Journal: bioRxiv

Article Title: Mapping ortholog-restricted ligandable cysteines in the wheat pathogen Zymoseptoria tritici

doi: 10.64898/2026.02.16.706151

Figure Lengend Snippet: a , Cysteine-directed ABPP data showing stereoselective liganding of C141 of Zt NMT1 (ZtIPO323_063250) by WX-03-60 (40 µM, 2 h) in Z. tritici cell lysates. b, Protein-directed ABPP data showing stereoselective liganding of Zt NMT1 by the alkyne/parent stereoprobe pair WX-03-349/WX-03-60 (10 µM, 1 h; 40 µM, 2 h pre-treatment) in Z. tritici cell lysates. For a , b , data represent mean values ± s.d. of four independent experiments. c, Sequence alignment of NMT1 orthologs showing lack of conservation of stereoprobe-liganded C141 of Zt NMT1 in human and wheat orthologs. d, Overlay of a predicted AlphaFold structure of Zt NMT1 (AF-F9XBB7-F1) and the crystal structure of Hs NMT1 in complex with a substrate peptide and myristoyl-CoA (Myr-CoA) cofactor (PDB: 5MU6). The location of C141 in Zt NMT1 is shown yellow. e, f, In situ protein-directed ( e ) and cysteine-directed ( f ) ABPP data showing stereoselective liganding of SAR1 (ZtIPO323_014820) by the alkyne/parent stereoprobe pair MY-11A/MY-1A (10 µM, 1 h; 40 µM, 2 h pre-treatment). Data represent mean values ± s.d. of four independent experiments. g, In vitro protein-directed ABPP data showing a lack of stereoselective enrichment of SAR1 in Z. tritici cell lysates by MY-11A (10 µM, 1 h). Data represent mean values ± s.d. of four independent experiments. h, Bar graph showing the extent of competitive blockade of MY-11A (10 µM, 1 h) enrichment of Zt SAR1 by pre-treatment with MY-1A (40 µM, 2 h) from protein-directed ABPP experiments performed in vitro or in situ. Data represent mean values ± s.d. of four independent experiments. i, Sequence alignment of SAR1 orthologs showing lack of conservation of stereoprobe-liganded C64 of Zt SAR1 in human and wheat orthologs. j, Gel-ABPP data showing engagement of recombinant WT- Zt SAR1, but a C64A- Zt SAR1 mutant by MY-11A (5 µM, 1 h) in HEK293T cells, as well as stereoselective blockade of MY-11A engagement of WT- Zt SAR1 by pre-treatment with MY-1A (20 µM, 2 h). Data are from a single experiment representative of three independent experiments. k, Gel-ABPP data showing engagement of recombinant WT- Zt SAR1 by MY-11A (5 µM, 1 h) in HEK293T cells, as well as stereoselective blockade of this engagement by pre-treatment with MY-1A (20 µM, 2 h) in HEK293T cells ( in situ ), but not cell lysates ( in vitro ). l, Quantification of gel-ABPP data shown in ( k ). Data represent mean values ± s.e.m. for three independent experiments; p values from unpaired two-tailed t tests.

Article Snippet: The full insert including 1kb of the genomic upstream and downstream flanking regions of Zt SAR1, selection cassettes (Hygromycin B and Isofetamid), TetOff promoter , Zt SAR1-WT-GFP or its Zt SAR1-C64A-GFP mutant were synthesized by GENEWIZ Inc. (Germany) and assembled into the binary plasmid pNOV2114 .

Techniques: Sequencing, In Situ, In Vitro, Recombinant, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Mapping ortholog-restricted ligandable cysteines in the wheat pathogen Zymoseptoria tritici

doi: 10.64898/2026.02.16.706151

Figure Lengend Snippet: Additional characterization of an ortholog-restricted, stereoprobe-liganded cysteine in Zt SAR1. a, Sequence alignment of Z. tritici SAR1 with other SAR1 orthologs from human and wheat ( T. aestivum ). Aligned residues showing identical (opaque) and similar (transparent) sequences are highlighted. b, Protein-directed ABPP data showing a lack of stereoselective enrichment of human SAR1A from Ramos cells treated with MY-11A or MY-11B (5 µM, 3 h). Data represent mean values ± s.d. for four independent experiments. c, Gel-ABPP data showing stereoselective engagement of recombinant WT- Zt SAR1 by MY-11A (5 µM, 1 h) and stereoselective blockade of this engagement by pre-treatment with MY-1A (20 µM, 2 h) in HEK293T cells. After stereoprobe treatments, cells were lysed and analyzed by gel-ABPP as either whole cell lysates or as soluble and particulate fractions. d, Quantification of gel-ABPP data shown in (c). e, Quantification of Zt SAR1 immunoblotting signals shown in (c). For d, e, data represent mean values ± s.e.m. for three independent experiments; two-way ANOVA.

Article Snippet: The full insert including 1kb of the genomic upstream and downstream flanking regions of Zt SAR1, selection cassettes (Hygromycin B and Isofetamid), TetOff promoter , Zt SAR1-WT-GFP or its Zt SAR1-C64A-GFP mutant were synthesized by GENEWIZ Inc. (Germany) and assembled into the binary plasmid pNOV2114 .

Techniques: Sequencing, Recombinant, Western Blot

Journal: bioRxiv

Article Title: Mapping ortholog-restricted ligandable cysteines in the wheat pathogen Zymoseptoria tritici

doi: 10.64898/2026.02.16.706151

Figure Lengend Snippet: a , Gel-ABPP data showing stereoselective blockade of MY-11A (5 µM, 1 h) engagement of WT- Zt SAR1 by pre-treatment with MY-1A (20 µM, 2 h) in HEK293T cells and concomitant accumulation of WT- Zt SAR1 in the particulate fraction. b, Quantification of gel-ABPP data shown in ( a ). c, Quantification of ZtSAR1 immunoblotting signals shown in ( a ). d , Gel-ABPP data showing the effect of GDP/GppCp (1 mM, 1 h) on the stereoselective engagement of WT- Zt SAR1 by MY-11A (5 µM, 1 h) in HEK293T cell lysates. e, Quantification of the gel-ABPP data shown in ( d ). For b , c , and e , data represent mean values ± s.e.m. for three independent experiments; p values from unpaired two-tailed t tests. f, Gel-ABPP data showing the concentration-dependent effects of GDP/GppCp on MY-11A (5 µM, 1 h) engagement of WT- Zt SAR1. g, Quantification of gel-ABPP data shown in ( f ). For g , data represent mean values ± s.d. of three independent experiments. h, Boltz-2-predicted conformational changes in predicted structures of Zt SAR1 in the apo form (pink, AF-F9X159-F1) or bound to GDP or GTP (brown and teal respectively, Boltz-2 predictions). Structures highlight that the positioning of C64 of Zt SAR1 in relation to the N-terminal helix (residues 1-23) is predicted to be affected by binding of GNP cofactor.

Article Snippet: The full insert including 1kb of the genomic upstream and downstream flanking regions of Zt SAR1, selection cassettes (Hygromycin B and Isofetamid), TetOff promoter , Zt SAR1-WT-GFP or its Zt SAR1-C64A-GFP mutant were synthesized by GENEWIZ Inc. (Germany) and assembled into the binary plasmid pNOV2114 .

Techniques: Western Blot, Two Tailed Test, Concentration Assay, Binding Assay

Journal: bioRxiv

Article Title: Mapping ortholog-restricted ligandable cysteines in the wheat pathogen Zymoseptoria tritici

doi: 10.64898/2026.02.16.706151

Figure Lengend Snippet: Additional characterization of an ortholog-restricted, stereoprobe-liganded cysteine in Zt SAR1. a, Gel-ABPP data showing concentration-dependent, stereoselective blockade of MY-11A (5 µM, 1 h) engagement of recombinant WT- Zt SAR1 by MY-1A (0.01-40 µM, 2 h), along with concomitant accumulation of WT- Zt SAR1 in the particulate fraction of HEK293T cells. b, Quantification of gel-ABPP data (top graph) and Zt SAR1 immunoblotting signals (bottom graph) shown in (a). Data represent mean values ± s.d. for three independent experiments. c, Gel-ABPP data showing engagement of recombinant WT- Zt SAR1 by MY-11A (5 µM, 1 h) and time-dependent stereoselective blockade of this engagement by MY-1A (20 µM) in HEK293T cells. After stereoprobe treatments, cells were lysed and analyzed by gel-ABPP as either whole cell lysates or as soluble and particulate fractions of these lysates. d, Quantification of gel-ABPP data shown in (c). e, Quantification of Zt SAR1 immunoblotting signals shown in (c). For d, e, data represent mean values ± s.e.m. for three independent experiments; two-way ANOVA. f, Gel-ABPP data showing increases in MY-11A (5 µM, 1 h) engagement of WT- Zt SAR1, but not C64A- Zt SAR1 in the presence of GDP cofactor (1 and 100 µM, 1 h). Data are from a single experiment representative of three independent experiments.

Article Snippet: The full insert including 1kb of the genomic upstream and downstream flanking regions of Zt SAR1, selection cassettes (Hygromycin B and Isofetamid), TetOff promoter , Zt SAR1-WT-GFP or its Zt SAR1-C64A-GFP mutant were synthesized by GENEWIZ Inc. (Germany) and assembled into the binary plasmid pNOV2114 .

Techniques: Concentration Assay, Recombinant, Western Blot

Journal: bioRxiv

Article Title: Mapping ortholog-restricted ligandable cysteines in the wheat pathogen Zymoseptoria tritici

doi: 10.64898/2026.02.16.706151

Figure Lengend Snippet: a , Confocal microscopy images of recombinant Zt SAR1-eGFP in HEK293T cells showing aggregation of WT-, but not C64A- Zt SAR1-eGFP, in ER-associated puncta following treatment with MY-1A, but not MY-1B (20 µM, 2 h); scale bar = 10 µm. b, Quantification of GFP puncta per cell from microscopy images represented by ( a ). Data represent mean values ± s.e.m. for n = 20 focal planes (125 µm x 125 µm area) and n = 18 cells on average per focal plane; one-way ANOVA. c, Confocal microscopy images of co-expressed recombinant human Halo-COPII vesicle proteins with WT- Zt SAR1-eGFP in HEK293T cells showing increased colocalization of Halo and eGFP signal in the presence of MY-1A, but not MY-1B (20 µM, 2 h). Merge channel is the overlay of GFP and Halo channels; scale bar = 10 µm. d, Quantification of Pearson correlation rates from microscopy images represented by ( c ). Data represent mean values ± s.e.m. for n = 5 focal planes (125 µm x 125 µm area) and n = 21 cells on average per focal plane; two-way ANOVA. e, Confocal microscopy images of Zt SAR1 WT- and C64A-eGFP in Z. tritici cells showing aggregation of WT-, but not C64A- Zt SAR1-eGFP, in ER-associated puncta following treatment with MY-1A, but not MY-1B (40 µM, 2 h). Merge channel is the overlay of GFP and ER channels; scale bar = 10 µm. f, Quantification of GFP puncta per cell from microscopy images represented in ( e ). Data represent mean values ± s.e.m. for n = 20 focal planes (125 µm x 125 µm area) and n = 24 cells on average per focal plane; two-way ANOVA. g, Growth assay for Z. tritici cells showing stereoselective effects of MY-1A (0–100 µM, 7 days) in WT-, but not C64A- Zt SAR1-expressing cells. Data represent mean values ± s.e.m. for four independent experiments.

Article Snippet: The full insert including 1kb of the genomic upstream and downstream flanking regions of Zt SAR1, selection cassettes (Hygromycin B and Isofetamid), TetOff promoter , Zt SAR1-WT-GFP or its Zt SAR1-C64A-GFP mutant were synthesized by GENEWIZ Inc. (Germany) and assembled into the binary plasmid pNOV2114 .

Techniques: Confocal Microscopy, Recombinant, Microscopy, Growth Assay, Expressing

Journal: bioRxiv

Article Title: Mapping ortholog-restricted ligandable cysteines in the wheat pathogen Zymoseptoria tritici

doi: 10.64898/2026.02.16.706151

Figure Lengend Snippet: Stereoprobe MY-1A promotes accumulation Zt SAR1 in ER puncta. a, Confocal microscopy images demonstrating concentration-dependent aggregation of WT- Zt SAR1-eGFP stably expressed in HEK293T cells treated with MY-1A, but not MY-1B (2 h treatments); scale bar = 10 µm. b, Quantification of GFP puncta per cell from microscopy images represented in (a). Data represent mean values ± s.e.m. n = 20 focal planes (125 µm x 125 µm area); n = 17 cells on average per focal plane; one-way ANOVA. c, Timelapse of confocal microscopy images of WT- Zt SAR1-eGFP and C64A- Zt SAR1-eGFP stably expressed in HEK293T cells following treatment with MY-1A (20 µM); scale bar = 10 µm.

Article Snippet: The full insert including 1kb of the genomic upstream and downstream flanking regions of Zt SAR1, selection cassettes (Hygromycin B and Isofetamid), TetOff promoter , Zt SAR1-WT-GFP or its Zt SAR1-C64A-GFP mutant were synthesized by GENEWIZ Inc. (Germany) and assembled into the binary plasmid pNOV2114 .

Techniques: Confocal Microscopy, Concentration Assay, Stable Transfection, Microscopy

Journal: bioRxiv

Article Title: Mapping ortholog-restricted ligandable cysteines in the wheat pathogen Zymoseptoria tritici

doi: 10.64898/2026.02.16.706151

Figure Lengend Snippet: Assessing stereoprobe ligandability of a neo-cysteine B. cinerea SAR1 variant. a, Left, Sequence alignment of Z. tritici SAR1 with other SAR1 orthologs ( B. cinerea , P. graminis , and H. sapiens ) showing conserved residues (yellow) that are proximal (<15 Å) to C64 of Zt SAR1. Right, AlphaFold-predicted structure of Zt SAR1 (AF-F9X159-F1) highlighting locations of conserved residues proximal (< 15 Å) to C64. b, Gel-ABPP data showing stereoselective engagement of V64C-, but not WT-BcSAR1 by MY-11A (5 µM, 1 h) and stereoselective blockade of this engagement by pre-treatment with MY-1A (20 µM, 2 h) in HEK293T cells. After stereoprobe treatments, cells were lysed and analyzed by gel-ABPP as either whole cell lysates or as soluble and particulate fractions of these lysates. c, Quantification of gel-ABPP data for the particulate fraction shown in (b). d, Quantification of Bc SAR1 immunoblotting signals (b). For c, data represent mean values ± s.e.m. n = 3; unpaired two-tailed t test. For d, data represent mean ± s.e.m. n = 3; two-way ANOVA. e, Confocal microscopy images of recombinant Bc SAR1-eGFP demonstrating stereoselective aggregation of V64C-, but not WT- Bc SAR1-eGFP, by MY-1A (20 µM, 2 h) in HEK293T cells; scale bar = 10 µm. f, Quantification of GFP puncta per cell from microscopy images represented in (e). Data represent mean values ± s.e.m. n = 20 focal planes (125 µm x125 µm area) and n = 15 cells on average per focal plane; one-way ANOVA.

Article Snippet: The full insert including 1kb of the genomic upstream and downstream flanking regions of Zt SAR1, selection cassettes (Hygromycin B and Isofetamid), TetOff promoter , Zt SAR1-WT-GFP or its Zt SAR1-C64A-GFP mutant were synthesized by GENEWIZ Inc. (Germany) and assembled into the binary plasmid pNOV2114 .

Techniques: Variant Assay, Sequencing, Western Blot, Two Tailed Test, Confocal Microscopy, Recombinant, Microscopy

Journal: bioRxiv

Article Title: Mapping ortholog-restricted ligandable cysteines in the wheat pathogen Zymoseptoria tritici

doi: 10.64898/2026.02.16.706151

Figure Lengend Snippet: Assessing stereoprobe ligandability of neo-cysteine P. graminis and H. sapiens SAR1 variants. a, b, Gel-ABPP data showing lack of MY-11A (5 µM, 1 h) engagement of WT or neo-cysteine variants of Pg SAR1 (a) and Hs SAR1A (b) proteins. Neo-cysteine variants; V64C- Pg SAR1, M69C- Hs SAR1A. Data are from a single experiment representative of three independent experiments. c-f, Confocal microscopy images demonstrating lack of stereoselective aggregation of WT- or neo-cysteine variants of Pg SAR1-eGFP and Hs SAR1A-eGFP by MY-1A (20 µM, 2 h) in HEK293T cells. c, e, representative confocal images; scale bar = 10 µm; d and f, quantification of GFP puncta per cell from microscopy images represented in (c) and (e), respectively. Data represent mean values ± s.e.m. n = 20 focal planes (125 µm x 125 µm area) and n = 16 ( Pg SAR1-eGFP) or 19 ( Hs SAR1A-eGFP) cells on average per focal plane; one-way ANOVA.

Article Snippet: The full insert including 1kb of the genomic upstream and downstream flanking regions of Zt SAR1, selection cassettes (Hygromycin B and Isofetamid), TetOff promoter , Zt SAR1-WT-GFP or its Zt SAR1-C64A-GFP mutant were synthesized by GENEWIZ Inc. (Germany) and assembled into the binary plasmid pNOV2114 .

Techniques: Confocal Microscopy, Microscopy