3 3 diaminobenzidine (dab)

CCCP, rotenone, oligomycin, and antimycin A were purchased from Sigma-Aldrich (St. Louis, MO). Cytochrome c from horse heart was supplied by Nacalai Tesque Inc. (Kyoto, Japan) and 3,3′-diaminobenzidine was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Furimazine was supplied by Promega (Madison, WI) and poly(I:C) was purchased from InvivoGen (San Diego, CA). Enzyme-linked immunosorbent assay (ELISA) kits for human and mouse IFN-β were supplied by Kamakura Techno-Science Inc. (Kanagawa, Japan) and PBL Assay Science (Piscataway, NJ), respectively. All other reagents were of biochemical research grade.

3,3′-Diaminobenzidine (DAB) staining was performed according to the procedures described by Fester and Hause (15 (link)) with the following modifications. To visualize hydrogen peroxide (H₂O₂), rice plants were placed in DAB solution (1 mg mL⁻¹ DAB; Tokyo Chemical Industry, Tokyo, Japan) buffered with 200 mM sodium phosphate, pH 6.5, at room temperature for 3–4 h. Photographs were taken using a Lumix DMC-G3 camera (Panasonic, Osaka, Japan). Quantitative measurements of H₂O₂ were analyzed as described previously (43 (link)). Briefly, DAB-stained root samples (100 mg) were washed in 80% ethanol for 20 min and homogenized immediately in 500 μL of 0.2 M perchloric acid (HClO₄) (Wako Pure Chemical Industries) in a pre-cooled mortar. The mixtures were incubated on ice for 5 min and then centrifuged (10,000×g, 4°C, 10 min). The absorbance of the supernatants was measured at 450 nm and H₂O₂ concentrations were obtained via a standard calibration with solutions of 0.2 M HClO₄ containing 5, 10, 25, and 50 μM H₂O₂ (Sigma-Aldrich, St. Louis, MO, USA).