Takashi Kikuma

Functional Analysis of the ATG8 Homologue Aoatg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae

ABSTRACT Autophagy is a well-known degradation system, induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. Recently, it was reported that autophagy is involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. In this study, we isolated the ATG8 gene homologue Aoatg8 from the filamentous fungus Aspergillus oryzae and visualized autophagy by the expression of DsRed2-AoAtg8 and enhanced green fluorescent protein-AoAtg8 fusion proteins in this fungus. While the fusion proteins were localized in dot structures which are preautophagosomal structure-like structures under normal growth conditions, starvation or rapamycin treatment caused their accumulation in vacuoles. DsRed2 expressed in the cytoplasm was also taken up into vacuoles under starvation conditions or during the differentiation of conidiophores and conidial germination. Deletion mutants of Aoatg8 did not form aerial hyphae and conidia, and DsRed2 was not localized in vacuoles under starvation conditions, indicating that Aoatg8 is essential for autophagy. Furthermore, Aoatg8 conditional mutants showed delayed conidial germination in the absence of nitrogen sources. These results suggest that autophagy functions in both the differentiation of aerial hyphae and in conidial germination in A. oryzae.

Development of a Versatile Expression Plasmid Construction System for Aspergillus oryzae and Its Application to Visualization of Mitochondria

We report here a development of the MultiSite GatewayTM-based versatile plasmid construction system applicable for the rapid and efficient preparation of Aspergillus oryzae expression plasmids. This system allows the simultaneous connection of the three DNA fragments inserted in entry clones along with a destination vector in a defined order and orientation. We prepared a variety of entry clones and destination vectors containing promoters, genes encoding carrier-proteins and fusion tags, and selectable markers, which makes it possible to generate 80 expression plasmids for each target protein. Using this system, plasmids for expression of the EGFP fused with the mitochondrial-targeting signal of citrate synthase (AoCit1) were generated. Tubular structures of mitochondria were visualized in the transformants expressing the AoCit1-EGFP fusion protein. This plasmid construction system allows us to prepare a large number of expression plasmids without laborious DNA manipulations, which would facilitate molecular biological studies on A. oryzae.

Macroautophagy-Mediated Degradation of Whole Nuclei in the Filamentous Fungus Aspergillus oryzae

Filamentous fungi consist of continuum of multinucleate cells called hyphae, and proliferate by means of hyphal tip growth. Accordingly, research interest has been focusing on hyphal tip cells, but little is known about basal cells in colony interior that do not directly contribute to proliferation. Here, we show that autophagy mediates degradation of basal cell components in the filamentous fungus Aspergillus oryzae. In basal cells, enhanced green fluorescent protein (EGFP)-labeled peroxisomes, mitochondria, and even nuclei were taken up into vacuoles in an autophagy-dependent manner. During this process, crescents of autophagosome precursors matured into ring-like autophagosomes to encircle apparently whole nuclei. The ring-like autophagosomes then disappeared, followed by dispersal of the nuclear material throughout the vacuoles, suggesting the autophagy-mediated degradation of whole nuclei. We also demonstrated that colony growth in a nutrient-depleted medium was significantly inhibited in the absence of functional autophagy. This is a first report describing autophagy-mediated degradation of whole nuclei, as well as suggesting a novel strategy of filamentous fungi to degrade components of existing hyphae for use as nutrients to support mycelial growth in order to counteract starvation.

Analysis of autophagy in Aspergillus oryzae by disruption of Aoatg13, Aoatg4, and Aoatg15 genes.

Autophagy is a degradation system in which cellular components are digested via vacuoles/lysosomes, and involved in differentiation in addition to helping cells to survive starvation. The autophagic process is composed of several steps: induction of autophagy, formation of autophagosomes, transportation to vacuoles, and degradation of autophagic bodies. To further understand autophagy in the filamentous fungus Aspergillus oryzae, we first constructed A. oryzae mutants defective for the Aoatg13, Aoatg4, and Aoatg15 genes and examined the resulting phenotypes. The ΔAoatg13 mutant developed conidiophores and conidia, although the number of conidia was decreased compared with the wild-type strain, while conidiation in the ΔAoatg4 and ΔAoatg15 mutants was not detected. The ΔAoatg15 mutants displayed a marked reduction of development of aerial hyphae. Moreover, autophagy in these mutants was examined by observation of the behavior of enhanced green fluorescent protein (EGFP)-AoAtg8. In the ΔAoatg13 mutant, the slight accumulation of EGFP-AoAtg8 in vacuoles, preautophagosomal structures (PAS), and autophagosomes was observed, whereas only PAS-like structures were detected in the ΔAoatg4 mutant. In the ΔAoatg15 mutant, autophagic bodies accumulated in vacuoles, suggesting that the uptake process proceeded. We therefore propose that the level of autophagy is closely correlated with the degree of differentiation in A. oryzae.

Vesicle trafficking, organelle functions, and unconventional secretion in fungal physiology and pathogenicity

Specific localization of appropriate sets of proteins and lipids is central to functions and integrity of organelles, which in turn underlie cellular activities of eukaryotes. Vesicle trafficking is a conserved mechanism of intracellular transport, which ensures such a specific localization to a subset of organelles. In this review article, we summarize recent advances in our understanding of how vesicle trafficking and related organelles support physiology and pathogenicity of filamentous fungi. Examples include a link between Golgi organization and polarity maintenance during hyphal tip growth, a new role of early endosomes in transport of translational machinery, involvement of endosomal/vacuolar compartments in secondary metabolite synthesis, and functions of vacuoles and autophagy in fungal development, nutrient recycling and allocation. Accumulating evidence showing the importance of unconventional secretion in fungal pathogenicity is also summarized.

Autophagy During Conidiation and Conidial Germination in Filamentous Fungi

Filamentous fungi form aerial hyphae on solid medium, and some of these differentiate into conidiophores for asexual sporulation (conidiation). In the filamentous deuteromycete, Aspergillus oryzae, aerial hyphae are formed from the foot cells and some differentiate into conidiophores, which are composed of vesicles, phialides and conidia. Recently, we isolated the yeast ATG8 gene homologue Aoatg8 from A. oryzae, and visualized autophagy by the expression of an EGFP (enhanced green fluorescent protein)–AoAtg8 fusion protein and DsRed2 protein in this fungus. Furthermore, by constructing the Aoatg8 deletion and conditional mutants, we demonstrated that autophagy functions during the process of differentiation of aerial hyphae, conidiation and conidial germination in A. oryzae. Here, we discuss the contribution of autophagy towards the differentiation and germination processes in filamentous fungi. Addendum to: Functional Analysis of the ATG8 Homologue Aoatg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae T. Kikuma, M. Ohneda, M. Arioka and K. Kitamoto Eukaryot Cell 2006; 5:1328-36

Autophagy delivers misfolded secretory proteins accumulated in endoplasmic reticulum to vacuoles in the filamentous fungus Aspergillus oryzae.

Autophagy is a conserved intracellular degradation process of eukaryotic cells. In filamentous fungi, although autophagy has been reported to have multiple physiological roles, it is not clear whether autophagy is involved in the degradation of misfolded proteins. Here, we investigated the role of autophagy in the degradation of misfolded secretory proteins accumulated in endoplasmic reticulum (ER) in the filamentous fungus Aspergillus oryzae. In late-phase cultures, a disulfide bond-deleted mutant of the secretory protein α-amylase AmyB fused with mDsRed that had accumulated in the ER was subsequently delivered to vacuoles, whereas wild-type AmyB-mDsRed was predominantly located at cell walls and septa. To examine the involvement of autophagy in the delivery of mutant AmyB to vacuoles, mutant AmyB-EGFP was expressed in an A. oryzae autophagy-deficient strain (ΔAoatg8). Microscopic examination revealed that the protein delivery to vacuoles did not occur in the absence of autophagic activity, with mutant AmyB-mDsRed forming large spherical structures surrounded by ER membrane. Hence, we conclude that autophagy is responsible for the delivery of misfolded secretory proteins accumulated in the ER to vacuoles for degradation during late-growth phase in A. oryzae. This is the first study to provide evidence that autophagy plays a role in the degradation of misfolded secretory proteins in filamentous fungi.

Enhanced Production of Bovine Chymosin by Autophagy Deficiency in the Filamentous Fungus Aspergillus oryzae

Aspergillus oryzae has been utilized as a host for heterologous protein production because of its high protein secretory capacity and food-safety properties. However, A. oryzae often produces lower-than-expected yields of target heterologous proteins due to various underlying mechanisms, including degradation processes such as autophagy, which may be a significant bottleneck for protein production. In the present study, we examined the production of heterologous protein in several autophagy (Aoatg) gene disruptants of A. oryzae. We transformed A. oryzae gene disruptants of Aoatg1, Aoatg13, Aoatg4, Aoatg8, or Aoatg15, with a bovine chymosin (CHY) expression construct and found that the production levels of CHY increased up to three fold compared to the control strain. Notably, however, conidia formation by the Aoatg gene disruptants was significantly reduced. As large amounts of conidia are necessary for inoculating large-scale cultures, we also constructed Aoatg gene-conditional expression strains in which the promoter region of the Aoatg gene was replaced with the thiamine-controllable thiA promoter. Conidiation by the resultant transformants was clearly enhanced in the absence of thiamine, while autophagy remained repressed in the presence of thiamine. Moreover, these transformants displayed increased CHY productivity, which was comparable to that of the Aoatg gene disruptants. Consequently, we succeeded in the construction of A. oryzae strains capable of producing high levels of CHY due to defects in autophagy. Our finding suggests that the conditional regulation of autophagy is an effective method for increasing heterologous protein production in A. oryzae.

Functional analysis of Abp1p-interacting proteins involved in endocytosis of the MCC component in Aspergillus oryzae

We have investigated the functions of three endocytosis-related proteins in the filamentous fungus Aspergillus oryzae. Yeast two-hybrid screening using the endocytic marker protein AoAbp1 (A.oryzae homolog of Saccharomyces cerevisiae Abp1p) as a bait identified four interacting proteins named Aip (AoAbp1 interacting proteins). In mature hyphae, EGFP (enhanced green fluorescent protein) fused to Aips colocalized with AoAbp1 at the hyphal tip region and the plasma membrane, suggesting that Aips function in endocytosis. aipA is a putative AAA ATPase and its function has been dissected (Higuchi et al., 2011). aipB, the homolog of A. nidulans myoA, encodes an essential class I myosin and its conditional mutant showed a germination defect. aipC and aipD do not contain any recognizable domains except some proline-rich regions which may interact with two SH3 (Src homology 3) domains of AoAbp1. Neither aipC nor aipD disruptants showed any defects in their growth, but the aipC disruptant formed less conidia compared with the control strain. In addition, the aipC disruptant was resistant to the triazole antifungal drugs that inhibit ergosterol biosynthesis. Although no aip disruptants showed any defects in the uptake of the fluorescent dye FM4-64, the endocytosis of the arginine permease AoCan1, one of the MCC (membrane compartment of Can1p) components, was delayed in both aipC and aipD disruptants. In A. oryzae, AoCan1 localized mainly at the plasma membrane in the basal region of hyphae, suggesting that different endocytic mechanisms exist in apical and basal regions of highly polarized cells.

Functional analysis of AoAtg11 in selective autophagy in the filamentous fungus Aspergillus oryzae

Autophagy is a highly conserved cellular degradation process in eukaryotes and consists of both non-selective and selective types. Selective autophagic processes include pexophagy, mitophagy, and the cytoplasm-to-vacuole targeting (Cvt) pathway of yeast, in which particular vacuolar proteins, such as aminopeptidase I (Ape1), are selectively transported to vacuoles. Although selective autophagy has been mainly studied in the yeasts Saccharomyces cerevisiae and Pichia pastoris, there is evidence for selective autophagy in filamentous fungi; however, the details are poorly understood. In S. cerevisiae, Atg11 is a selective autophagy-specific protein that recognizes and transports substrates to the pre-autophagosomal structure (PAS). Here, we first identified an ATG11 homologue in the filamentous fungus Aspergillus oryzae and analyzed the localization of the corresponding protein, designated AoAtg11, fused to enhanced green fluorescent protein (EGFP). Imaging analysis revealed that AoAtg11-EGFP was localized to PAS-like structures. We next constructed an Aoatg11 disruptant of A. oryzae and showed that AoAtg11 is involved in pexophagy and mitophagy. In addition, AoAtg11 was found to be dispensable for non-selective autophagy and for transporting AoApe1 to vacuoles. Taken together, these results suggest that AoAtg11 is a selective autophagy-specific protein in A. oryzae, and has distinct molecular functions from that of S. cerevisiae Atg11.