Phosphorylation-dependent routing of RLP44 towards brassinosteroid or phytosulfokine signalling

Abstract

Plants rely on a complex network of 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 acts to promote brassinosteroid and phytosulfokine signalling, which orchestrate a wide variety of cellular responses. However, it is unclear how these activities are coordinated. Here, we show that RLP44 is phosphorylated in its highly conserved C-terminal cytosolic tail and that this post-translational modification governs its subcellular localization. RLP44 variants in which phosphorylation is blocked enter endocytosis prematurely, leading to an almost entirely intracellular localization, whereas mimicking phosphorylation results in preferential RLP44 localization at the plasma membrane. Phosphorylation is crucial for regulating RLP44's interaction with the brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1, and thus its function in BR signalling activation. In contrast, the interaction of RLP44 with PHYTOSULFOKINE RECEPTOR 1 is not affected by its phospho-status. Analysis of the contribution of individual amino acid modifications suggests that routing of RLP44 to its target receptor complexes is controlled by its phosphorylation pattern, providing a framework to understand how a common component of different receptor complexes can get specifically engaged in a particular signalling pathway.

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

“…In theory, the lower expression of FLS2 could mask a possible interaction between RLP44 and FLS2 in intensity-based FRET measurements. However, no interaction between them was observed in the more sensitive FRET-FLIM measurements and previously, no interaction was found in BiFC (Garnelo Gomez et al, 2019). Our observation that BRI1 and FLS2 are located in distinct sub-compartments in the PM is in accordance with previous evidence by variable angle epifluorescence microscopy (VAEM) using BRI1-eGFP and FLS2-RFP (Bücherl et al, 2017).…”

“…It was shown previously, that BRI1-BAK1, RLP44-BRI1 and RLP44-BAK1 form heterodimers (Nam and Li, 2002;Wolf et al, 2014;Holzwart et al, 2018). As a negative control, the immune-response mediating FLAGELLIN SENSING2 FLS2 was used as it is a plasma membrane-localized LRR-RLK like BRI1, that did not interact with RLP44 or BRI1 previously (Bücherl et al, 2017;Garnelo Gomez et al, 2019). The spectra were acquired in transiently transformed N. benthamiana leaves.…”

“…A recent study also showed that RLP44 plays a role in vascular development, which involves phytosulfokine hormone signalling (Holzwart et al, 2018). The phospho-state of the short, C-terminal tail of RLP44 is important for the interaction with BRI1 and determines, whether RLP44 is located at the PM or internalized into intracellular compartments (Garnelo Gomez et al, 2019). This suggests, that RLP44 directly modulates BR-signalling upon input from cell wall integrity signalling cues.…”

Nanobody-dependent delocalization of endocytic machinery in Arabidopsis root cells dampens their internalization capacity

“…This twd40-2-3 mutant (Bashline et al, 2015) is however likely merely a knockdown as twd40-2-1 and twd40-2-2 mutants are pollen lethal (Gadeyne et al, 2014). Knockdowns of TML and TPLATE resulted in seedling lethality with a reduced internalization of FM4-64, BRI1, RECEPTOR-LIKE PROTEIN 44 (RLP44) and the cellulose synthase subunit CESA6 (Irani et al, 2012; Gadeyne et al, 2014; Sánchez-Rodríguez et al, 2018; Gómez et al, 2019). Silencing works on the messenger level and phenotypes only become apparent following degradation of pre-made proteins.…”

Regulation and activation of SOBIR1-containing receptor complexes involved in plant immune signalling

“…Additionally, overlapping components involved in multiple signalling pathways can define their specific roles via the employment of differential phospho-sites, such as phosphorylation of BAK1 at residues Y403, S602, T603, S604 and S612 is required for its role in FLS2 signalling, but not in BRI1 signalling (Perraki et al, 2018;Schwessinger et al, 2011). Furthermore, strikingly, RLP44 is phosphorylated in vivo, and the phospho-site S268 is important for RLP44 to activate BRI1 signalling, but not PSKR1 signalling (Gómez et al, 2019;Wolf et al, 2014).…”