Measurement of callose deposition

Measurement of plant callose deposition in response to bacterial flagellin peptide was performed on 2-wk-old Col-0 seedlings germinated on 1/2 MS medium. Whole seedlings were individually placed in six-well plates with 4 ml of liquid 1/2 MS supplemented with DMSO or 25 μM DSF for 24 h at 22°C, and then elicited for 24 h with 1 μM of flg22. Challenged plants were destained with acetic acid:ethanol (1:3) overnight, and then washed in 150 mM K₂HPO₄ for 30 min. Destained leaves were incubated in 0.01% aniline blue (dissolved in 150 mM K₂HPO₄) in the dark for 2 h, and then mounted on microscope slides in 50% glycerol. Callose deposition were observed on a Zeiss ELYRA PS.1 + LSM780 system using a DAPI filter (excitation:emission, 370:509 nm) with a 10× objective. The experiment was repeated twice, each involved eight individual plants and at least six images were taken from each plant. For callose deposition response to DSF alone, the plants were incubated with increasing concentrations of DSF for 24 h in 1/2 MS, and then stained with aniline blue and visualized as indicated above. The experiment was performed once with n ≥ 18 images taken from three individual plants. Callose deposition was quantified using Fiji software.