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

Abstract

The role of cytosolic pH (pHc ) 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 pHc 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 pHc in growing germ tubes was consistently pH 7.2±0.1 during all developmental stages analysed. Only slight changes in pHc (<0.1 pH unit) were found in response to alkaline external pH (pH 8.0). No changes in pHc 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 pHc in growing germ tubes accompanying conidium germination, germ tube growth or early appressorium formation.