, 2010). In NAE1 cells, EGFP fluorescence was not detected in vacuoles under growth conditions that were sufficient for the observation of the Cvt pathway in WT (Fig. 3b, NAE1). This result indicated that AoApe1–EGFP was mainly transported to vacuoles via the Cvt pathway. To further investigate the apparent link between autophagy and differentiation of filamentous fungi, including aerial hyphal growth, conidiation, and sclerotial formation, we assayed
for differentiation in an Aoatg1-overexpressing strain (A1-OE), in which Aoatg1 was expressed under control of the amyB promoter. When strain A1-OE strain was grown on PD and CD agar plates, the colonies appeared slightly white in color (Fig. 4a). Moreover, aerial hyphae were longer compared with those formed by WT (Fig. 4b). To determine whether conidiation was repressed in A1-OE, we counted the number of conidia that were harvested from the A1-OE Tacrolimus mw and WT strains grown on CD agar plates for 3 days at 30 °C. The number of conidia formed by A1-OE was decreased by 10% compared to WT (Fig. 4c). These findings suggested that increased levels of AoAtg1 protein facilitated aerial hyphae growth and the repression of conidiation. Finally, we evaluated sclerotial formation in three autophagy-related gene disruptants (ΔAoatg1, ΔAoatg8, and ΔAoatg13) and the Aoatg1-overexpressing strain A1-OE (Fig. 5).
When these strains were grown on DPY agar medium for 9 days at 30 °C, sclerotial formation was increased in A1-OE compared with WT. For ΔAoatg1 and ΔAoatg8, no sclerotia were formed, whereas Bioactive Compound Library a few sclerotia were formed by ΔAoatg13. Taken together, these results suggested that sclerotial formation
was GNAT2 dependent on the degree of autophagy. To investigate the induction of autophagy in A. oryzae, we first analyzed the localization of AoAtg1 fused to EGFP. In S. cerevisiae, Atg1 complexes and many Atg proteins localize to PAS (Suzuki et al., 2001). We found that AoAtg1–EGFP localized to PAS-like structures, as reported for S. cerevisiae Atg1, and that these punctate structures increased when cells were shifted to starvation conditions. This result suggests that AoAtg1 has similar functions to Atg1 in yeast. No differences were observed between ΔAoatg1 and WT with respect to vegetative growth, but marked inhibition of conidiation and aerial hyphal growth were detected. Aspergillus oryzae Aoatg4 and Aoatg8 disruptants are defective in autophagy and display the same phenotype as ΔAoatg1, which is characterized by aerial hyphae formation (Kikuma & Kitamoto, 2011), suggesting a relationship exists between autophagy and aerial hyphae growth. This speculation is consistent with evidence indicating that aerial hyphae grow by reconstructing basal hyphae (Kikuma et al., 2006).