Cytoplasmic Membrane Depolarization Assay

Cytoplasmic membrane depolarization activity of the PGP-C peptide was determined using a potential sensitive dye, DiSC₃-(5), as previously described (Wu and Hancock, 1999; Papo and Shai, 2005; Kim et al., 2018). Briefly, X. oryzae pv. oryzae and R. solanacearum were grown at 30°C to mid-log phase (OD_{600 nm} = 0.7) and were harvested by centrifugation. Cells were washed 3 times with buffer solution (5 mM HEPES, 20 mM glucose, pH 7.4) and gently resuspended to an OD_{600 nm} of 0.15 in the same buffer solution. Then, the cell suspension was incubated with 2 μM of DiSC₃-(5) at room temperature for 1 h. DiSC₃-(5) was used with 2 mM stock in dimethylsulfoxide (DMSO). Membrane depolarization was monitored using a F-4500 FL fluorescence spectrophotometer (Hitachi, Japan), with filter wavelengths of 620 and 670 nm for excitation and emission, respectively. The peptide was treated after 100 s and the peptide-free cells were used as a negative control.