Mechanisms of RALF peptide perception by a heterotypic receptor complex

Abstract

Receptor kinases of the Catharanthus roseus RLK1-like (CrRLK1L) family have emerged as important regulators of plant reproduction, growth and responses to the environment1. Endogenous RAPID ALKALINIZATION FACTOR (RALF) peptides2 have previously been proposed as ligands for several members of the CrRLK1L family1. However, the mechanistic basis of this perception is unknown. Here we report that RALF23 induces a complex between the CrRLK1L FERONIA (FER) and LORELEI (LRE)-LIKE GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEIN 1 (LLG1) to regulate immune signalling. Structural and biochemical data indicate that LLG1 (which is genetically important for RALF23 responses) and the related LLG2 directly bind RALF23 to nucleate the assembly of RALF23-LLG1-FER and RALF23-LLG2-FER heterocomplexes, respectively. A conserved N-terminal region of RALF23 is sufficient for the biochemical recognition of RALF23 by LLG1, LLG2 or LLG3, and binding assays suggest that other RALF peptides that share this conserved N-terminal region may be perceived by LLG proteins in a similar manner. Structural data also show that RALF23 recognition is governed by the conformationally flexible C-terminal sides of LLG1, LLG2 and LLG3. Our work reveals a mechanism of peptide perception in plants by GPI-anchored proteins that act together with a phylogenetically unrelated receptor kinase. This provides a molecular framework for understanding how diverse RALF peptides may regulate multiple processes, through perception by distinct heterocomplexes of CrRLK1L receptor kinases and GPI-anchored proteins of the LRE and LLG family.

CrRLK1L receptor‐like kinases HERK1 and ANJEA are female determinants of pollen tube reception

“…We hypothesise that LRE could act as co-receptor for FER and HERK1 or ANJ at the filiform apparatus, forming tripartite HERK1-LRE-FER or ANJ-LRE-FER complexes that sense pollen-derived ligands such as RALF peptides or cell wall ▸ Figure 6. Confirmation of the role of CrRLK1Ls and LRE proteins as RALF peptide sensors has been recently obtained through an elegant combination of crystallographic and biochemical techniques [35]. components, in a mechanism analogous to that described for pollen tube growth through BUPS1/2-ANX1/2-LLG2/3-RALF4/19 signalling.…”

“…LRE functions in the maternal control of fertilisation and early seed development [31,57], whereas its homolog LLG1 is restricted to vegetative growth and plant-pathogen interactions [34]. LRE proteins are thought to stabilise their receptor partners in the plasma membrane and act as direct co-receptors for the extracellular cues sensed by the RLK [18,35]. LRE proteins are thought to stabilise their receptor partners in the plasma membrane and act as direct co-receptors for the extracellular cues sensed by the RLK [18,35].…”

“…Model of HERK1/ANJ involvement in pollen tube reception. By solving the structure of a FER-LLG1-RALF23 complex, Xiao et al [35] have demonstrated that (i) LRE proteins play a central role in the recognition of RALFs; (ii) the N-terminal region of a subgroup of RALFs is sufficient to induce the interaction between LRE proteins and FER; (iii) while LLG1-3 proteins are capable of binding RALF23, interaction between LRE and RALF23 was not detected; and (iv) specific amino acid differences between LRE and LLG1-3 proteins are responsible for such affinity differences. Further verification of the protein-protein interactions described here could be done via Förster resonance energy transfer (FRET) analysis, cryo-electron microscopy [59], or super-resolution microscopy techniques such as Stimulated Emission-Depletion Measurements (STED [60]).…”

“…LRE and its homolog LORELEI-LIKE GPI-ANCHORED PROTEIN 1 (LLG1) physically interact with RLKs FER, FLAGELLIN SENSING 2 (FLS2) and EF-TU RECEPTOR (EFR) [18,34]. Mutations in these GPI-anchored proteins and their associated RLKs result in similar phenotypes, with LRE and LLG1 regarded as co-receptors and/or stabilisers of RLK function [18,34,35]. HERK1, ANJ and FER are closely related RLKs and, given the similarities in reproduction defects and subcellular localisation in synergid cells ( Fig 3A), we hypothesised that HERK1 and ANJ may act in complex with LRE and/or FER at the filiform apparatus.…”

Rapid Alkalinization Factor (RALF) gene family genomic structure and transcriptional regulation during host-pathogen crosstalk in Fragaria vesca and Fragaria x ananassa strawberry

“…RALF genes are translated as pre- pro-peptides and activated in the apoplast through proteolytic cleavage. Besides the signal sequence necessary for extracellular extrusion, canonical RALF proteins contain distinctive amino acid motifs, such as the RRILA motif for S1P protease recognition [7] and the YISY motif, important for signaling cascade activation [8, 9]. In addition, four conserved cysteines form two disulfide bonds stabilise the mature RALF proteins.…”