2a–c), similar to the ΔAoatg8 disruptant (Kikuma et al., 2006). The ΔAoatg13 and ΔAoatg8 disruptants exhibit decreased levels of autophagy, particularly strain ΔAoatg8, in which autophagy is completely inhibited (Kikuma et al., 2006; Kikuma & Kitamoto, 2011) (Fig. 2b), indicating that the level of autophagic activity correlates with Akt inhibitor drugs the degree of conidiation and aerial hyphal growth (Kikuma & Kitamoto, 2011). Based on the lack of aerial hyphae and conidiation in ΔAoatg1, autophagy was likely completely inhibited in ΔAoatg1. To confirm the above speculation,

we generated a ΔAoatg1 strain expressing EGFP–AoAtg8 (ΔA1EA8). We previously demonstrated that the Atg8 ortholog in A. oryzae, AoAtg8, is a useful marker for detecting autophagy in A. oryzae (Kikuma et al., 2006). When the ΔA1EA8 strain was cultured in CD + m medium (growth condition), EGFP–AoAtg8 was localized in PAS-like structures, but was also diffused in the cytoplasm (Fig. 3a). After shifting the mutant to nitrogen-deprived medium (CD − N) to induce autophagy, EGFP–AoAtg8 fluorescence was observed in PAS-like structures, but could not be detected in vacuoles (Fig. 3a). Moreover, punctate structures with larger diameters than typical PAS-like structures were observed (Fig. 3a, arrows), and no cup-shaped isolation membranes or Y-27632 ring-like structures were detected. These observations indicated that the autophagic process was completely defective in the

ΔAoatg1 disruptant. To determine whether the Cvt pathway exists in A. oryzae and to evaluate the role of AoAtg1 in this pathway, we constructed strains expressing Aspartate AoApe1, which is an A. oryzae homolog of prApe1, fused to EGFP in the wild type (WT) and ΔAoatg1 backgrounds (Ku70aApe1EG and ΔA1Ape1EG, respectively). We selected prApe1 as it has been used as marker for the visualization of the Cvt pathway in S. cerevisiae (Harding et al.,

1995). Under normal growth conditions, prApe1 oligomerizes into homo-dodecamers and is then delivered to vacuoles by autophagic machinery, where it is cleaved to form the mature peptide. When the Ku70aApe1EG and ΔA1Ape1EG strains were cultured in CD medium for 20 h at 30 °C, AoApe1–EGFP was localized to vacuoles in WT, but appeared as punctate structures in ΔA1Ape1EG (Fig. 3b). These observations indicated that the Cvt pathway was functional in A. oryzae, but was completely defective in ΔAoatg1. PAS-like structures are normally observed at the periphery of vacuoles in yeast and filamentous fungi (Shintani et al., 2002); however, in strain ΔA1EA8 expressing EGFP–AoAtg8 and strain ΔA1Ape1EG expressing AoApe1–EGFP in the ΔAoatg1 background, the punctate structures observed in the perivacuolar region of ΔAoatg1 were also localized diffusely in the cytoplasm. Therefore, we consider that the structures observed in ΔAoatg1 were not normal PAS-like structures, but aggregates of AoAtg8 or AoApe1 oligomers.