3 3 diaminobenzidine (dab)

To observe the H₂O₂ accumulation in rice plants, the leaves or the 5-cm-long leaf sheaths of the three- to five-leaf-stage seedlings were inoculated with M. oryzae strain Guy11 at the concentration of 5 × 10⁵ conidia ml^–1 as described previously (Kankanala et al., 2007 (link)). Then, the inoculated leaves or the excised epidermal layer of the leaf sheaths were incubated in 1 mg/ml 3,3′-diaminobenzidine (DAB; Sigma–Aldrich, Germany) at 22°C for 8 h. The DAB-stained leaves and leaf sheaths were cleaned in 95% ethanol and then observed under a microscope (Zeiss Imager A2, Carl Zeiss, Germany).

Cellular ROS accumulation in fungal infected plant cells was analyzed by using DAB (Sigma, D-8001) staining method^{88 (link)}. Briefly, infected tissues were immersed in DAB solution (1 mg/ml, pH 3.8) at room temperature for 8 h and destained with decolorant solution (ethanol: acetic acid = 94: 4, v/v) for 1 h. The DAB-stained plant tissues were examined under a Zeiss LSM780 confocal microscope (Gottingen, Niedersachsen, Germany).
To assess NO production, fungal hyphae or plant tissue were stained using 10 μM DAF-FM DA (Sigma, Shanghai, China). To confirm genuine reaction between DAF-FM DA and NO, a cell-permeant NO scavenger cPTIO (Sigma) at 100 μM was included as a control. DAF-FM DA combined with or without cPTIO was directly applied towards plant tissues with or without Fg infection. Plant tissues or fungal hyphae were stained with DAF-FM DA dye for 30 min at room temperature and visualized in a bight/fluorescence field of view under a Zeiss LSM780 confocal microscope (Gottingen) at the excitation/emission wavelengths of 488/525 nm. Each experiment was repeated three times.