Autophagy is a well-known degradation system, induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. Recently, it was reported that autophagy is involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. In this study, we isolated the ATG8 gene homologue Aoatg8 from the filamentous fungus Aspergillus oryzae and visualized autophagy by the expression of DsRed2-AoAtg8 and enhanced green fluorescent protein-AoAtg8 fusion proteins in this fungus. While the fusion proteins were localized in dot structures which are preautophagosomal structure-like structures under normal growth conditions, starvation or rapamycin treatment caused their accumulation in vacuoles. DsRed2 expressed in the cytoplasm was also taken up into vacuoles under starvation conditions or during the differentiation of conidiophores and conidial germination. Deletion mutants of Aoatg8 did not form aerial hyphae and conidia, and DsRed2 was not localized in vacuoles under starvation conditions, indicating that Aoatg8 is essential for autophagy. Furthermore, Aoatg8 conditional mutants showed delayed conidial germination in the absence of nitrogen sources. These results suggest that autophagy functions in both the differentiation of aerial hyphae and in conidial germination in A. oryzae.Autophagy is a protein degradation system that is conserved in eukaryotic cells and used to recycle macromolecules and aid cell survival under nutritional starvation conditions (12, 13). When autophagy is induced, bulk cytoplasm and/or organelles are sequestered within double-membrane vesicles termed autophagosomes. The outer membranes of the autophagosomes then fuse to the vacuolar/lysosomal membrane and deliver single-membrane vesicles, called autophagic bodies, into the lumina of the vacuoles/lysosomes. The subsequent breakdown of the vesicle membranes allows the degradation of the contents of the autophagic bodies by vacuolar hydrolases. ATG8 is an autophagy-related gene found in Saccharomyces cerevisiae that plays an important role in the formation of autophagosomes (9). Atg8 is localized in the membranes of preautophagosomal structures (PAS), autophagosomes, and autophagic bodies and has therefore been used as a marker of these organelles (33).In addition to helping cells to survive starvation, autophagy is involved in stress-induced differentiation and development. In S. cerevisiae diploid cells, atg mutations block starvationinduced sporulation (34). In Dictyostelium discoideum, starvation, overcrowding, and high temperature induce the formation of fruiting bodies and atg mutations block these multicellular developmental processes (25). Additionally, in Caenorhabditis elegans, atg mutations result in abnormal dauer development, which is also induced by starvation, overcrowding, high temperature and so on (17). Recent studies have suggested that autophagy might participate in diseases such as cancer, liver disease, muscula...

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The vacuolar protein sorting (vps) system in the filamentous fungus Aspergillus oryzae, which has unique cell polarity and the ability to secrete large amounts of proteins, was evaluated by using mutants that missort vacuolar proteins into the medium. Vacuolar carboxypeptidase Y (CPY) fused with enhanced green fluorescent protein (EGFP) was used as a vacuolar marker. Twenty dfc (dim EGFP fluorescence in conidia) mutants with reduced intracellular EGFP fluorescence in conidia were isolated by fluorescence-activated cell sorting from approximately 20,000 UV-treated conidia. Similarly, 22 hfm (hyper-EGFP fluorescence released into the medium) mutants with increased extracellular EGFP fluorescence were isolated by using a fluorescence microplate reader from approximately 20,000 UV-treated conidia. The dfc and hfm mutant phenotypes were pH dependent, and missorting of CPY-EGFP could vary by 10-to 40-fold depending on the ambient pH. At pH 5.5, the dfc-14 and hfm-4 mutants had an abnormal hyphal morphology that is consistent with fragmentation of vacuoles and defects in cell polarity. In contrast, the hyphal and vacuolar morphology of the dfc-14 and hfm-4 mutants was normal at pH 8.0, although CPY-EGFP accumulated in perivacuolar dot-like structures similar to the class E compartments in Saccharomyces cerevisiae vps mutants. In hfm-21, CPY-EGFP localized at the Spitzenkörper when the mutant was grown at pH 8.0 but not in vacuoles, suggesting that hfm-21 may transport CPY-EGFP via a novel pathway that involves the Spitzenkörper. Correlations between vacuolar protein sorting, pH response, and cell polarity are reported for the first time for filamentous fungi.