MAPK Activation Assays

Twelve-days-old Arabidopsis seedlings grown on liquid ½ Murashige and Skoog media (Duchefa) were treated with PcBMM spores extracts or 100 mM flg22 for 0, 5, 15, and 30 min. PcBMM spores are stored at -80° in 20% glycerol. To obtain spores extracts, a suspension of 4 × 10⁶ spores/ml were spinned down, resuspended in sterilized water and grinded in liquid nitrogen. 100 μl of the spores extract was added to 12–15 Arabidopsis seedlings grown in 2 ml of liquid ½ Murashige and Skoog media. Protein extraction and detection of activated MAPKs were performed as described (Ranf et al., 2011): the activated MAP kinases were detected using anti-P44/42 (Anti-Erk1-Erk2; Thr202-Tyr204) MAPK Rabbit primary antibody overnight at 4°C (1:1000; Cell Signaling Technology) rinsed four times for 5 min, followed by treatment with HRP conjugate Goat anti-rabbit IgG secondary antibody for 2 h (1: 5000; Fisher Scientific). Before ECL (Pierce) detection membranes were rinsed four times with 0.1 TBST for 5 min each.