Wheat germ agglutinin fluorescein isothiocyanate

For chitin exposure analysis, cells were stained with 1mg/ml wheat germ agglutinin-fluorescein isothiocyanate (Sigma-Aldrich, St. Louis, MO, United States), for 60min at room temperature. Flow cytometry was performed in a MoFlo XDP apparatus (Beckman Coulter), collecting 50,000 singlet events. Fluorescence of positive events was recovered from the compensated FL3 (green) channel using unlabeled yeast cells. Total population densities were gated and analyzed using FlowJo (version 10.0.7) software.
The heat-killed (HK) cells were prepared by incubating at 60°C for 2h. The cellular death was confirmed by incubating aliquots of the preparations in YPD plates at 37°C for 5days.

Fungal‐infected tissue was stained with wheat germ agglutinin–fluorescein isothiocyanate (Sigma‐Aldrich, St. Louis) and visualized under blue light (Ayliffe et al., 2011). Histochemical detection of H₂O₂ in infected leaf tissue was undertaken by 3,3′‐diaminobenzidine (DAB) staining (Sigma‐Aldrich, St. Louis) (Thordal‐Christensen et al., 1997). H₂O₂ levels were determined in ground leaf tissues using an Amplex Red H₂O₂ assay kit as described by the manufacturer (ThermoFisher).