Involvement of AAA ATPase AipA in endocytosis of the arginine permease AoCan1 depending on AoAbp1 in Aspergillus oryzae

AAA ATPases widely exist in many organisms and function in various organelles. However, there is little information about AAA ATPase functioning in endocytosis. In Aspergillus oryzae, we previously discovered a putative AAA ATPase AipA that would be involved in endocytosis. Here, we further examined the function of AipA and AoAbp1 in endocytosis using enhanced green fluorescent protein (EGFP)-tagged arginine permease AoCan1 as an endocytic marker. In the ΔaipA strain, endocytosis of AoCan1-EGFP was more facilitated than the control strain, suggesting that AipA negatively regulates endocytosis. In contrast, in the ΔAoabp1 strain, endocytosis of AoCan1-EGFP was delayed compared with the control strain, suggesting that AoAbp1 positively functions in endocytosis. In addition, in the ΔaipAΔAoabp1 strain, endocytosis of AoCan1-EGFP was delayed. AipA localized at the endocytic collar of the hyphal tip, only in the presence of AoAbp1, suggesting AipA functions downstream of AoAbp1 in endocytosis. Moreover, we investigated the aipA-overexpressing strain, and found that endocytosis of AoCan1-EGFP was inhibited. Furthermore, we examined strains expressing aipA^K542A or aipA^E596Q, which decreased ATPase activity, in the backgrounds of complementation or overexpression, respectively, and found that AoCan1-EGFP endocytosis was promoted. These results suggested that AAA ATPase activity of AipA is important for its function in endocytosis.     

Septum-directed secretion in the filamentous fungus Aspergillus oryzae

Although exocytosis in fungal cells takes place at hyphal tips, there also seems a line of circumstantial evidence suggesting the occurrence of exocytosis at other sites of cells, such as septa. To investigate whether exocytosis takes place at fungal septa, we monitored dynamics of EGFP‐fused α‐amylase (AmyB–EGFP), the representative secretory enzyme of the filamentous fungus Aspergillus oryzae. We found that AmyB–EGFP accumulates in Spitzenkörper at hyphal tips as well as septal periplasm between the plasma membrane and cell walls. The septal accumulation of AmyB–EGFP was a rapid process, and required microtubules but not F‐actin. Thus, this process is independent of exocytosis at hyphal tips that requires both microtubules and F‐actin. In addition, fluorescence recovery after photobleaching (FRAP) analysis of EGFP‐fused AoSnc1 revealed that secretory vesicles constitutively fuse with the septal plasma membrane. These results demonstrated that exocytosis takes place at septa in addition to hyphal tips. Analysis of two plasma membrane transporters, AoUapC and AoGap1, revealed that they preferentially accumulate at septa and the lateral plasma membrane with no clear accumulation at apical Spitzenkörper, suggesting that non‐tip directed exocytosis is important for delivery of these proteins.     

Systematic analysis of SNARE localization in the filamentous fungus Aspergillus oryzae

In spite of their great importance for both applied and basic biology, studies on vesicular trafficking in filamentous fungi have been so far very limited. Here, we identified 21 genes, which might be a total set, encoding putative SNARE proteins that are key factors for vesicular trafficking, taking advantage of available whole genome sequence in the filamentous fungus Aspergillus oryzae. The subsequent systematic analysis to determine the localization of putative SNAREs using EGFP-fused chimeras revealed that most putative SNAREs show similar subcellular distribution to their counterparts in the budding yeast. However, there existed some characteristic features of SNAREs in A. oryzae, such as SNARE localization at/near the septum and the presence of apparently non-redundant plasma membrane Qa-SNAREs. Overall, this analysis allowed us to provide an overview of vesicular trafficking and organelle distribution in A. oryzae.     

Visualization of the endocytic pathway in the filamentous fungus Aspergillus oryzae using an EGFP-fused plasma membrane protein

Endocytosis is an important process for cellular activities. However, in filamentous fungi, the existence of endocytosis has been so far elusive. In this study, we used AoUapC-EGFP, the fusion protein of a putative uric acid-xanthine permease with enhanced green fluorescent protein (EGFP) in Aspergillus oryzae, to examine whether the endocytic process occurs or not. Upon the addition of ammonium into the medium the fusion protein was internalized from the plasma membrane. The internalization of AoUapC-EGFP was completely blocked by sodium azide, cold, and cytochalasin A treatments, suggesting that the internalization possesses the general features of endocytosis. These results demonstrate the occurrence of endocytosis in filamentous fungi. Moreover, we discovered that the endosomal compartments appeared upon the induction of endocytosis and moved in a microtubule-dependent manner.     

Vacuolar membrane dynamics in the filamentous fungus Aspergillus oryzae

Vacuoles in filamentous fungi are highly pleomorphic and some of them, e.g., tubular vacuoles, are implicated in intra- and intercellular transport. In this report, we isolated Aovam3, the homologue of the Saccharomyces cerevisiae VAM3 gene that encodes the vacuolar syntaxin, from Aspergillus oryzae. In yeast complementation analyses, the expression of Aovam3 restored the phenotypes of both Deltavam3 and Deltapep12 mutants, suggesting that AoVam3p is likely the vacuolar and/or endosomal syntaxin in A. oryzae. FM4-64 [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)pyridinium dibromide] and CMAC (7-amino-4-chloromethylcoumarin) staining confirmed that the fusion protein of enhanced green fluorescent protein (EGFP) with AoVam3p (EGFP-AoVam3p) localized on the membrane of the pleomorphic vacuolar networks, including large spherical vacuoles, tubular vacuoles, and putative late endosomes/prevacuolar compartments. EGFP-AoVam3p-expressing strains allowed us to observe the dynamics of vacuoles with high resolutions, and moreover, led to the discovery of several new aspects of fungal vacuoles, which have not been discovered so far with conventional staining methods, during different developmental stages. In old hyphae, EGFP fluorescence was present in the entire lumen of large vacuoles, which occupied most of the cell, indicating that degradation of cytosolic materials had occurred in such hyphae via an autophagic process. In hyphae that were not in contact with nutrients, such as aerial hyphae and hyphae that grew on a glass surface, vacuoles were composed of small punctate structures and tubular elements that often formed reticulum-like networks. These observations imply the presence of so-far-unrecognized roles of vacuoles in the development of filamentous fungi.     

Characterization and functional analysis of ERAD-related AAA+ ATPase Cdc48 in Aspergillus oryzae

Aspergillus oryzae can secrete large amounts of enzymes. However, the production of abundant secretory proteins triggers the unfolded protein response (UPR) in the endoplasmic reticulum (ER), and it is not clear how ER-associated protein degradation (ERAD) contributes to bulk protein production in A. oryzae. Here we identified AoCdc48, the sole A. oryzae ortholog of Saccharomyces cerevisiae AAA+ ATPase Cdc48, a component of the ERAD machinery. We found that AoCdc48 localizes in both nuclei and cytoplasm. Generation of an Aocdc48 conditional mutant showed that Aocdc48 repression leads to reduced cell growth and aberrant hyphal morphology. When Aocdc48-repressed cells were cultured on starch-containing plates, the α-amylase-encoding gene amyB was about 1.3-fold higher expressed. Indeed, a halo produced by secreted amylase was seen on potato starch-containing plates even when there was almost no growth under Aocdc48 repression. Fluorescence microscopy revealed that although AmyB seemed to be secreted, various organelle distributions were aberrant in Aocdc48-repressed cells. We found that D1 AAA domain is crucial for cell viability. Finally, we show that Aocdc48-overexpression also causes defects of cell growth, colonial morphology and conidial formation. Collectively, our results suggest that AoCdc48 is essential for growth and organelle distribution but dispensable for amylase secretion.     

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.     

Presence and functionality of mating type genes in the supposedly asexual filamentous fungus Aspergillus oryzae

The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the reference strain RIB40. We now report the existence of a complementary MAT1-2 gene and the sequencing of an idiomorphic region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of the MAT1-1 and MAT1-2 genotypes among 180 strains assayed, including industrial tane-koji isolates. Strains used for sake and miso production showed a near-1:1 ratio of the MAT1-1 and MAT1-2 mating types, whereas strains used for soy sauce production showed a significant bias toward the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and gene expression was compared by DNA microarray and quantitative real-time PCR (qRT-PCR) methodologies under conditions in which MAT genes were expressed. Thirty-three genes were found to be upregulated more than 10-fold in either the MAT1-1 host strain or the MAT1-2 gene replacement strain relative to each other, showing that both the MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating type-dependent manner, the first such report for a supposedly asexual fungus. MAT1-1 expression specifically upregulated an α-pheromone precursor gene, but the functions of most of the genes affected were unknown. The results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed.     

Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins

Proteolytic degradation by secreted proteases into the culture medium is one of the significant problems to be solved in heterologous protein production by filamentous fungi including Aspergillus oryzae. Double (tppA, and pepE) and quintuple (tppA, pepE, nptB, dppIV, and dppV) disruption of protease genes enhanced human lysozyme (HLY) and bovine chymosin (CHY) production by A. oryzae. In this study, we used a quintuple protease gene disruptant and performed successive rounds of disruption for five additional protease genes (alpA, pepA, AopepAa, AopepAd, and cpI), which were previously investigated by DNA microarray analyses for their expression. Gene disruption was performed by pyrG marker recycling with a highly efficient gene-targeting background (∆ligD) as previously reported. As a result, the maximum yields of recombinant CHY and HLY produced by a decuple protease gene disruptant were approximately 30% and 35%, respectively, higher than those produced by a quintuple protease gene disruptant. Thus, we successfully constructed a decuple protease gene disruptant possessing highly improved capability of heterologous protein production. This is the first report on decuple protease gene disruption that improved the levels of heterologous protein production by the filamentous fungus A. oryzae.     

Three-dimensional image analysis of plugging at the septal pore by Woronin body during hypotonic shock inducing hyphal tip bursting in the filamentous fungus Aspergillus oryzae

We observed that the filamentous fungus, Aspergillus oryzae, grown on agar media burst out cytoplasmic constituents from the hyphal tip soon after flooding with water. Woronin body is a specialized organelle known to plug the septal pore adjacent to the lysed compartment to prevent extensive loss of cytoplasm. A. oryzae Aohex1 gene homologous to Neurospora crassa HEX1 gene encoding a major protein in Woronin body was expressed as a fusion with DsRed2, resulting in visualization of Woronin body. Confocal microscopy and three-dimensional reconstruction of images visualized the septal pore as a dark region surrounded by green fluorescence of EGFP-fused secretory protein, RNase T1, on the septum. Dual fluorescent labeling revealed the plugging of the septal pores adjacent to the lysed apical compartments by Woronin bodies during hypotonic shock. Disruption of Aohex1 gene caused disappearance of Woronin bodies and the defect to prevent extensive loss of cytoplasm during hypotonic shock.     

Characterisation of CwpA, a putative glycosylphosphatidylinositol-anchored cell wall mannoprotein in the filamentous fungus Aspergillus niger

Glycosylphosphatidylinositol (GPI)-anchored proteins in fungi are found at the cell surface, either as plasma membrane proteins (GPI-PMPs) or attached by a remnant of the GPI-anchor to the cell wall (GPI-CWPs). GPI-CWPs can be extracted from the cell wall by treatment with hydrofluoric acid (HF), which cleaves the phosphodiester bond that is present in the remnant of the GPI-anchor. The filamentous fungus Aspergillus niger contains at least seven HF-extractable cell wall mannoproteins. One gene encoding an HF-extractable cell wall mannoprotein, cwpA, was cloned and further characterised. The protein sequence of CwpA indicated the presence of two hydrophobic signal sequences both at the N-terminus and C-terminus of the protein, for entering the ER and the addition of a GPI-anchor, respectively. A CwpA-specific antiserum was raised and in combination with fractionation experiments, we show that this protein was abundantly present as an HF-extractable protein in the cell wall of A. niger.     

Distinct enzymatic and cellular characteristics of two secretory phospholipases A2 in the filamentous fungus Aspergillus oryzae

Microbial secretory phospholipases A₂ (sPLA₂s) are among the last discovered and least known members of this functionally diverse family of enzymes. We analyzed here two sPLA₂s, named sPlaA and sPlaB, of the filamentous ascomycete Aspergillus oryzae. sPlaA and sPlaB consist of 222 and 160 amino acids, respectively, and share the conserved Cys and catalytic His-Asp residues typical of microbial sPLA₂s. Two sPLA₂s differ in pH optimum, Ca²⁺ requirement and expression profile. The splaA mRNA was strongly upregulated in response to carbon starvation, oxidative stress and during conidiation, while splaB was constitutively expressed at low levels and was weakly upregulated by heat shock. Experiments with sPLA₂ overexpressing strains demonstrated that two enzymes produce subtly different phospholipid composition variations and also differ in their subcellular localization: sPlaA is most abundant in hyphal tips and secreted to the medium, whereas sPlaB predominantly localizes to the ER-like intracellular compartment. Both sPLA₂ overexpressing strains were defective in conidiation, which was more pronounced for sPlaB overexpressors. Although no major morphological abnormality was detected in either ΔsplaA or ΔsplaB mutants, hyphal growth of ΔsplaB, but not that of ΔsplaA, displayed increased sensitivity to H₂O₂ treatment. These data indicate that two A. oryzae sPLA₂ enzymes display distinct, presumably non-redundant, physiological functions.     

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.     

Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori

Many transformation methods have been developed to introduce DNA into filamentous fungi. One of these methods is Agrobacterium-mediated transformation (AMT). Here, we describe an efficient protocol for AMT of Aspergillus awamori. This protocol has been used to determine the function of Agrobacterium virulence genes during AMT, to identify factors influencing transformation frequencies, to generate insertional mutants and to generate A. awamori gene knockout transformants. This protocol in not only applicable to A. awamori, but can be used as a more general guideline for AMT of other filamentous fungi. Conidiospores are incubated with induced Agrobacterium, and, after a cocultivation and selection period, hygromycin-resistant transformants are obtained with a frequency of 200-250 transformants per 1 x 10(6) conidiospores. Using this protocol, transformants can be obtained within 10-12 d.