Seed Cell Wall Staining Protocol

The staining procedure was performed as previously described [24 (link)], with slight modifications, as follows. After neutralization of potassium hydroxide (KOH) treatment, the seeds were stained with 40 µg/mL WGA-Alexa fluor-488 (Alexa-488) solution (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min and 20 µg/mL Calcofluor White solution (Sigma-Aldrich, St. Louis, MO, USA) for 5 min in the dark at room temperature. A seed coat of the stained intact seed was removed under a stereomicroscope (SZX16; Olympus, Tokyo, Japan). Stained cells were observed under a fluorescence microscope (DM2500; Leica, Wetzlar, Germany) with an excitation filter of 480/40 nm for Alexa-488-conjugated fungal hyphae and 340–380 nm for Calcofluor White-stained plant cell walls.