Development of Approaches and Identifying Limitations toward Functional Yeast Surface Display of FLS2

Abstract Pattern recognition receptors such as FLAGELLIN SENSING 2 (FLS2) are central to plant immunity and attractive targets for engineering broader detection of bacterial phytopathogens for application in pest management (and diagnostics) in food crops and sustainable agriculture practices. However, evaluating numerous FLS2 variants for altered pathogen sensing specificity directly in plants is slow and has low throughput and has been seldom optimized for heterologous display systems. Here, we established conditions that enabled Arabidopsis thaliana FLS2 ectodomain expression on the surface of Saccharomyces cerevisiae and evaluated 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 against non-FLS2 proteins 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 with serine. We identified a key glycan-site variant, N388S, which lies proximal to the flg22 binding interface and increased the binding population size under stress conditions. Binding assays against FLS2 variants and a reported non-binding variant affirmed that FLS2 selectivity was specific to FLS2 display, showing that all variants maintained a low affinity interaction with flg22. Together, these results point to FLS2 display conditions, not only glycosylation state, as an underlying limitation to detect true flg22 interactions, which will require more sensitive approaches to confidently resolve true binding populations.