Confocal ratio imaging of cytoplasmic pH during germ tube growth and appressorium induction by Magnaporthe grisea

The role of cytosolic pH (pH_c) in growing germ tubes of the filamentous fungus Magnaporthe grisea was analysed by confocal ratio imaging of the pH-sensitive fluorescent dye 5(6)-carboxyseminaphthorhodafluor-1 (SNARF-1). The cytosol of these cells was successfully loaded with the acetoxymethyl ester of the dye and the pH_c was visualized and quantified during conidium germination, germ tube growth and appressorium induction by simultaneous dual-emission confocal ratio imaging. Calibrations of the free acid in vitro and calibrations in vivo produced results indicating a similar dynamic response in the pH range 6.0–8.0 for both methods. The pH_c in growing germ tubes was consistently pH 7.2±0.1 during all developmental stages analysed. Only slight changes in pH_c (<0.1 pH unit) were found in response to alkaline external pH (pH 8.0). No changes in pH_c occurred in response to an acidic extracellular pH (pH 6.0) or to the presence of nutrients. There was no observation of either pronounced gradients or changes in pH_c in growing germ tubes accompanying conidium germination, germ tube growth or early appressorium formation.