Fig. 5: C₁₈-SMe₂⁺-induced ROS production and programmed cell death in Z. tritici.

a Quantitative comparison of DHR-123 staining of mROS in Z. tritici cells, treated for 30 min with solvent (Control), the complex I inhibitor rotenone, or C₁₂-G⁺ and C₁₈-NMe₃⁺. b DHR-123 staining of mROS in Z. tritici cells, treated with solvent (Control) and C₁₈-SMe₂⁺. The plasma membrane is labeled by mCherry-Sso1. Note that the red-fluorescence images were scaled identically. Scale bars = 10 µm. c mROS production in Z. tritici in presence of solvent control (Control), 30 min treatment with C₁₈-SMe₂⁺ (C₁₈-SMe₂⁺, 0.5 h), and 24 h treatment with C₁₈-SMe₂⁺ (C₁₈-SMe₂⁺, 24 h), and co-treatment with rotenone and C₁₈-SMe₂⁺ (C₁₈-SMe₂⁺+Roten., 0.5 h). d mROS production in Z. tritici after treatment with solvent (Control), C₁₂-SMe₂⁺, C₁₆-SMe₂⁺, and C₁₈-SMe₂⁺, at 5 µg ml⁻¹ or 20 µg ml⁻¹. See also Supplementary Fig. 6. e Apoptotic Z. tritici cells, detected by CaspACE FITC-VAD-fmk. Healthy cells exclude the dye (open white arrowheads), whereas apoptotic cells are filled with green-fluorescence (open blue arrowheads). Post-apoptotic cells that contain propidium iodide were not included in the analysis (yellow open arrowhead). The plasma membrane is visualized by the marker mCherry-Sso1 (red). Scale bar = 10 µm. f Number of early apoptotic Z. tritici cells (CaspACE FITC-VAD-fmk-positive, but propidium iodide-negative), treated with the solvent, C₁₂-G⁺, C₁₈-NMe₃⁺, and C₁₈-SMe₂⁺. g Apoptotic Z. tritici cells, detected by Annexin-V-fluorescein staining. Healthy cells are unstained (left panel), whereas apoptotic cells expose phosphatidylserine and show green-fluorescence at their plasma membrane (right panel; plasma membrane in red, labeled with mCherry-Sso1). Post-apoptotic cells were identified by double staining with propidium iodide and excluded from the analysis. Scale bars = 10 µm (left panel), 5 µm (insert, right panel). h Number of early apoptotic Z. tritici cells (Annexin-V-fluorescein-positive, but propidium iodide-negative), treated with the solvent control, C₁₂-G⁺, C₁₈-NMe₃⁺ and C₁₈-SMe₂⁺. a, c, d Whiskers’ plots, with 25th/75th percentiles (blue lines), medians (red line), and minimum and maximum (whiskers ends). Values f, h are shown as mean ± SEM. Red dots represent data points. Sample size n is indicated in each panel. Non-parametric Mann–Whitney testing (a, c, d); n.s. non-significant difference to control (P > 0.05) (d), ***P < 0.0001 (a, c, d). Student’s t-testing with Welch correction (f, h); n.s. non-significance (f, h) and ***P = 0.0002 (f, h). All P-values are two-tailed. See Supplementary Table 7 for experimental conditions. All source data are provided as a Source Data file.