Functional analysis of Aoatg1 and detection of the Cvt pathway in Aspergillus oryzae

Abstract

Autophagy is a degradation system in which cellular components are digested via vacuoles/lysosomes. In the budding yeast Saccharomyces cerevisiae, the induction of autophagy results from inactivation of target of rapamycin complex 1 (TORC1), promoting formation of the serine/threonine kinase Atg1, which is one of the key autophagy-related (Atg) proteins required for both nonselective and selective autophagy such as the cytoplasm-to-vacuole targeting (Cvt) pathway. Here, to understand the induction mechanism of autophagy in filamentous fungi, we first identified the ATG1 homolog Aoatg1 in Aspergillus oryzae and then analyzed the localization of an enhanced green fluorescent protein (EGFP)-AoAtg1 fusion protein. AoAtg1-EGFP localized to pre-autophagosomal structure (PAS)-like structures, similar to Atg1 localization in S. cerevisiae. The function of AoAtg1 was evaluated by constructing an Aoatg1 disruptant, ΔAoatg1. Conidiation and development of aerial hyphae were scarcely observed in ΔAoatg1. Moreover, autophagy in the disruptant was examined by observation of the localization of EGFP-AoAtg8 and AoApe1-EGFP, with the results indicating that AoAtg1 is essential for nonselective autophagy and the Cvt pathway. Furthermore, we demonstrated that the overexpression of Aoatg1 results in decreased conidiation and the excessive development of aerial hyphae and sclerotia. Taken together, our findings provide evidence for the existence of the Cvt pathway in A. oryzae. © 2012 Federation of European Microbiological Societies.