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

Tuning Yeast Glycosylation Proximal to the FLS2-flg22 Binding Interface enables Functional Yeast Surface Display under Induced ER Stress | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Tuning Yeast Glycosylation Proximal to the FLS2-flg22 Binding Interface enables Functional Yeast Surface Display under Induced ER Stress View ORCID Profile Benedikt Dolgikh , View ORCID Profile Samantha Schulte , View ORCID Profile Daniel R. Woldring doi: https://doi.org/10.1101/2025.11.25.690463 Benedikt Dolgikh 1 Department of Biochemistry and Molecular Biology, Michigan State University 3 Institute for Quantitative Health Science and Engineering, Michigan State University Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Benedikt Dolgikh Samantha Schulte 2 Department of Chemical Engineering and Materials Science, Michigan State University 3 Institute for Quantitative Health Science and Engineering, Michigan State University Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Samantha Schulte Daniel R. Woldring 2 Department of Chemical Engineering and Materials Science, Michigan State University 3 Institute for Quantitative Health Science and Engineering, Michigan State University Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Daniel R. Woldring For correspondence: woldring{at}egr.msu.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF 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. 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. Download figure Open in new tab Introduction Global climate change and rapid pathogen evolution threaten sustainable agriculture by increasing disease pressure on major crops ( 1 – 3 ). For instance, Huanglongbing (citrus greening) has contributed to a ∼90% decline in Florida citrus production since the mid-2000s ( 4 – 6 ). Many bacterial pathogens are detected by orthologs of the pattern recognition receptor (PRR) FLAGELLIN SENSING 2 (FLS2), a leucine-rich repeat receptor-like kinase that perceives conserved microbe-associated molecular patterns in bacterial flagellin (flg22). This detection initiates pattern-triggered immunity via BAK1 co-receptor recruitment and downstream signaling ( 7 – 11 ). FLS2-flg22 recognition exhibits clear signatures of co-evolution: single amino acid changes in flg22 sequences can abolish perception by specific FLS2 variants, allowing pathogens to evade detection, whereas compensatory mutations in specific FLS2 orthologs restore or broaden responsiveness to previously unrecognized flg22 peptides ( 7 , 12 – 15 ). This tunability makes FLS2 a powerful candidate for protein engineering aimed at expanding the spectrum of detectable bacterial pathogens. High-throughput engineering of FLS2 variants in planta remains challenging. Even with advances in CRISPR-Cas and nanoparticle-based delivery ( 16 , 17 ), generating and phenotyping transgenic plants typically supports libraries of only 10 2 -10 3 variants over several months ( 18 ). Heterologous hosts (bacteria, insect cells, or mammalian cells) offer alternative routes for protein production, but often lack plant-like glycosylation or are not optimized for large-scale surface display campaigns ( 19 – 25 ). Yeast ( Saccharomyces cerevisiae and Pichia pastoris ) occupy a useful middle ground: they are eukaryotic, genetically tractable, and support yeast surface display platforms that routinely screen >10⁷ variants in weeks ( 26 – 30 ). However, FLS2 is an unusually large, heavily glycosylated receptor, and prior attempts to display its ectodomain on yeast failed to yield detectable ligand binding ( 31 ). Non-optimized binding conditions and differences in protein folding between plants and yeast, such as during endoplasmic reticulum quality control (ERQC) and N-linked glycosylation ( 23 , 25 , 32 – 36 ), may complicate functional expression. Thus, we set out to address the previous shortcomings in establishing a functional FLS2 ectodomain display system. Here, we systematically investigate how yeast N-linked glycosylation and ER stress influence surface display and ligand binding of the A. thaliana FLS2 ectodomain. We show that FLS2 expressed on the yeast surface is extensively modified with high-mannose glycans, that complete removal of N-glycosylation motifs impairs trafficking, and that partial inhibition of glycosylation combined with thermal stress yields a subpopulation of cells with detectable flg22 binding. Guided by structural modeling of high-mannose glycans on the FLS2 ectodomain, we identify the N388 glycan site, which is proximal to the flg22 binding interface, to be particularly disruptive to FLS2 functional expression. These findings define a set of parameters for achieving functional yeast surface display of FLS2 and provide design principles for expressing other plant PRRs in yeast. Results and Discussion FLS2 is hyperglycosylated and non-functional when surface displayed in yeast To establish a high-throughput platform for screening FLS2 variants, we first displayed the A. thaliana FLS2 ectodomain on the surface of S. cerevisiae as a standard Aga2 fusion ( Figure S1– S2 ), hereafter termed FLS2LRR. Upon induction, flow cytometry showed that a high fraction of cells (>50%) expressed the fusion protein on the surface, and Western blot analysis confirmed robust protein expression ( Figure 1A,C ; Figure S3 ). Surprisingly, despite this high expression level, we did not detect binding to the cognate ligand flg22 under these standard induction and labeling conditions ( Figure S3 ). Download figure Open in new tab Figure 1. Yeast N-linked glycosylation supports maximal surface expression. (A) Western blot analysis of Aga2-HA-FLS2 (FLS2LRR) after Endo H treatment under denaturing or non-denaturing conditions attributes large kDa shift to yeast-specific glycosylation. (B) Diagram of expected mass change for non-glycosylated Aga2-HA-FLS2 (NtoS) with N-linked glycan associated asparagines (22 sites) mutated to serine. (C) The mean percentage of FLS2LRR and NtoS populations with surface display expression.(D) Western blot analysis of surface displayed (YSD) or cell lysate extracted (cytosolic) FLS2LRR and NtoS with or without denaturing Endo H treatment. For western blots, FLS2LRR and NtoS are detected using rat anti-HA-biotin (3F10) and streptavidin-647 antibodies. Cytometry data (C) shown is the mean and standard error for n=4 biological replicates per group (each bar) where p-value of * = < 0.05. We reasoned that differences between plant and yeast ERQC and post-translational modification pathways could yield a non-functional FLS2 fold. In particular, FLS2 orthologs contain numerous highly conserved N-linked glycosylation motifs (NXS/T, where X ≠ Pro) ( 37 ) ( Figure S4 ). In contrast to the complex/hybrid, often xylose- and/or fucose-containing plant glycans, yeast N-linked glycans are predominantly high-mannose structures ( Figure S5 )( 38 ). These lineage-specific differences motivated us to examine how yeast glycosylation affects the functional expression of FLS2LRR. Given the 22 putative N-linked glycosylation sites on the FLS2 ectodomain, we anticipated that high-mannose glycans would add ∼30–67 kDa to the ectodomain mass ( Figure S6 ), consistent with hyperglycosylation observed for other heterologous glycoproteins in S. cerevisiae ( 39 ). To test this, we expressed FLS2LRR on the yeast surface, treated cells with DTT to cleave the disulfide bond between Aga1 and Aga2–FLS2LRR, and collected the supernatant containing the released fusion protein. We then treated this extract with endoglycosidase H (Endo H), which cleaves high-mannose N-linked glycans between the two terminal GlcNAc residues ( Figure S5 ), under either denaturing or native conditions. Under denaturing conditions, we expected glycans to be fully accessible, allowing near-complete de-glycosylation. Under native conditions, incomplete removal of glycans would indicate steric occlusion of Endo H cleavage sites and thereby provide indirect information about protein folding ( 40 ). Western blot analysis revealed that untreated FLS2LRR migrated as a broad band at >140 kDA, which collapsed to a sharper band at ∼115 kDa after Endo H treatment under denaturing conditions ( Figure 1A ). In contrast, Endo H treatment under non-denaturing conditions resulted in an intermediate pattern, with partial molecular weight reduction consistent with only partial de-glycosylation. The large apparent molecular weight and incomplete glycan removal under native conditions together indicate that FLS2LRR is heavily modified with high-mannose N-glycans in yeast and that at least some folding has occurred to partially shield glycan sites. Consequently, hyperglycosylation and partial folding could help explain why the ectodomain does not adopt a fully functional conformation. Thus, to probe this hypothesis further, we implemented multiple approaches to manipulate ectodomain glycosylation. Complete removal of N-glycans impairs FLS2 trafficking and surface expression Because non-functional FLS2LRR was heavily modified with high-mannose N-glycans, we next asked whether glycosylation is strictly required for FLS2 expression and trafficking, or whether reduced glycosylation could improve functional display. We anticipated that genetic removal of N-glycan sites via substitution of asparagine residues within NxS/T motifs could influence both expression (folding and translocation to the surface) and function (flg22 binding). To systematically identify amino acid substitutions compatible with FLS2 structure, we evaluated alternative residues at all 22 putative glycosylation-site asparagines guided by the frequency of amino acid substitution at a given residue in nature (BLOSUM62 substitution frequencies) and modeled each sequence using AlphaFold2 (AF2) Multimer ( 41 )( Figure S7A ). AF2-predicted structures were analyzed with Rosetta stability calculations ( 42 – 44 ), and asparagine-to-serine substitutions at all 22 sites yielded the most similar stability to the wild-type (WT) structure ( Figure S7B ). Based on these analyses, we chose the asparagine-to-serine variant to generate a fully de-glycosylated FLS2 ectodomain Aga2 fusion (NtoS) ( Figure 1B ). This may be advantageous compared to the asparagine-to-aspartate substitutions described in the literature ( 32 ), given that serine physicochemical properties are more suitable for maintaining continuous hydrogen bonding to preserve fold at LRR motifs ( 45 ). We compared surface expression of FLS2LRR and NtoS using flow cytometry. Across biological replicates (n = 3), the mean fraction of expression-positive cells was ∼60% for FLS2LRR and ∼40% for NtoS ( Figure 1C ). Since properly folded proteins are more likely to be trafficked to the surface rather than routed to degradation pathways or aggregation ( 46 , 47 ), this reduction in surface expression is consistent with a role for N-glycans in promoting FLS2 folding and/or trafficking. Western blot analysis of equivalent quantities of total protein from surface-extracted and cytosolic fractions confirmed that a majority of both FLS2LRR and NtoS remained trapped inside the cell during induction ( Figure 1D ). Notably, a larger fraction of NtoS than FLS2LRR protein was present in the cytosolic fraction, consistent with impaired secretion and/or surface trafficking when all ectodomain N-glycosylation motifs are removed. Together, these results indicate that while yeast high-mannose glycosylation contributes to a non-functional FLS2 fold under baseline conditions, complete removal of all N-glycans also impairs FLS2 trafficking and reduces surface expression. Thus, some degree of glycosylation appears necessary for productive folding and export of Aga2–FLS2 in yeast, suggesting that an intermediate “reduced but not absent” glycosylation state might be optimal for functional display ( Figure S8 ). Tunicamycin-mediated partial glycosylation and thermal stress promote weak but selective flg22 binding Although our data showed that yeast glycosylation promotes FLS2 expression, multiple studies suggest that glycosylation is not strictly required for flg22 recognition ( 32 , 33 , 48 ). Sun et al. (2012) reported that a panel of FLS2 glycosylation site variants, ranging from single to octuple glycan-site substitutions, largely retained flg22 responsiveness ( 32 ). Only two of four octuple mutants showed partial loss of responsiveness. In that work it was not clear whether reduced responsiveness arose from poor expression of the mutants in T1 seedlings or from weakened binding. Additionally, Tang et al. (2019) crystallized the FLS2–flg22–BAK1 complex after removing FLS2 glycans with endoglycosidases following recombinant insect expression ( 48 ). These findings imply that reduced glycosylation should not inherently block flg22 binding as long as overall folding is preserved. To test whether partial reduction of yeast glycosylation could improve flg22 binding while maintaining expression, we used tunicamycin, a nucleoside antibiotic that inhibits N-linked glycan formation by competitively inhibiting DPAGT1 (ALG7 in yeast) early in lipid-linked oligosaccharide (LLO) biosynthesis ( 49 , 50 ). Tunicamycin concentration controls the availability of LLOs for oligosaccharyltransferase (OST) complex–mediated glycosylation, thereby tuning the number of occupied glycosylation sites and influencing glycoprotein heterogeneity and expression levels ( 51 – 53 ). We induced FLS2LRR-expressing cells in the presence of 0, 0.2, or 2 µg/mL tunicamycin and collected DTT-released supernatant protein for Western blot analysis. Increasing tunicamycin concentrations decreased, but did not completely eliminate, fully glycosylated FLS2LRR species, with 2 µg/mL producing the most pronounced shift toward lower molecular weight forms ( Figure S9A ). Because tunicamycin globally affects glycoprotein synthesis, it can also compromise cell viability and potentially increase non-specific peptide binding by altering protein surface chemistry ( 49 , 54 , 55 ). To account for this, we compared flg22 binding in FLS2LRR-displaying cells to unrelated surface-displayed proteins (Aga2–HA–HaloTag and Aga2–HA–RIXI) across the same tunicamycin treatments using flow cytometry. We quantified both the percentage of flg22-binding cells and the percentage of expression-positive cells across independent replicates (n = 3). For FLS2LRR, the mean fraction of flg22-positive cells increased from 0.2% at 0 µg/mL to 5.4% and 11.2% at 2 µg/mL tunicamycin ( Figure 2A,B ), whereas HaloTag-and RIXI-displaying populations showed smaller increases (2.6% to 3.4% and 3.3% to 5.8%, respectively). The consistently larger and statistically significant increase in flg22-positive events for FLS2LRR relative to non-binder controls at 2 µg/mL tunicamycin indicates that partial inhibition of glycosylation enhances selective flg22 recognition by FLS2LRR, rather than merely increasing non-specific peptide binding. The variation in FLS2LRR flg22-positive percentages between biological replicates (5.4–11.2%) is consistent with tunicamycin generating a heterogeneous distribution of under-glycosylated FLS2 molecules, where replicate-to-replicate differences in the subset of occupied sites could influence the fraction of receptors capable of binding flg22 ( Figure S8B ). Download figure Open in new tab Figure 2. flg22 is selective for FLS2LRR even after tunicamycin treatment during induction. Fraction of FLS2LRR – or HTWT (A) or RIXI (B) – expressing cells that bind flg22 upon induction with increasing concentrations of tunicamycin. Cytometry data (A,B) shown are the mean and standard error for n=3 biological replicates after background subtraction (see methods) per group (each bar) with Bonferroni Correction factor of 0.025; * < 0.025, ** < 0.0125, *** < 0.00625. HTWT: HaloTag, Engineered R. rhodochrous haloalkane dehalogenase (UNIPROT ID: P0A3G2, DHAA_RHORH; PDB ID: 5Y2X). RIXI: Rice GH11 Xylanase Inhibitor (UNIPROT ID: Q7GCM7, XIP_ORYSJ). Tunicamycin treatment also intersects with unfolded protein response (UPR) and ER stress pathways, which can either suppress or enhance recombinant protein production depending on the stimulus and context ( 52 , 56 , 57 ). To further probe the role of ER stress and secretory capacity in FLS2 functional expression, we combined tunicamycin with thermal stress during induction. In yeast, heat shock responses promote UPR activation and chaperone expression, which can aid folding and prevent aggregation of difficult-to-express proteins ( 58 , 59 ). Given that a majority of both FLS2LRR and NtoS remained intracellular under standard conditions ( Figure 1D ), we hypothesized that exogenous stress could modulate ERQC and improve the fraction of FLS2 molecules trafficked in a functional state. We induced FLS2LRR- and NtoS-displaying cells under three stress conditions: tunicamycin alone (2 µg/mL), heat alone (37 °C), and combined tunicamycin plus heat (“additive stress”). Then, with three independent biological replicates, we analyzed the mean flg22 binding and expression from flow cytometry experiments ( Figure 3 ). For FLS2LRR, the fraction of flg22-binding cells increased from 0.4% under no stress to 3.0% with heat, 13.4% with tunicamycin, and 13.8% with additive stress ( Figure 3A ). For NtoS, flg22 binding showed a significant increase under heat alone (5.4% to 14.7%), whereas tunicamycin alone had a smaller effect ( Figure 3C ). Median fluorescence intensity (MFI) for FLS2LRR expression decreased ∼4.2-fold upon tunicamycin treatment ( Figure 3B ), and NtoS expression decreased ∼1.9-fold under additive stress ( Figure 3D ), indicating a trade-off between expression level and functional binding. Density plots further revealed that NtoS flg22-binding/expression-positive events were detectable with equal or fewer stress conditions than FLS2LRR, while tightly clustered double-positive populations for FLS2LRR were most prominent under additive stress ( Supporting Material 2 – Supplementary Information 2 ). Download figure Open in new tab Figure 3. Tunicamycin and thermal stress impact FLS2LRR flg22 binding differently than NtoS. The mean fraction of background subtracted binding+ve/expression+ve FLS2LRR (A) and NtoS (C) populations after induction with tunicamycin, thermal stress, or both. The means of median fluorescence intensity from total surface expression+ve FLS2LRR (B) and NtoS (D) populations after induction with tunicamycin, thermal stress or both. Cytometry data (A-D) shown is the mean and standard error for n=3 biological replicates after background subtraction (see methods) per group (each bar) with Bonferroni Correction factor of 0.0125; * < 0.0125, ** < 0.00313, *** < 0.00078. To compare the apparent binding strength under these optimized conditions with values reported in planta, we next measured flg22–FLS2 binding across a range of ligand concentrations. Chinchilla et al. (2006) reported IC₅₀ values of 5–10 nM for competition of radiolabeled flg22 by unlabeled flg22 in plant membrane preparations ( 60 ). In contrast, our cytometry experiments had used relatively high flg22-TAMRA concentrations (1–10 µM) to saturate binding. High ligand concentrations can exaggerate weak or non-specific interactions in yeast surface display ( 61 ), so we anticipated that the apparent affinity in our system would be substantially weaker than in planta . We induced FLS2LRR and NtoS under optimized stress conditions (37 °C, 2 µg/mL tunicamycin) and incubated cells with 100, 500, 1000, 3000, or 5000 nM flg22-TAMRA prior to flow cytometry. For each construct, MFI increased with ligand concentration and approached a plateau at low micromolar ligand levels ( Figure 4 ). Fitting these curves with MFI as a proxy for receptor occupancy yielded apparent KD values of ∼5–10 µM for both FLS2LRR and NtoS. These estimates are several orders of magnitude weaker than the 5–10 nM IC₅₀ values reported in planta , likely reflecting residual misfolding or suboptimal glycosylation in yeast, differences in assay format (fluorescent ligand binding to yeast-displayed receptors versus radioligand binding to plant membranes), and the contribution of low-level non-specific interactions at high ligand concentrations. To mitigate background, we subtracted the fraction of TAMRA signal associated with FLS2-negative cells from the FLS2-positive population for all analyses ( Supporting Material 2 – Supplementary Information 1 ), but considerable background remained at >1 µM ligand. Download figure Open in new tab Figure 4. On the surface of yeast, FLS2LRR and NtoS show weak affinity for flg22-TAMRA. The binding affinity of FLS2LRR and NtoS after 2 ug/mL tunicamycin, 37 °C induction. Surface display cells were treated with 100, 500, 1000, 3000, and 5000 nM flg22-TAMRA and median fluorescence intensity was obtained to determine binding affinity. Affinity (K D ) was estimated by fitting flg22-TAMRA concentration and median fluorescence intensity with a nonlinear least-squares regression curve showing mean and standard error for n=3 biological replicates per group (each point). Despite these limitations, the consistent increase in flg22 binding for FLS2LRR and NtoS under stress conditions, relative to non-binding controls, indicates that tunicamycin-mediated partial glycosylation and thermal stress together promote a subpopulation of yeast-displayed FLS2 molecules that weakly but selectively recognize flg22. Magnetic bead enrichment confirms flg22 selectivity despite low affinity Because flow cytometry–based KD estimates indicated weak binding and substantial background, we sought an orthogonal, more sensitive method to confirm flg22 selectivity for yeast-displayed FLS2. We reasoned that magnetic bead–based enrichment should be capable of capturing weak-affinity interactions (>1 µM) if selections are conducted under saturating ligand and bead conditions ( 62 , 63 ). We adapted a previously reported yeast display selection workflow ( 63 ), using streptavidin-coated magnetic beads that were either bare (depletion of bare bead binders), coated with biotin– IgG (depletion of non-target protein binders), or biotin–flg22 (enrichment of flg22 target protein binders) ( Figure S10 ). FLS2LRR-expressing cells (“binder”) and RIXI-expressing cells (“non-binder”) were induced under either additive stress or no stress, mixed at a 1:1000 binder:non-binder ratio, and subjected to sequential negative and positive selections. Bead-bound cells were recovered and analyzed after each round, and clones were genotyped to determine the fraction of FLS2LRR versus RIXI in the enriched populations ( Figure S10–S11 ). After the first round of selection, all screened clones from each induction condition were RIXI-positive (non-binders), indicating that a single round was insufficient to overcome the initial 1:1000 dilution and weak affinity of FLS2–flg22 ( Figure S11A ). However, after the second round of selection, FLS2LRR clones were detected in almost all replicate selection and induction groups, with enrichment factors between ∼330-and 830-fold for additive stress–induced cells in two independent experiments ( Figure S11B, Supporting Material 1 - Supplementary Information 1 ). The only condition that failed to enrich FLS2LRR clones was the bare-bead negative selection without stress, which is consistent with the requirement for stress-optimized conditions to produce functionally competent receptors. These enrichment results provide additional evidence of flg22 selectively, recognizing yeast-displayed FLS2 despite the low apparent affinity measured by flow cytometry. In particular, the ability to recover FLS2LRR clones from a 1:1000 excess of non-binding cells using flg22-coated beads indicates that a subset of yeast-displayed receptors engages flg22 with sufficient specificity to support functional enrichment, even when detection at the single-cell level is limited by background and weak binding. Structure-guided removal of a proximal N-glycan (N388) enhances functional expression Tunicamycin-based partial glycosylation improved flg22 binding but did so by globally perturbing glycoprotein biosynthesis and generating a heterogeneous distribution of glycan occupancy across FLS2LRR molecules ( Figure S9B ). To more precisely dissect how individual glycosylation sites influence functional expression, we turned to structure-guided design of N-glycan site variants. We modeled yeast high-mannose glycans onto the FLS2 ectodomain crystal structure (PDB ID 4MN8)( 11 ) using the GlycoShape Re-glyco tool ( 64 ) ( Figure 5A ). The modeled structure was aligned to the original crystal structure in PyMOL ( 65 ), focusing on FLS2 (chain A) and flg22 (chain C). We then analyzed the spatial relationship between modeled glycans and the flg22 binding interface, as well as regions where multiple glycans appeared to cluster. We defined two classes of sites: (i) “proximal” sites where glycan atoms approached within 5 Å of flg22 and could plausibly occlude the binding surface, and (ii) “aggregated” sites where modeled high-mannose glycans from multiple nearby N-glycan motifs appeared to crowd local regions of the ectodomain, potentially hindering optimal folding. From these analyses, we identified three proximal sites (N347, N371, N388) and two aggregated sites (N631, N704). Download figure Open in new tab Figure 5. Glycosylation site variants including Var1 (N388S) typically outperform FLS2LRR after additive stress induction. (A) GlycoShape Re-Glyco modeled FLS2 ectodomain crystal structure (PDB ID 4MN8) with high-mannose glycans (GlyTouCan ID G92042VQ). Transparent glycans are modified for that specific FLS2LRR glycan variant. (B-C) The fraction binding/expression positive populations for FLS2LRR, four select site NtoS variants, and NtoS after induction of protein expression at either (B) 37 °C or (C) 37 °C with 2 ug/mL tunicamycin. (D) Comparison of events between both stress condition sample groups for the four select site NtoS variants plotted to visualize the significance of tunicamycin on functional expression. Cytometry data (B-D) shown is the mean and standard error for n=3 biological replicates after background subtraction (see methods) per group (each bar) with Bonferroni Correction factor of 0.0125 (4 comparisons) or 0.01 (5 comparisons): * < 0.0125, 0.01; ** < 0.00313, 0.002; *** < 0.00063, 0.0005. Based on these positions, we designed four NtoS-derived variants, each retaining a subset of these sites as serine substitutions: Var1 (N388S), Var2 (N347S/N371S), Var3 (N347S/N371S/N388S), and Var4 (N347S/N371S/N388S/N631S/N704S) ( Figure 5A ). Each variant was expressed on the yeast surface as an Aga2 fusion, and we evaluated flg22 binding and expression by flow cytometry under thermal stress alone (37 °C) and under additive stress (37 °C, 2 µg/mL tunicamycin) ( Figure 5B–D ). Under thermal stress alone, Var3 (N347/N371/N388S) and the fully de-glycosylated NtoS construct showed the highest fraction of flg22-binding cells (∼12.4% and 11.5%, respectively), whereas Var1 (N388) and Var2 (N347S/N371S) displayed significantly lower binding (∼4.8% and ∼3.6%, respectively) ( Figure 5B ). Var3 (N347S/N371S/N388S) exhibited flg22 binding similar to NtoS as indicated by a tighter cluster of double-positive (binding/expression) events compared to Var1 and Var2 in raw density plots ( Supporting Material 2 - Supplementary Information 2 ). This indicates that removal of N388 in combination with N347 and N371 can restore binding to levels comparable to the fully de-glycosylated construct under these conditions. After additive stress (37 °C, 2 µg/mL tunicamycin) flg22 binding increased for all variants ( Figure 5C ), except this increase was not significant for Var2 between additive stress and thermal stress alone ( Figure 5D ). Var1 consistently showed a robust and statistically significant increase in flg22-binding/expression-positive events relative to thermal stress alone ( Figure 5D ). Importantly, in raw density plots, Var1 displayed a higher percentage and tighter cluster of double-positive (binding/expression) events than Var2 and Var3 under additive stress ( Supporting Material 2 - Supplementary Information 2 ), suggesting that N388S promotes a larger fraction of properly folded, ligand-accessible receptors than other proximal-site combinations. Collectively, these data highlight N388 as a critical glycosylation site whose removal improves functional expression of yeast-displayed FLS2, particularly under optimized ER stress conditions. N388 is the most proximal glycosylation site to the flg22 binding interface, meaning that it is the most likely high-mannose glycosylation site to block flg22. Conversely, removing glycans only at N347 and N371 (Var2) did not confer similar benefits and, in some conditions, appeared detrimental to binding and/or expression. Extending de-glycosylation to additional proximal and aggregated sites (Var3, Var4) did not further enhance binding and sometimes reduced surface expression, consistent with a trade-off between alleviating steric hindrance near the binding interface and maintaining glycan-mediated folding and stability. Overall, the performance of Var1 (N388S) supports a model in which selective de-glycosylation at N388 relieves local steric obstruction of the flg22 binding site while preserving sufficient glycosylation at other positions to support productive folding and trafficking of FLS2 in the yeast secretory pathway. Conclusion We set out to develop a yeast surface display platform for the plant pattern recognition receptor FLS2, motivated by the need for high-throughput engineering of receptors with broadened detection of evading bacterial flagellin epitopes. Initial attempts yielded high levels of FLS2 ectodomain expression but no detectable flg22 binding, prompting a systematic dissection of how yeast ER quality control and N-linked glycosylation shape functional display. We show that FLS2 is hyperglycosylated with high-mannose glycans in yeast and that complete removal of N-glycan motifs impairs trafficking. By partially inhibiting glycosylation with tunicamycin and engaging stress-responsive pathways via thermal induction, we identified conditions under which a subpopulation of yeast displays FLS2 with detectable, albeit weak, flg22 binding. Nevertheless, this platform has the potential to aid the rapid identification of evading flg22 variants and performing competitive binding assays with agonist and antagonist peptides from commensal communities. Structure-guided removal of an N-glycan at residue N388, proximal to the flg22 binding interface, further enhances functional expression, revealing this site as a key determinant of ligand access in the yeast context. Together, our results demonstrate that lineage-specific glycosylation and ER stress responses are critical levers for expressing plant pattern recognition receptors in yeast. This provides a roadmap for refining yest surface display systems to support high-throughput engineering of FLS2 and related immune receptors. Materials and Methods Materials and Media preparation We purchased yeast extract, peptone, and dextrose from Fisher for YPD growth media preparation. We purchased SD/-trp packets (clonetech) for SD/-trp selection media preparation. We purchased sodium phosphate dibasic, sodium phosphate monobasic, galactose from Sigma-Aldrich; dextrose and casamino acids from Fisher; and yeast nitrogen base from DOT Scientific for SG-CAA induction media preparation. We purchased LB broth (Miller) for bacterial growth media. For growth or selection agar plate preparation, we purchased agar from fisher. Agar plates were prepared in 500 mL batches including reagents specified for growth or selection media and 8 g agar, then autoclaved, cooled, and poured onto 100x15mm petri dishes from Fisher (∼20-25 mL media + agar per plate). For short-term storage (1-2 months), ampicillin (Sigma-Aldrich) was added to cooled media for a final concentration of 1 mg/mL ampicillin. Bacterial media and agar plates always contained 1 mg/mL ampicillin for plasmid selection. For western blot wash buffer, 500 mL of 1X TBS was prepared using 10% 10X TBS (24 g Tris, 88 g NaCl). For flow cytometry working buffer (washing, staining), 500 mL of 1X PBS + 0.1% (w/v) BSA was prepared using 10% 10X PBS w/o calcium, w/o magnesium (VWR) and 0.5g bovine serum albumin (Fisher), then sterile filtered. Yeast and Bacterial Strains and Plasmids All surface display constructs were designed in-house, except the plasmid pCT80-FnLoopHp, which is routinely used for our YSD applications. Insert sequences were codon optimized ( S. cerevisiae ) and cloned into pCT80-FnLoopHp by GenScript, except FLS2LRR. pCT80-FnLoopHp is a modified version of the pCTcon2 ( 66 ) surface display shuttle vector that was used to design FLS2LRR, NtoS, 4 NtoS glycan variants, RIXI, and HaloTag yeast display constructs ( Figure S2, Supporting Material 1 - Supplementary Information 2,3 ). Briefly, S. cerevisiae codon-optimized recombinant FLS2LRR insert sequence with homologous overlapping ends ( Supporting Material 1 – Supplementary Table 1 ) was inserted via homologous recombination or Gibson cloning at NheI and BamHI restriction enzyme cut sites in the GAL1 open reading frame of restriction enzyme linearized vector. All constructs were transformed into the EBY100 yeast strain (genotype MATa AGA1::GAL1-AGA1::URA3 ura3-52 trp1 leu2-delta200 his3-delta200 pep4::HIS3 prbd1.6R can1 GAL). For plasmid propagation, NEB5a (NEB) E. coli strain was used. Yeast outgrowth, storage, transformation, selection, and induction of protein expression WT EBY100 yeast passage was performed in YPD media and all stocks were stored at - 80 °C in 25% Glycerol. Yeast transformation was performed using the Frozen-EZ Yeast Transformation II Kit (Zymo) or electroporation was performed to acquire recombinant yeast clones by homologous recombination of linearized display vector with recombinant inserts containing homologous overlapping ends ( 67 ). Transformants were plated on SD/-trp plates for selection. Yeast transformants were grown in SD/-trp selective media at 30 °C/225 rpm/overnight and induced in SG-CAA induction media at 30 °C/225 rpm/overnight. To confirm successful transformation, single colony clones were picked and outgrown in selective media for DNA extraction using the Zymoprep Yeast Plasmid Miniprep II kit (Zymo). Then, genomic extract was transformed by heat shock into NEB5a chemically competent E. coli (following High Efficiency Transformation Protocol from NEB) for plasmid propagation and extraction (QIAprep Spin Miniprep Kit, QIAGEN). Plasmid concentration was determined using a UV/Vis Spectrophotometer (NanoDrop One, Fisher). DNA plasmids were submitted to Plasmidsaurus for High Copy Whole Plasmid Sequencing service to confirm successful yeast transformants. For induction under stress conditions, after outgrowth in SD/-trp, yeast were resuspended in SG media with or without tunicamycin (Sigma-Aldrich) at a starting OD of 0.2-0.5 and incubated at either 30 or 37 °C/225 rpm/overnight. Due to growth disruption and cell death caused by tunicamycin treatment, it is recommended to grow in a larger volume of induction media for sufficient cell quantities in downstream experiments (e.g. 2-3x induction cultures). FLS2 Computational Modeling and Stability Calculation Figure S7 summarizes the workflow for identifying amino acids to substitute putative N-glycan asparagines by AlphaFold2 (AF2) modeling of de-glycosylated FLS2 ectodomains and metrics/tools for evaluating prediction performance, modeling atomic deviation, and modeling ΔEnergy. Briefly, AF2 provides an interface predicted template modelling (ipTM) score, which measures how well AlphaFold2-Multimer predicts the overall structural complex ( 41 ). Using the Rosetta molecular modeling software ( 68 ), modeled structures are relaxed (Fast Relax) and total_score is calculated using the Rosetta scoring function (REF15), which is compared to the AF2-predicted WT structure total_score as described previously ( 42 , 68 ). Root mean-squared deviation (RMSD, Å) is obtained after PyMOL ( 65 ) structural alignment of relaxed modeled structures to the crystal structure (PDB ID 4MN8, only FLS2 ectodomain (chain A) and flg22 (chain C)). Evaluation metrics (ipTM, RMSD, total_score) were tabulated for WT and substituted modeled structures to determine which amino acid change at all putative N-glycan sites (substituted structures) resulted in the fewest deviations from values for the WT modeled structure ( Figure S7 ). This FLS2LRR de-glycosylated variant clone was established in yeast as described in Yeast outgrowth, storage, transformation, selection, and induction of protein expression . For choosing specific-site NtoS variants, FLS2 ectodomain crystal structure (PDB ID 4MN8) was modelled with yeast-type high mannose glycans using GlycoShape ( 64 ). Prior to choosing N-glycan sites for removal, the modeled structure was aligned to the original crystal structure (only FLS2 ectodomain (chain A) and flg22 (chain C)) in PyMOL. To determine N-glycan sites chosen for removal, we considered the proximity within 5 Å of modelled high-mannose glycan atoms to flg22 (proximal sites) and locations where GlycoShape predicted glycan aggregation (aggregated sites) using PyMOL and commands described previously ( 42 ). Based on these constraints we chose 3 proximal glycan sites (N388, N347, N371) and 2 aggregated glycan sites (N631, N704S). Four variant constructs were designed using combinations of these identified sites – Var1 :N388S, Var2 :N347S/N371S , Var3 :N388S/N347S/N371S, Var4 :N388S/N347S/N371S/N631S/N704S. FLS2LRR variant clones were established in yeast as described in Yeast outgrowth, storage, transformation, selection, and induction of protein expression . Protein Extraction, SDS-PAGE, and Western Blot Assays To obtain protein extracts containing surface displayed protein from EBY100 transformant clones – after overnight induction of protein expression – cell cultures were treated with 2 mM DTT (GOLDBIO) for 45 minutes at 30 °C/225 rpm. DTT disrupts disulfide bridges formed between Aga1 and Aga2 that allow for yeast surface display ( 69 ). For obtaining cytosolic extracts, after surface extraction with DTT, we performed horn-type sonication of a high cell density mixture as described previously ( 70 ). Briefly, sonication per sample was performed 20 s on, 30 s off, for 10 minutes. After DTT treatment and sonication, cells were pelleted at 4000 xg/ 10 minutes and supernatant was collected for Amicon Ultra 15-30K concentration. Concentrated supernatant protein density was determined by Bradford Assay (Quick Start Bradford Protein Assay, Bio-Rad) against freshly prepared BSA standard dilution panel using 595 nm setting on a spectrophotometer. After determining protein concentration, samples were snap frozen and stored at - 80 °C as 50-100 uL 0.5-2 mg/mL aliquots in 10% glycerol and 1X Protease Inhibitor solution (Halt Protease Inhibitor Cocktail (100X), Fisher). For protein extracts treated with endoglycosidase H (Endo H, NEB) or PNGase F (NEB), 10-20 ug concentrated supernatant protein was processed following the manufacturer recommended instructions for denaturing (PNGase F only) and non-denaturing Endo H treatment. Briefly, non-denaturing reaction samples were incubated with Glyco 3 Buffer and Endo H at 37 °C overnight, prior to SDS-PAGE and Western Blot. Non-denaturing incubations did not include a the initial incubation with denaturing buffer. To prepare samples for SDS-PAGE, 10-20 ug of concentrated supernatant protein was prepared with 2.5 uL LDS (4X LDS Sample Buffer, GenScript) and 1 uL 500 mM DTT, then denatured at 100 °C/ 10 minutes. Pre-made 4-12% Bis-Tris gel (SurePAGE, Bis-Tris, 10x8, 4-12%, 12 well, GenScript) was prepared in polyacrylamide gel electrophoresis box with Tris-MOPS-SDS Running Buffer (GenScript). Samples and PageRuler Prestained protein ladder (Fisher) were loaded onto the gel so that sample protein quantity was equivalent for each sample regardless of prior treatments (e.g. loading 20 uL endo-H treated sample vs 10 uL un-treated sample both contain 10 ug of concentrated supernatant protein from the same initial batch extraction). Electrophoresis was conducted at 200V/40min. After SDS-PAGE, gel was prepared for wet transfer using the sandwich method ( 71 ) in 0.1% SDS, 1X Transfer (Towbin) Buffer (10% v/v 10X Transfer Buffer (Fisher), 20% v/v Methanol (Sigma-Aldrich)). Briefly, sandwich was prepared for gel to activated PVDF membrane transfer and loaded into Criterion Blotter (Bio-Rad) for wet transfer at 4 °C/ 90 V/ 60 minutes. After wet transfer, membrane was carefully removed and washed twice with 1X TBS, then blocked with 1X TBS + 3% (w/v) BSA for 60 minutes/room temperature (RT)/slow shaking. After blocking, membrane was washed once with 1X TBS, then twice with 1X TBS-T (0.1% v/v Tween-20), before incubation in primary antibody solution (1:1000 anti-HA-biotin (3F10, Millipore Sigma), 1X TBS + 3% BSA) overnight/4 °C/slow shaking. After primary antibody incubation, membrane was washed twice with 1X TBS-T before incubation in secondary antibody solution (1:10000 streptavidin-AlexaFluor647 (Fisher), 1X TBS + 3% BSA) for 60 minutes/RT/slow shaking. After secondary antibody incubation, membrane was washed four times with 1X TBS-T and detected with laser compatible for AlexaFluor647 detection using a ChemiDoc Imager (BioRad). Flow Cytometry Assays To prepare samples for flow cytometry analysis, induced cells optical density was measured using a spectrophotometer set to measure OD600. For each sample replicate, 4x10 6 were transferred to microfuge tubes and washed with 1X PBS + 0.1% BSA. After washing, each sample was incubated with primary antibody at a final concentration of 5 ug/mL in 1X PBS + 0.1% BSA for 20 minutes at room temperature. After primary antibody incubation, samples were washed and resuspended in secondary antibody solution at a final concentration of 10 ug/mL in 1X PBS + 0.1% BSA for 20 minutes at room temperature. Finally, after secondary antibody incubation, samples were washed and resuspended in 1-10 uM TAMRA-flg22 (BIOMATIK) in 1X PBS + 0.1% BSA solution with concentration consistent between experiments unless otherwise specified. Incubation with TAMRA-flg22 was conducted for 20 minutes at room temperature. After final incubation, samples were washed once and resuspended in 100 or 500 uL 1X PBS + 0.1% BSA for flow cytometry analysis using either the Accuri C6 (BD Biosciences) or Attune Cytpix (Fisher), respectively. The median fluorescence intensity from flow cytometry measurements for FLS2LRR and variant populations was used to calculate the binding affinity (K D ) as described previously ( 72 , 73 ). FCS Express 7 (De Novo Software) was used for statistical analyses, data extraction, and figure preparation of cytometry data. Magnetic Bead Enrichment of FLS2LRR-displaying cells Figure S9 diagrams the workflow for the first round of enrichment, subsequent rounds of enrichment, and enrichment analysis using a modified approach described in Ackerman et al. 2009 ( 63 ). Briefly, modifications to this approach include: using Dynabeads Biotin Binder (Fisher) magnetic streptavidin-coated beads. Beads are coated with negative (biotin conjugated goat-IgG, Rockland) and positive (biotin-flg22, GenScript) selection agent prior to incubation with cells (direct approach). Selections are conducted for 1.5 hrs/4 °C/rotating before bead binding cells are transferred for enrichment analysis. Prior to induction of positive selection cells before subsequent rounds, beads from previous round are removed. To wash, remove unbound cells, and remove previous round beads, the bead/cell mixture was placed on a DynaMag-2 (Fisher) magnetic tube rack for 2-5 minutes before removal of suspension without disrupting beads. Bead/cell mixtures are washed three times between selections using 1X PBS + 0.1% BSA. Statistical Analysis and Reproducibility We determined the statistical significance with two-tailed T-test with unequal variance for all cytometry data and used Bonferroni correction (corrected α provided in figure descriptions) when >1 comparisons were made. All collected data were used for the calculation of means and standard error. The fraction binding for cytometry data bar plots represents the mean of n=3 biological replicates after background subtraction performed as described in Supporting Material 2 - Supplementary Information 1 . Author Information Author Contributions B.D. and D.R.W. contributed equally to the conception, design, and interpretation of the study. B.D. and S.S. contributed to the execution of experiments. All authors contributed to writing review, and approval of the final manuscript. Notes The authors declare no competing financial interest. Acknowledgement We thank Benjamin Orlando for his contribution to conceptualization of the FLS2LRR specific site glycosylation variant panel. We are thankful to Jens Schmidt for suggestions on analyzing flow cytometry data. We thank Eric Patterson and Michael Feig for critical assessment of findings throughout. We are thankful to the Michigan State University Flow Cytometry Core for training and super-user access to flow cytometry instrumentation used in the experiments described. Lastly, we thank Joelle Eaves, Annie Needs, and Logan Garland for providing critical feedback during the writing process. B.D. and D.R.W. acknowledge funding from USDA-NIFA-AFRI-009003 (GRANT13700968). B.D. also acknowledges partial support by a fellowship from Michigan State University under the Training Program in Plant Biotechnology for Health and Sustainability (T32-GM152798). Funder Information Declared National Institute of Food and Agriculture , GRANT13700968 Abbreviations FLS2 FLAGELLIN SENSING 2 PRR Pattern Recognition Receptor ERQC Endoplasmic Reticulum Quality Control LLO Lipid-linked Oligosaccharide OST Oligosaccharyltransferase DTT Dithiothreitol UPR Unfolded Protein Response TAMRA Tetramethylrhodamine MFI Median Fluorescence Intensity YSD Yeast Surface Display References 1. ↵ Arulbalachandran , D. ; Mullainathan , L. ; Latha , S. Food security and sustainable agriculture . Sustainable Agriculture towards Food Security 2017 , 3 – 13 . doi: 10.1007/978-981-10-6647-4_1/FIGURES/1 . OpenUrl CrossRef 2. Roberts , D. P. ; Mattoo , A. K . Sustainable Agriculture—Enhancing Environmental Benefits, Food Nutritional Quality and Building Crop Resilience to Abiotic and Biotic Stresses . Agriculture 2018, Vol. 8, Page 8 2018 , 8 ( 1 ), 8 . doi: 10.3390/AGRICULTURE8010008 . OpenUrl CrossRef 3. ↵ Singh , P. P. ; Kujur , A. ; Yadav , A. ; Kumar , A. ; Singh , S. 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