For the phenotypic analysis of all disruptants, hyphae or conidia were point inoculated on M+m, dextrin–polypeptone–yeast extract (DPY), ABT737 and potato dextrose (PD) (Nissui, Japan) agar media, and plates were then incubated for 4 days at 30 °C. NSRku70-1-1A was used as a control. To visualize autophagy, the pgEGA8 plasmid containing the A. oryzae niaD gene as a selection marker and the egfp gene-linked Aoatg8 gene (Kikuma et al., 2006) were introduced into the disruption
mutants. Conidia or hyphae from the disruption mutants were cultured in a glass-based dish (Asahi Techno Glass Co., Japan) using 100 μL CD+m medium for 24 h at 30 °C. The medium was then replaced with either fresh CD+m medium (control) or CD+m−N (for the induction of autophagy), and buy FK228 the cells were further incubated for 4 h at 30 °C. The strains were then observed
with an IX71 confocal laser scanning microscope (Olympus Co., Japan). To investigate the effects of defects in signal transduction in autophagy, we first identified the ATG13 homologue in A. oryzae, Aoatg13, from the A. oryzae genome database (http://www.bio.nite.go.jp/dogan/MicroTop?GENOME_ID=ao) using the blast algorithm. Aoatg13 (DDBJ accession number AB586123) contained two introns and three exons, and encoded a predicted polypeptide of 974 amino acids with a calculated molecular mass of 104 kDa. AoAtg13 displayed 24% identity to Atg13 of S. cerevisiae, and an Atg13 family domain was identified in the Pfam database (http://pfam.sanger.ac.uk/) (Fig. S1). To determine the function of Aoatg13, we disrupted Aoatg13 by replacement with the selective marker adeA, which was confirmed by Southern blot analysis (Fig. S4). When Galactosylceramidase the ΔAoatg13
mutant was grown on PD and DPY agar media, the colonies appeared slightly green in color (Fig. 1a) and generated conidia, unlike the ΔAoatg8 mutant (Kikuma et al., 2006). This result suggested that autophagy occurs in the ΔAoatg13 mutants. To confirm this speculation, we generated an ΔAoatg13 strain expressing EGFP–AoAtg8 (DA13EA8). Saccharomyces cerevisiae Atg8 and its orthologues, which are anchored in the membranes of autophagosomes and autophagic bodies, have been used as markers for visualization of autophagy in various organisms (Kabeya et al., 2000; Pinan-Lucarréet al., 2003; Yoshimoto et al., 2004; Monastyrska et al., 2005; Kikuma et al., 2006). In a previous study, we showed that the A. oryzae Atg8 orthologue, AoAtg8, was a useful marker for detecting autophagy in A. oryzae (Kikuma et al., 2006). When strain DA13EA8 was cultured in CD+m medium, EGFP–AoAtg8 was localized in PAS-like structures, but was also diffused in cytoplasm. After growth for 24 h at 30 °C in CD+m medium, the mutant was shifted to nitrogen-deprived medium (CD+m−N) to induce autophagy. Following the induction of autophagy under starvation conditions, the fluorescence of EGFP–AoAtg8 was predominantly observed in PAS-like structures, but could also be seen to a lesser extent in vacuoles (Fig. 1b, CD+m−N).