MAPK Activity Assays

Immunokinase and IP assays were performed using submergence-treated rice seedlings that were harvested at the stipulated time point and were ground in liquid nitrogen. Proteins were isolated using kinase extraction buffer (50 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, 10 mM Na₃VO₄, 10 mM NaF, 50 mM β-glycerolphosphate, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail, and 10% glycerol) and were quantified using Bradford assay (Bradford, 1976). Total protein (30 to 40 µg) was separated by 10% SDS-PAGE, and later, immunoblotting analysis was performed using anti-pTEpY (Cell Signaling Technology; catalog #9101, lot #28).

Crude protein (300 µg) was used for immunoprecipitation using protein A-sepharose beads with the respective antibodies. Anti-pTEpY, rabbit polyclonal anti-MPK6 (catalog #7104, lot #124K4857), anti-MPK4 (catalog #A6979, lot # 124K4855), and anti-MPK3 (catalog #M8318, lot #124K4856) all from Sigma-Aldrich were used for IP. The OsMPK3 antibody was raised in rabbit against the PVAEFRPTMTHGGR polypeptide of OsMPK3. For immunoblot analysis, a 1:7500 dilution of the OsMPK3 antibody was used. As depicted in Supplemental Figure 2D, the anti-AtMPK3, -AtMPK4, and -AtMPK6 antibodies specifically react with the homologous rice counterparts. Also, using the anti-OsMPK3 antibody, a single discrete band was observed for GST-OsMPK3 and in crude protein extracts of N. benthamiana leaf cells overexpressing MPK3, establishing the specificity of the antibody (Supplemental Figure 2E).

In-gel kinase activity assays were performed as described previously (Raina et al., 2012). Briefly, 30 μg total protein was fractionated on a 10% polyacrylamide gel containing 0.1% SDS and 0.5 mg mL⁻¹ bovine brain MBP (Sigma Aldrich). After electrophoresis, the SDS from the gel was removed with buffer (25 mM Tris-HCl, pH 7.5, 0.5 mM DTT, 5 mM Na₃VO₄, 0.1 mM NaF, 0.5 mg mL BSA, and 0.1% Triton X 100) followed by renaturation in buffer (25 mM Tris-HCl, pH 7.5, 0.5 mM DTT, 5 mM Na₃VO₄, and 0.1 mM NaF) at 4°C overnight. MBP phosphorylation was performed by incubating the gel in 20 mL reaction buffer (25 mM Tris-HCl, pH 7.5, 2 mM EGTA, 12 mM MgCl₂, 1 mM DTT, 0.1 mM Na₃VO₄, 1 μM ATP, and 50 μCi [γ-³²P]ATP [3000 Ci mmol⁻¹]) for 60 min at room temperature. The gel was washed three times with 5% trichloroacetic acid and 1% sodium pyrophosphate and visualized by autoradiography using a phosphor imager (Typhoon; GE Healthcare).