Vacuolar membrane dynamics in the filamentous fungus Aspergillus oryzae

Vacuoles in filamentous fungi are highly pleomorphic and some of them, e.g., tubular vacuoles, are implicated in intra- and intercellular transport. In this report, we isolated Aovam3, the homologue of the Saccharomyces cerevisiae VAM3 gene that encodes the vacuolar syntaxin, from Aspergillus oryzae. In yeast complementation analyses, the expression of Aovam3 restored the phenotypes of both Deltavam3 and Deltapep12 mutants, suggesting that AoVam3p is likely the vacuolar and/or endosomal syntaxin in A. oryzae. FM4-64 [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)pyridinium dibromide] and CMAC (7-amino-4-chloromethylcoumarin) staining confirmed that the fusion protein of enhanced green fluorescent protein (EGFP) with AoVam3p (EGFP-AoVam3p) localized on the membrane of the pleomorphic vacuolar networks, including large spherical vacuoles, tubular vacuoles, and putative late endosomes/prevacuolar compartments. EGFP-AoVam3p-expressing strains allowed us to observe the dynamics of vacuoles with high resolutions, and moreover, led to the discovery of several new aspects of fungal vacuoles, which have not been discovered so far with conventional staining methods, during different developmental stages. In old hyphae, EGFP fluorescence was present in the entire lumen of large vacuoles, which occupied most of the cell, indicating that degradation of cytosolic materials had occurred in such hyphae via an autophagic process. In hyphae that were not in contact with nutrients, such as aerial hyphae and hyphae that grew on a glass surface, vacuoles were composed of small punctate structures and tubular elements that often formed reticulum-like networks. These observations imply the presence of so-far-unrecognized roles of vacuoles in the development of filamentous fungi.