The staining methods for callose and dead cells were based on Wang et al. [41 (link)] and were repeated three times. To observe callose deposition and dead cells, 20 freshly inoculated young leaves or JA + M. oryzae-infected 36 hpi and JA + M. oryzae-infected 48 hpi young leaves of each sample were studied. To analyze callose deposition, fresh leaves were cleared with 95% ethanol and incubated in an ethanol-emulsifiable solution (glycerol:phenol:water:ethanol:lactic acid = 1:1:1:8:1) at 65 °C until the removal of the green color. The leaves were then washed with 50% ethanol, followed by sterilized water. The number of deposited callose was counted in 20 fields of vision after staining the treated leaves with 0.1% aniline blue for 1 h. Observations and photographs were taken using a DMI4000B fluorescence inverted microscope (Leica Camera AG, Wetzlar, Germany).
To observe dead cells, fresh leaves were cleaned with sterile water and incubated in 100% ethanol until the complete removal of the green color. The leaves were then stained for 24 h in DAB (3,3-diaminobezidin, 1 mg/mL, pH 5.8). The number of dead cells was measured through photographic analysis of 20 separate visual fields.
To observe dead cells, fresh leaves were cleaned with sterile water and incubated in 100% ethanol until the complete removal of the green color. The leaves were then stained for 24 h in DAB (3,3-diaminobezidin, 1 mg/mL, pH 5.8). The number of dead cells was measured through photographic analysis of 20 separate visual fields.
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