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

Shin Yanagisawa , Takashi Kikuma , Katsuhiko Kitamoto
DOI: http://dx.doi.org/10.1111/1574-6968.12047 168-176 First published online: 1 January 2013

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 italiclamentous fungi, we italicrst identiitaliced 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 italicndings provide evidence for the existence of the Cvt pathway in A. oryzae.

Keywords
  • autophagy
  • filamentous fungi
  • differentiation
  • sclerotia

Introduction

Macroautophagy (hereafter autophagy) is a highly conserved degradation pathway that mediates the turnover of bulk cytoplasmic protein and organelles induced under nutritional starvation conditions (Nakatogawa et al., 2009). Autophagy plays a number of roles associated with quality and quantity control of cytoplasmic components, including the killing of intracellular microorganisms (Deretic & Levine, 2009) and removal of damaged or depolarized mitochondria (Apostolova et al., 2011). The autophagic process consists of several sequential steps: the induction of autophagy, autophagosome formation, fusion of autophagosomes to lysosomes/vacuoles, and degradation of autophagic bodies (Mizushima, 2007). The basic machinery of autophagy appears to be evolutionarily conserved as it is found in all eukaryotic cells (Di Bartolomeo et al., 2010; Avin-Wittenberg et al., 2012). The induction of autophagy elicits the formation of cup-shaped isolation membranes that elongate and sequester cytosol and/or organelles within double-membrane vesicles termed autophagosomes. Autophagosomes subsequently fuse with lysosomes/vacuoles, into which the inner single-membrane vesicle is released. The membrane of the resulting autophagic body is lysed to allow the contents to be broken down (Suzuki et al., 2001).

In the budding yeast Saccharomyces cerevisiae, autophagy is induced by the inactivation of target of rapamycin complex 1 (TORC1), allowing formation of the Atg1 kinase complex, which is composed of the autophagy-related (Atg) proteins Atg1, Atg13, and Atg17 (Kabeya et al., 2005). Atg13 directly associates with the serine/threonine kinase Atg1, and the formation of this complex correlates with an increase in autophagic activity (Yeh et al., 2011). Atg1 is a key Atg protein, as it is required for both nonselective and selective autophagy such as the cytoplasm-to-vacuole targeting (Cvt) pathway. In the Cvt pathway, the substrates prApe1 (precursor of aminopeptidase) and Ams1 (α-mannosidase) form homo-oligomers in the cytoplasm and are then enwrapped by the autophagosomal membrane, forming the Cvt vesicle. Under conditions suitable for growth, the interaction between Atg1 and Atg13 is inhibited by the phosphorylation of Atg13 in a TORC1-dependent manner, leading to the activation of the Cvt pathway. In contrast, under starvation conditions, Atg13 is dephosphorylated due to the inactivation of TORC1, allowing Atg13 to associate with Atg1 (Kamada et al., 2000). To date, it is not clear whether the Cvt pathway exists in filamentous fungi.

Although the study of autophagic machinery has mainly been performed in S. cerevisiae, autophagy has also been studied in the filamentous fungi Podospora anserina, Fusarium graminearum, Magnaporthe oryzae, Trichoderma reesei, Penicillium chrysogenum, Aspergillus fumigatus, Aspergillus nidulans, and Aspergillus oryzae (Liu et al., 2007, 2010, 2011; Richie et al., 2007; Bartoszewska et al., 2011; Kikuma & Kitamoto, 2011; Kim et al., 2011a, b; Nguyen et al., 2011). In A. fumigatus, ΔAfatg1 disruptants are deficient in autophagy and exhibit reduced conidiation, resulting from the formation of abnormal conidiophores (Richie et al., 2007). Autophagy also contributes to the recycling of essential metal ions in A. fumigatus under nutrient-starved conditions (Richie et al., 2007). To date, however, detailed analyses of autophagy induction in filamentous fungi have not performed, and thus, the autophagic process remains poorly understood in these organisms.

In previous studies of A. oryzae, we identified and analyzed the autophagy-related proteins AoAtg8 (Kikuma et al., 2006), AoAtg13, AoAtg4, and AoAtg15 (Kikuma & Kitamoto, 2011). AoAtg8 is essential for autophagy and serves as a useful marker for detecting autophagy in A. oryzae, while AoAtg4 and AoAtg15 are required for autophagosome formation and the lysis of autophagic bodies, respectively. Disruption of the genes coding for AoAtg4, AoAtg8, and AoAtg15 causes severe defects in the formation of aerial hyphae and conidia, resulting from the impairment of autophagic flux. In contrast, disruptants of Aoatg13 form a few aerial hyphae and conidia, suggesting that these disruptants still possess autophagic activity, unlike S. cerevisiae ATG13 disruptants. Therefore, the underlying mechanism and components involved in autophagy in A. oryzae remain incompletely understood.

In the present study, we identified a homolog of Atg1 in A. oryzae (AoAtg1) that appears to participate in the first stage of autophagy induction. To evaluate the function of AoAtg1 in the autophagy process, we generated an Aoatg1 disruptant (ΔAoatg1) expressing EGFP–AoAtg8 and AoApe1–EGFP revealing that AoAtg1 has an essential function in the autophagy process. We also found evidence for the Cvt pathway in A. oryzae by observing the transportation of AoApe1 to vacuoles, suggesting that AoAtg1 also plays an essential role in the Cvt pathway.

Materials and methods

Strains and growth media

The A. oryzae strains used in this study are listed in Table 1. The A. oryzae wild-type strain RIB40 was used as a DNA donor, and strain NSRku70-1-1 (niaD sC adeA argB Δku70::argB) (Takahashi et al., 2006) was used to disrupt the Aoatg1 gene. Strain NSRku70-1-1 transformed with adeA (NSRku70-1-1A) (Higuchi et al., 2009) was used as a control for the phenotypic assay. Strain niaD300 was used to overexpress the Aoatg1 gene. Czapek-Dox (CD) medium [0.3% NaNO3, 0.2% KCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.002% FeSO4·7H2O, and 2% glucose (pH 5.5)] supplemented with 0.0015% methionine (CD + m) was used as a selective medium for identifying positive clones of ΔAoatg1 disruptants expressing EGFP–AoAtg8 and AoApe1–EGFP. CD medium lacking sodium nitrate (CD N) was used for inducing autophagy. Dextrin–polypeptone–yeast extract (DPY) agar medium was used for the sclerotial formation assay.

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Table 1

Aspergillus oryzae strains used in this study

StrainGenotypeReference
RIB40WT
Ku70-1-1niaD sC adeA argB Δku70::argBTakahashi et al. (2006)
Ku70-1-1AniaD sC adeA argB Δku70::argB adeAHiguchi et al. (2009)
ΔAoatg8niaD sC adeA Aoatg8::adeAKikuma et al. (2006)
ΔAoatg13niaD sC adeA argB Δku70::argB ΔAoatg13::adeAKikuma & Kitamoto (2011)
niaD300niaDMinetoki et al. (1996)
A1EGniaD sC adeA argB Δku70::argB (Aoatg1 C-terminal 1.6 kb-egfp-amyB-adeA-Aoatg1 downstream 1.6 kb, adeA)This study
ΔAoatg1niaD sC adeA argB Δku70::argB ΔAoatg1::adeAThis study
ΔA1EA8niaD sC adeA argB Δku70::argB adeA (PAoatg8-egfp-Aoatg8, niaD)This study
Ku70EGA8niaD sC adeA argB Δku70::argB adeA (PAoatg8-egfp-Aoatg8, niaD)This study
ΔA1Ape1EGniaD sC adeA argB Δku70::argB ΔAoatg1::adeAThis study
Ku70aApe1EGniaD sC adeA argB Δku70::argB adeA (PamyB-AoApe1–egfp, niaD)This study
NAE1niaD (PamyB–egfp, niaD)Kimura et al. (2010)
A1-OEniaD (PamyB-Aoatg1, niaD)This study

Construction of Aoatg1 disruptants

To disrupt the Aoatg1 gene, the plasmid pTΔAoatg1 was constructed using fusion PCR and pCR®4Blunt-TOPO® (Invitrogen, Carlsbad, CA). The upstream and downstream 1.5-kb regions of the Aoatg1 gene and the adeA genes were amplified by PCR using the following primer pairs, which contained overlapping sequences (underlined) at the 5′ terminus: 5′-TGGAGGCAAGTCCTTGGAAG-3′ and 5′-CTGTTGCGCAAAGAATCAACCACACCCCGG-3′, 5′-GTTGATTCTTTGCGCAACAGCATACGAGTC-3′ and 5′-AATCTCATGCCATGCCGTCATGTCCAGGAA-3′, 5′-TGACGGCATGGCATGAGATTAGTCGTTCCACGTT-3′ and 5′-CAACCCAATGCCACGTTGGT-3′, respectively. The amplified fragments were introduced into pCR®4Blunt-TOPO® by ligation to generate pTΔAoatg1. Using plasmid pgΔAoatg1 as a template, the sequence containing the Aoatg1 deletion cassette, which consisted of the 1.5-kb upstream region of Aoatg1, adeA gene (2.0 kb), and 1.5-kb downstream region of Aoatg1, was amplified by PCR with the primers upAoatg1-F and downAoatg1-R and then transformed into A. oryzae NSRku70-1-1. Disruption of the Aoatg1 gene was confirmed by Southern blotting using a 1.5-kb fragment of the region upstream of Aoatg1 as a probe, which was generated by PCR with the primers upAoatg1-F and upAoatg1-R.

Visualization of nonselective autophagy and the Cvt pathway in the Aoatg1 disruptant

To visualize autophagy in A. oryzae, the plasmid pgEGA8 containing the A. oryzae niaD gene as a selection marker and the egfp gene linked to the Aoatg8 gene (Kikuma et al., 2006) was introduced into the ΔAoatg1 disruptant. To visualize the Cvt pathway, the gene encoding AoApe1 (DDBJ accession no. AB698488), Aoape1, was amplified by PCR using the primer pairs pgE_Aoape1_F (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTAAATGACCAAAGGAGTGCCTTG-3′) and pgE_Aoape1_R_nonstop (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCGAAATCTGCAAATTCCTTGTCCAC-3′), which contained Multisite Gateway attB recombination sites (underlined). The amplified attB-flanked fragment was introduced into pDONR™221 using the Gateway BP Clonase Reaction Mix (Invitrogen, Japan), generating the center Entry Clone plasmid pgEAoApe1. Three entry clones containing the amyB promoter, Aoape1, and egfp, respectively, were integrated into a destination vector containing the niaD marker gene using the Multisite Gateway system (Mabashi et al., 2006). The resulting plasmid, pgaApe1EG, was then transformed into the ΔAoatg1 disruptant and NSRku70-1-1A. Conidia or hyphae from the ΔAoatg1 strain expressing EGFP–AoAtg8 (ΔA1EA8) or expressing AoApe1–EGFP (ΔA1Ape1EG) 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 − N (for the induction of autophagy), and 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).

Construction of strains expressing AoAtg1–EGFP

To determine the coding region of AoAtg1 (DDBJ accession no. AB698487), rapid amplification of cDNA ends (RACE) analysis was performed using a GeneRacer™ kit (Invitrogen) as directed by the manufacturer. The plasmid pgA1EG was constructed to express AoAtg1–EGFP protein under control of the native AoAtg1 promoter using the Multisite Gateway cloning system. Briefly, a 0.8-kb fragment of the C-terminal side of AoAtg1, a 1.5-kb downstream region of the Aoatg1 gene, and egfp and adeA genes were amplified by PCR using the primer pairs pg5′aoatg1locusF (5′-GGGGACAACTTTGTATAGAAAAGTTGAATGGTCCCGGAAGAACCGTGG-3′) and pg5′aoatg1locusR_no-tag (5′-GGGGACTGCTTTTTTGTACAAACTTGATTTGGGCGTTGTCCCGACGG-3′), DA1_fusion_F_2 (5′-GTTGATTCTTTGCGCAACAGCATACGAGTC-3′) and DA1_fusion_R_2 (5′-AATCTCATGCCATGCCGTCATGTCCAGGAA-3′), pg3′aoatg1-locusdownF (5′-GGGGACAGCTTTCTTGTACAAAGTGGAAAACGTGGAACGACTAATCTCATGCATGCA-3′) and pg3′aoatg1-locusdownR (5′-GGGGACAACTTTGTATAATAAAGTTGATAAACGTACTTCGGGATAGCAGTACCC-3′), respectively, which contained Multisite Gateway attB recombination sites (underlined). The amplified attB-flanked upstream and downstream fragments were introduced into pDONR™P4-P1R and pDONR™P2R-P3, respectively, using the Gateway BP Clonase Reaction Mix (Invitrogen, Japan), generating the Entry Clone plasmids pg5′CAoatg1 and pg3′downAoatg1, respectively. The plasmids pg5′CAoatg1, pg3′downAoatg1, the Entry Clone plasmid containing the A. oryzae adeA gene as a selective marker (constructed in our laboratory), and the destination vector pDEST™R4-R3 (Invitrogen) were then subjected to the Gateway LR reaction using the Gateway LR Clonase Reaction Mix (Invitrogen) to generate plasmid pgA1EG. Using plasmid pgΔAoatg1 as a template, the sequence containing the deletion cassette, which consisted of the C-terminal region of Aoatg1 (0.8 kb), egfp and adeA genes (2.9 kb), and 1.5-kb downstream region of Aoatg1, was amplified by PCR with the primers pg5′aoatg1locusF and pg3′aoatg1-locusdownR, and then transformed into A. oryzae NSRku70-1-1. The recombination of the Aoatg1 and egfp genes was confirmed by Southern blotting using a 2.0-kb fragment of the region downstream of Aoatg1 as a probe, which was generated by PCR with the primers downAoatg1-F and downAoatg1-R.

Construction of an Aoatg1-overexpressing strain

The plasmid pgaA1, which harbored the amyB promoter, Aoatg1 gene, and selection marker niaD, was constructed to overexpress AoAtg1 under control of the amyB promoter using the Multisite Gateway cloning system. The pgaA1 plasmid was transformed into A. oryzae niaD300.

Results

EGFP–AoAtg1 localizes to PAS

We first identified an A. oryzae ATG1 homolog, Aoatg1, in the A. oryzae genome database (http://www.bio.nite.go.jp/dogan/project/view/AO) using the BLAST algorithm. 5′-and 3′-RACE analyses revealed that Aoatg1 contained one intron and two exons, and encoded a predicted polypeptide of 986 amino acids with a calculated molecular mass of 107 kDa. AoAtg1 displayed 25% identity to Atg1 of S. cerevisiae and, as determined from the Pfam database, had an Atg1 kinase domain identified in the Pfam database (http://pfam.sanger.ac.uk/) (Supporting Information, Fig. S1).

To determine the localization of AoAtg1, we constructed strain A1EG, which expressed the fusion protein AoAtg1–EGFP under control of the native promoter. After culturing A1EG for 24 h at 30 °C in CD + m medium to promote growth, the strain was transferred to nitrogen-deprived medium (CD − N) and further cultured for 4 h to induce autophagy. In CD + m medium, AoAtg1–EGFP localized to PAS-like structures and in the cytoplasm (Fig. 1, left). After starvation in CD − N medium, the number of punctate fluorescent spots had clearly increased (Fig. 1, right). These results were consistent with the reported localization of Atg1–GFP in S. cerevisiae, in which the number of the PAS increased after the induction of autophagy (Cheong et al., 2008).

Localization of AoAtg1–EGFP under starvation conditions. The A1EG strain expressing AoAtg1–EGFP was cultured on CD medium (growth condition) for 24 h at 30 °C (left panel) and then shifted to nitrogen-deprived CD medium (CD − N) for 4 h at 30 °C to induce autophagy (right panel). After incubation, hyphae were observed by differential interference contrast (DIC) and fluorescence microscopy. The arrowheads indicate PAS-like structures. Scale bar, 5 µm.

AoAtg1 is essential for autophagy

To investigate the function of AoAtg1, we disrupted Aoatg1 by the replacement with the selective marker adeA and confirmed the mutation by Southern blot analysis (Fig. S2). When ΔAoatg1 was grown on PD agar, the colonies were white in color and defective in aerial hyphae growth (Fig. 2ac), 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 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.

Disruption of the Aspergillus oryzae Aoatg1 gene. (a) Image of strains ΔAoatg1, ΔAoatg8, ΔAoatg13, and Ku70-AA (WT) grown on a PD agar plate, as indicated in the left diagram, for 4 days at 30 °C. (b) Side view of the WT, ΔAoatg1, ΔAoatg8, and ΔAoatg13 strains grown on a PD plate. (c) Examination of conidiation by the WT and ΔAoatg1 strains using a stereomicroscope. Scale bar, 500 µm.

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 ring-like structures were detected. These observations indicated that the autophagic process was completely defective in the ΔAoatg1 disruptant.

Autophagy and the Cvt pathway in ΔAoatg1. (a) Strains ΔA1EA8 and Ku70EGA8 expressing EGFP–AoAtg8 were grown on CD medium for 24 h at 30 °C and then shifted to CD − N medium for 4 h. Hyphae were then visualized by differential interference contrast (DIC) and confocal laser scanning microscopy. The arrowheads and arrows point to PAS-like structures and larger punctate structures, respectively. (b) Strains Ku70aApe1EG, ΔA1Ape1EG, and NAE1 were grown on CD medium for 20 h at 30 °C, and then, hyphae were visualized by DIC and confocal laser scanning microscopy. The black arrowheads point to vacuoles. Scale bars, 5 µm.

The Cvt pathway functions in A. oryzae

To determine whether the Cvt pathway exists in A. oryzae and to evaluate the role of AoAtg1 in this pathway, we constructed strains expressing 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. Moreover, we confirmed that the accumulation of AoApe1–EGFP in vacuoles was not due to nonselective autophagy by observation of the NAE1 strain expressing only EGFP (Kimura et al., 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.

Overexpression of Aoatg1 increases the development of aerial hyphae and sclerotia, but decreases conidiation

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 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.

Phenotype of the Aoatg1-overexpressing strain A1-OE. (a, b) Images of strain A1-OE after growth on PD and CD agar plates for 3 days at 30 °C. (c) The number of conidia in the colony formed by A1-OE after growth on CD agar plates for 3 days at 30 °C. Error bars represent the SD for three independent experiments. *P < 0.05.

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 a few sclerotia were formed by ΔAoatg13. Taken together, these results suggested that sclerotial formation was dependent on the degree of autophagy.

Sclerotia-forming ability of the Aoatg gene disruptants and Aoatg1-overexpressing strain. Conidia harvested from these strains were grown on an DPY agar plate, as indicated in the left diagram, for 9 days at 30 °C. The black arrowheads in the WT, A1-OE, and ΔAoatg13 regions of growth indicate sclerotia.

Discussion

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). To determine the function of AoAtg1 in autophagy and the Cvt pathway, we analyzed EGFP–AoAtg8 or AoApe1–EGFP expression in the ΔA1EGA8 and ΔA1Ape1EG strains, respectively, which indicated that both nonselective autophagy and the Cvt pathway are completely defective in these disruptant strains. 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 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 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 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). On the other hand, AoAtg13 is not essential for autophagy (Kikuma & Kitamoto, 2011). Our present results suggest that AoAtg1 has a similar function to Atg1. Taken together, these findings indicate that the components involved in autophagy or its regulation in A. oryzae differ from those of S. cerevisiae. The existence of other functional Atg13 homologs in A. oryzae is possible, as it is clear that AoAtg1 is a key regulator of autophagy and the Cvt pathway.

In S. cerevisiae and Drosophila melanogaster, the overexpression of Atg1 and DmAtg1 (D. melanogaster Atg1 homolog) increases autophagic activity (Scott et al., 2007; Ma et al., 2007). Thus, we predicted that the overexpression of AoAtg1 would lead to excessive growth of aerial hyphae and conidiation. Surprisingly, however, conidiation in the Aoatg1-overexpressing strain was suppressed, although long aerial hyphae were formed. In deuteromycetes, conidia are important for dispersion and serve as safe structures for genomic storage during adverse environmental conditions, such as nutrient starvation. In addition, it is thought that aerial hyphae that are not in contact with the growth medium might acquire nutrients through the recycling of intracellular components by autophagy. Therefore, we speculated that excessive autophagy resulting from AoAtg1 overexpression would increase available nutrients in cells as compared to WT, resulting in decreased conidiation and longer aerial hyphae, and that the regulatory mechanism of aerial hyphae formation was different from that controlling the development of conidiophores and conidiation. Moreover, we analyzed the formation of sclerotia in the Aoatg gene disruptants and the Aoatg1-overexpressing strain, with the results suggesting that autophagy is an important factor affecting differentiation into sclerotia, as well as the formation of aerial hyphae.

In conclusion, we found that although AoAtg1 has a similar function to Atg1 of S. cerevisiae, the induction system of autophagy in the filamentous fungus A. oryzae does not appear identical to that of yeast. In addition, we have provided evidence for the existence of the Cvt pathway in A. oryzae. As A. oryzae has a high capacity for protein secretion, studies of vacuolar degradation systems, such as autophagy and the Cvt pathway, are important for industrial heterologous protein production.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Fig. S1. Alignment of A. oryzae AoAtg1 and S. cerevisiae Atg1 homolog of other microorganisms.

Fig. S2. Schema for the integration of the adeA gene, and Southern blotting for the Aoatg1 gene in the disruptant.

Acknowledgements

This study was supported by a Grant-in-Aid for Challenging Exploratory Research to K. Kitamoto from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References

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