These findings suggest that AoAtg1 plays an essential role in these pathways in A. oryzae. Based on the observed localization of EGFP–AoAtg8 in the ΔAoatg1 disruptant, EGFP Protease Inhibitor Library high throughput fluorescence was not detected in vacuoles even under starvation conditions, whereas EGFP puncta were formed under both nutrient-rich

and starvation conditions. It appears that the punctate structures observed in the strain expressing EGFP–AoAtg8 differed from the PAS-like structures observed in WT. Although PAS is generally localized to the periphery of vacuoles in WT, the punctate structures in ΔA1EGA8 were observed not only around vacuoles but were also found in the cytoplasm and, in addition, were larger in size than the PAS of WT. This result is consistent with the finding in S. cerevisiae that PAS-like punctate structures in an atg1 mutant are larger than those of WT and that an overabundance of Atg proteins is assembled to PAS, suggesting that Atg1 functions in the formation of autophagosomes from PAS (Suzuki et al., 2001). In a temperature-sensitive atg1 mutant (apg1ts), punctate structures of GFP–Atg8, which are excessively assembled at restrictive temperatures, are transported to vacuoles after a shift to permissive temperature (Suzuki et al., 2001). This phenomenon suggests that the punctate structures observed in

Aoatg1 disruptants are an assembly AZD6244 mw of AoAtg proteins that results due to inhibition of autophagosome

formation from PAS. Our localization analysis of the EGFP-fused prApe1 homolog in A. oryzae represents the first such analysis in a filamentous fungus. After incubation of the WT and ΔA1Ape1EG strains for 20 h at 30 °C, we observed AoApe1–EGFP fluorescence in vacuoles Tobramycin in WT, whereas that in ΔA1Ape1EG was not detected in vacuoles, but appeared as punctate structures in the cytoplasm. The localization pattern did not change even when ΔA1Ape1EG was shifted to starvation conditions. These results suggest that A. oryzae has a functional Cvt pathway and that the punctate structures observed in ΔAoatg1 were not Cvt vesicles, but rather were clusters of AoApe1 proteins. Genome-wide functional analysis in M. oryzae revealed that nonselective autophagy was an important factor of plant infection, and clear orthologues of S. cerevisiae genes required for the Cvt pathway, such as ATG19, were not found in the M. oryzae genome sequence (Kershaw & Talbot, 2009). Therefore, the Cvt pathway is considered to be missing in M. oryzae. Also, the Cvt pathway -specific proteins in S. cerevisiae are poorly conserved in A. oryzae, suggesting the existence of a functional homolog that is unable to be identified by homology searches. Atg13 plays an essential role in autophagy, as demonstrated by the disruption of atg13 in yeast, which results in the impaired ability to form Atg1 kinase complexes to induce autophagy (Kabeya et al., 2005).