and phytosulfokine, to control cell fate in the root vasculature. Furthermore, we show that the brassinosteroid receptor has functions that are independent from the responses to its hormone ligands and reveal that phytosulfokine signaling promotes procambial cell identity. These results provide a mechanistic framework for the integration of multiple signaling pathways at the plasma membrane by shifting associations of receptors in multiprotein complexes.

Plants depend on various cell surface receptors to integrate extracellular signals with developmental programs. One of the beststudied receptors is BRASSINOSTEROID INSENSITIVE 1 (BRI1) in Arabidopsis (Arabidopsis thaliana). Upon binding of its hormone ligands, BRI1 forms a complex with a shape-complementary coreceptor and initiates a signal transduction cascade, which leads to a variety of responses. At the macroscopic level, brassinosteroid (BR) biosynthetic and receptor mutants have similar growth defects, which initially led to the assumption that the signaling pathways were largely linear. However, recent evidence suggests that BR signaling is interconnected with several other pathways through various mechanisms. We recently described that feedback from the cell wall is integrated at the level of the receptor complex through interaction with RECEPTOR-LIKE PROTEIN 44 (RLP44). Moreover, BRI1 is required for another function of RLP44: the control of procambial cell fate. Here, we report a BRI1 mutant, bri1 cnu4 , which differentially affects canonical BR signaling and RLP44 function in the vasculature. Although BR signaling is only mildly impaired, bri1 cnu4 mutants show ectopic xylem in place of procambium. Mechanistically, this is explained by an increased association between RLP44 and the mutated BRI1 protein, which prevents the former from acting in vascular cell fate maintenance. Consistent with this, the mild BR response phenotype of bri1 cnu4 is a recessive trait, whereas the RLP44-mediated xylem phenotype is semidominant. Our results highlight the complexity of plant plasma membrane receptor function and provide a tool to dissect BR signaling-related roles of BRI1 from its noncanonical functions. Plant cells perceive a multitude of extracellular signals through a battery of plasma membrane-bound receptors that are crucial for the integration of environmental and developmental signals. The response to the growth-regulatory brassinosteroid (BR) phytohormones is mediated by one of the best-characterized plant signaling pathways (Singh and Savaldi-Goldstein, 2015) initiated by a receptor complex containing the Leu-rich repeat receptor-like kinase BRASSINOSTE-ROID INSENSITIVE 1 (BRI1; Li and Chory, 1997) and its coreceptors of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family (Ma et al., 2016; Hohmann et al., 2017). Binding of the brassinosteroid ligand mediates hetero-dimerization of BRI1 and SERK family members such as BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1; Li et al., 2002; Nam and Li, 2002), which in turn triggers extensive autoand transphosphorylation of the intracellular BAK1 and BRI1 kinase domains (Hohmann et al., 2017). The activated kinases recruit and activate downstream BR signaling components, which eventually leads to vast changes in gene expression mediated by BR signalingregulated transcription factors such as BRASSINA-ZOLE-RESISTANT 1 (BZR1; Wang et al., 2002) and BRI1-EMS-SUPPRESSOR 1 (BES1; Yin et al., 2002). Among the transcriptional targets of these transcription factors, cell...

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.

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.

Plants rely on cell surface receptors to integrate developmental and environmental cues into behaviour adapted to the conditions. The largest group of these receptors, leucine-rich repeat receptor-like kinases, form a complex interaction network that is modulated and extended by receptor-like proteins. This raises the question of how specific outputs can be generated when receptor proteins are engaged in a plethora of promiscuous interactions. RECEPTOR-LIKE PROTEIN 44 (RLP44) acts to promote both brassinosteroid and phytosulfokine signalling, which orchestrate diverse cellular responses. However, it's unclear how these activities are coordinated. Here, we show that RLP44 is phosphorylated in its highly conserved cytosolic tail and that this post-translational modification governs its subcellular localization. Whereas phosphorylation is essential for brassinosteroid-associated functions of RLP44, its role in phytosulfokine signalling is not affected by phospho-status. Detailed mutational analysis suggests that phospho-charge, rather than modification of individual amino acids determines routing of RLP44 to its target receptor complexes, providing a framework to understand how a common component of different receptor complexes can get specifically engaged in a particular signalling pathway.