Root tissue of barley control and mutant plants colonized by *S. indica* was harvested at 6 dpi and then stained as previously described.46 Briefly, roots were incubated at 95 °C for 2 min in 10% KOH, washed 3 times for 30 min in deionized water and 3 times for 30 min in PBS (pH 7.4). The roots were stained for 5 min under vacuum and then washed three times with deionized water. Fungal structures were visualized using 10 μg·mL-1 fluorescently labeled wheat germ agglutinin (WGA-AF488, Invitrogen, Thermo Fisher Scientific, Schwerte, Germany) in PBS (pH 7.4) and imaging was conducted with an excitation wavelength of 488 nm and emission detection between 500-540 nm. Papillae and root cell wall appositions were stained with 10 μg·mL-1 fluorescently labeled Concanavalin A (ConA-AF633, Invitrogen, Thermo Fisher Scientific, Schwerte, Germany) in PBS (pH 7.4) and imaged by excitation at 633 nm and detection at 650-690 nm.
Callose was stained by aniline blue according to a protocol adapted from Mason and coworkers.89 Roots were incubated at 95 °C for 2 min in 10% KOH, washed 3 times for 30 min in deionized water and 3 times for 30 min in PBS (pH 7.4). Roots were washed for 1 h at RT under continuous shaking in 67 mM K2HPO4 (pH 12). The samples were then incubated for 1 h at RT under continuous shaking in an aniline blue solution composed of 0.01% aniline blue (w/v) in 67 mM K2HPO4 (pH 12). Roots were washed again for 1 h at RT under continuous shaking in 67 mM K2HPO4 (pH 12). Imaging was conducted with an excitation wavelength of 405 nm and emission detection from 490-510 nm.
Images were taken with a Leica TCS SP8 confocal microscope (Wetzlar, Germany). The percentage area of ConA staining was quantified using ImageJ81 in maximum intensity projections of 10-slice Z-stacks with an image depth of 10 μm. At least 24 different root regions of each genotype were analyzed.
