Structural basis for recognition of RALF peptides by LRX proteins during pollen tube growth

Abstract

Plant reproduction relies on the highly regulated growth of the pollen tube for sperm delivery. This process is controlled by secreted RALF signaling peptides, which have previously been shown to be perceived by Catharanthus roseus RLK1-like (CrRLK1Ls) membrane receptor-kinases/LORELEI-like GLYCOLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEINS (LLG) complexes, or by leucine-rich repeat (LRR) extensin proteins (LRXs). Here, we demonstrate that RALF peptides fold into bioactive, disulfide bond-stabilized proteins that bind the LRR domain of LRX proteins with low nanomolar affinity. Crystal structures of LRX2–RALF4 and LRX8–RALF4 complexes at 3.2- and 3.9-Å resolution, respectively, reveal a dimeric arrangement of LRX proteins, with each monomer binding one folded RALF peptide. Structure-based mutations targeting the LRX–RALF4 complex interface, or the RALF4 fold, reduce RALF4 binding to LRX8 in vitro and RALF4 function in growing pollen tubes. Mutants targeting the disulfide-bond stabilized LRX dimer interface fail to rescue lrx infertility phenotypes. Quantitative biochemical assays reveal that RALF4 binds LLGs and LRX cell-wall modules with drastically different binding affinities, and with distinct and mutually exclusive binding modes. Our biochemical, structural, and genetic analyses reveal a complex signaling network by which RALF ligands instruct different signaling proteins using distinct targeting mechanisms.

Overlapping functions and protein-protein interactions of LRR-extensins in Arabidopsis

“…RALFs and modifies their binding to interaction partners as demonstrated for the CrRLK1L THE1 and its interaction partner RALF34 [20]. Similarly, binding of RALF4 to LRX8 is pH-dependent and stronger under acidic conditions [51]. In vivo and in vitro analyses have shown that dimerization of LRXs is necessary for biological activity [51].…”

“…It is possible that RALFs present in tobacco support the formation of the LRX1-FER interaction observed in Co-IP experiments and additional RALF1 does not further improve the formation of this protein complex. While the amino acid residues of the LRR domain involved in LRX-RALF interaction have been identified for some LRX/RALF pairs [51], the details on the LRX-FER interaction requires further analyses.…”

OsPEX1, a leucine-rich repeat extensin protein, plays an important role in the regulation of caryopsis development in rice

“…6B). A cysteine-rich domain (CRD) might act to stabilize the structure of the LRX protein through forming disul de bonds with cysteine residues of the LRR domain (Herger et al, 2019;Moussu et al, 2020). None of the OsLRXs genes except for OsPEX2 has introns (Fig.…”

Genomic structure and transcript analysis of the Rapid Alkalinization Factor (RALF) gene family during host-pathogen crosstalk in Fragaria vesca and Fragaria x ananassa strawberry

“…Binding RALF peptides to CrRLK1L receptors also involves other interacting partners such as Lorelei-like-Glycosylphosphatidylinositol-Anchored proteins (LLG1,2,3) [9,15] and Leucine-Rich Repeat Extensins (LRX). The latter has been reported to bind RALF4/19 in the pollen tube [16,17] and to interact with FER, as part of cell wall sensing system responsible for vacuolar expansion and cellular elongation [18]. Binding of RALFs to their receptors leads to a number of different intracellular signaling events involving different molecular components, mostly still unidentified.…”