Tuning Yeast Glycosylation Proximal to the FLS2-flg22 Binding Interface enables Functional Yeast Surface Display under Induced ER Stress

Pattern recognition receptors such as FLAGELLIN SENSING 2 (FLS2) are central to plant immunity and attractive targets for engineering broader detection of bacterial phytopathogens. However, evaluating numerous FLS2 variants directly in plants is slow and low throughput, and have been seldom optimized for heterologous display systems. Here, we established conditions that enabled Arabidopsis thaliana FLS2 ectodomain functional expression on the surface of Saccharomyces cerevisiae and binding to its cognate ligand, flg22. We show how yeast high-mannose glycosylation of the FLS2 ectodomain contributes to inefficient folding and loss of detectable flg22 binding in standard yeast surface display conditions. Substitutions at all N-glycosylation motifs compromised surface expression, indicating that some glycosylation is required for trafficking. We tuned the extent of glycosylation using tunicamycin, an N-linked glycosylation inhibitor, in combination with thermal stress to modulate ER quality control. Under these conditions, we observed a reproducible subpopulation of cells with improved flg22 binding despite reduced overall expression, and we confirmed flg22 selectivity using both flow cytometry and magnetic bead–based enrichment. Guided by structural modeling of high-mannose glycans on the FLS2 ectodomain, we then substituted asparagines at selected N-glycan sites. We identified a key glycan site variant, N388, which lies proximal to the flg22 binding interface and enhanced functional expression under stress conditions. Together, these results reveal that lineage-specific glycosylation patterns and ER stress responses critically shape the functional display of FLS2 in yeast. This approach provides a framework for engineering yeast surface display platforms for high-throughput screening of FLS2 and related immune receptors.