Yeast surface display reveals structure-dependent β-glucan association with the plant immune receptor AtIGP1

Abstract

Cell wall-derived oligosaccharides can function as immune-active signals in plants and trigger pattern-triggered immunity (PTI) through pattern recognition receptors (PRRs). In Arabidopsis thaliana, the leucine-rich repeat malectin (LRR-MAL) receptor kinase IGP1 (impaired in glycan perception 1) contributes to immune responses induced by cellulose- and mixed-linkage {beta}-1,3/1,4-glucan (MLG)-derived oligosaccharides, although whether MLG-derived oligosaccharides are directly recognized by AtIGP1 needs further clarification. We established a Saccharomyces cerevisiae surface-display system in which the AtIGP1 extracellular domain (ECD) is presented at the yeast cell surface, allowing oligosaccharide recognition to be examined separately from downstream plant signaling and without receptor purification. Previously, cellulose-derived oligosaccharides have been shown to elicit plant defense responses via AtIGP1. Here, surface-displayed AtIGP1 retained the ability to recognize fluorescently labeled cellotriose (CEL3) and cellopentaose (CEL5) ligands, supporting functional presentation of the receptor for investigating glycan recognition in a heterologous context. Using this platform, we investigated the association of AtIGP1 with fluorescently labeled MLG-derived oligosaccharides differing in degree of polymerization (DP) and glycosidic linkage arrangement. This analysis revealed a specific, competitively displaceable association with MLG DP2 (G3G; laminaribiose), whereas the DP3 (G4G3G) and DP4 (G4G4G3G) structures tested showed no detectable association. To test whether this association is relevant in planta, we treated A. thaliana seedlings with unlabeled MLG DP2, which induced a cytosolic Ca2+ response, an early readout of immune activation. This response was markedly reduced in the igp1-3 mutant. Together, these results identify laminaribiose as an additional candidate ligand of AtIGP1. The established yeast surface-display platform thus provides an approach for testing candidate oligosaccharides for association with the extracellular domains of plant PRRs.