Hyphal heterogeneity in Aspergillus oryzae is the result of dynamic closure of septa by Woronin bodies

Hyphae of higher fungi are compartmentalized by septa. These septa contain a central pore that allows for inter‐compartmental and inter‐hyphal cytoplasmic streaming. The cytoplasm within the mycelium is therefore considered to be a continuous system. In this study, however, we demonstrate by laser dissection that 40% of the apical septa of exploring hyphae of Aspergillus oryzae are closed. Closure of septa correlated with the presence of a peroxisome‐derived organelle, known as Woronin body, near the septal pore. The location of Woronin bodies in the hyphae was dynamic and, as a result, plugging of the septal pore was reversible. Septal plugging was abolished in a ΔAohex1 strain that cannot form Woronin bodies. Notably, hyphal heterogeneity was also affected in the ΔAohex1 strain. Wild‐type strains of A. oryzae showed heterogeneous distribution of GFP between neighbouring hyphae at the outer part of the colony when the reporter was expressed from the promoter of the glucoamylase gene glaA or the α‐glucuronidase gene aguA. In contrast, GFP fluorescence showed a normal distribution in the case of the ΔAohex1 strain. Taken together, it is concluded that Woronin bodies maintain hyphal heterogeneity in a fungal mycelium by impeding cytoplasmic continuity.     

Twinfilin regulates actin assembly and Hexagonal peroxisome 1 (Hex1) localization in the pathogenesis of rice blast fungus Magnaporthe oryzae

Actin assembly at the hyphal tip is key for polar growth and pathogenesis of the rice blast fungus Magnaporthe oryzae. The mechanism of its precise assemblies and biological functions is not understood. Here, we characterized the role of M. oryzae Twinfilin (MoTwf) in M. oryzae infection through organizing the actin cables that connect to Spitzenkörper (Spk) at the hyphal tip. MoTwf could bind and bundle the actin filaments. It formed a complex with Myosin2 (MoMyo2) and the Woronin body protein Hexagonal peroxisome 1 (MoHex1). Enrichment of MoMyo2 and MoHex1 in the hyphal apical region was disrupted in a ΔMotwf loss-of-function mutant, which also showed a decrease in the number and width of actin cables. These findings indicate that MoTwf participates in the virulence of M. oryzae by organizing Spk-connected actin filaments and regulating MoHex1 distribution at the hyphal tip.     

Invasion of paranasal sinuses by Aspergillus oryzae

A 24-year-old male patient receiving chemotherapy for acute promyelocytic leukemia developed fever, right periorbital swelling and mild right proptosis. A head scan showed opacification of the right maxillary and ethmoid sinuses with adjacent soft tissue swelling. Biopsy of the nasal mucosa demonstrated the typical septate hyphae of Aspergillus species which was later shown on culture to be Aspergillus oryzae. A. oryzae has only rarely been reported in human disease and there is confusion as to its precise identification and role. We would like to confirm the pathogenicity of A. oryzae with this uncommon presentation of aspergillosis and also emphasize the need to take adequate and multiple cultures in suspected cases so that the possibility of species identification will be maximized.     

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

Chitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.     

A Large Nonconserved Region of the Tethering Protein Leashin Is Involved in Regulating the Position,Movement, and Function of Woronin Bodies in Aspergillus oryzae

The Woronin body is a Pezizomycotina-specific organelle that is typically tethered to the septum, but upon hyphal wounding, it plugs the septal pore to prevent excessive cytoplasmic loss. Leashin (LAH) is a large Woronin body tethering protein that contains highly conserved N- and C-terminal regions and a long (∼2,500-amino-acid) nonconserved middle region. As the involvement of the nonconserved region in Woronin body function has not been investigated, here, we functionally characterized individual regions of the LAH protein of Aspergillus oryzae (AoLAH). In an Aolah disruptant, no Woronin bodies were tethered to the septum, and hyphae had a reduced ability to prevent excessive cytoplasmic loss upon hyphal wounding. Localization analysis revealed that the N-terminal region of AoLAH associated with Woronin bodies dependently on AoWSC, which is homologous to Neurospora crassa WSC (Woronin body sorting complex), and that the C-terminal region was localized to the septum. Elastic movement of Woronin bodies was observed when visualized with an AoLAH N-terminal-region–enhanced green fluorescent protein (EGFP) fusion protein. An N- and C-terminal fusion construct lacking the nonconserved middle region of AoLAH was sufficient for the tethering of Woronin bodies to the septum. However, Woronin bodies were located closer to the septum and exhibited impaired elastic movement. Moreover, expression of middle-region-deleted AoLAH in the Aolah disruptant did not restore the ability to prevent excessive cytoplasmic loss. These findings indicate that the nonconserved middle region of AoLAH has functional importance for regulating the position, movement, and function of Woronin bodies.     

AoAtg26, a putative sterol glucosyltransferase,is required for autophagic degradation of peroxisomes,mitochondria, and nuclei in the filamentous fungus Aspergillus oryzae

Autophagy is a conserved process in eukaryotic cells for degradation of cellular proteins and organelles. In filamentous fungi, autophagic degradation of organelles such as peroxisomes, mitochondria, and nuclei occurs in basal cells after the prolonged culture, but its mechanism is not well understood. Here, we functionally analyzed the filamentous fungus Aspergillus oryzae AoAtg26, an ortholog of the sterol glucosyltransferase PpAtg26 involved in pexophagy in the yeast Pichia pastoris. Deletion of Aoatg26 caused a severe decrease in conidiation and aerial hyphae formation, which is typically observed in the autophagy-deficient A. oryzae strains. In addition, cup-shaped AoAtg8-positive membrane structures were accumulated in the Aoatg26 deletion strain, indicating that autophagic process is impaired. Indeed, the Aoatg26 deletion strain was defective in the degradation of peroxisomes, mitochondria, and nuclei. Taken together, AoAtg26 plays an important role for autophagic degradation of organelles in A. oryzae, which may physiologically contribute to the differentiation in filamentous fungi.     

Cell biology of the Koji mold Aspergillus oryzae

Koji mold, Aspergillus oryzae, has been used for the production of sake, miso, and soy sauce for more than one thousand years in Japan. Due to the importance, A. oryzae has been designated as the national micro-organism of Japan (Koku-kin). A. oryzae has been intensively studied in the past century, with most investigations focusing on breeding techniques and developing methods for Koji making for sake brewing. However, the understanding of fundamental biology of A. oryzae remains relatively limited compared with the yeast Saccharomyces cerevisiae. Therefore, we have focused on studying the cell biology including live cell imaging of organelles, protein vesicular trafficking, autophagy, and Woronin body functions using the available genomic information. In this review, I describe essential findings of cell biology of A. oryzae obtained in our study for a quarter of century. Understanding of the basic biology will be critical for not its biotechnological application, but also for an understanding of the fundamental biology of other filamentous fungi.     

Increased enzyme production under liquid culture conditions in the industrial fungus Aspergillus oryzae by disruption of the genes encoding cell wall α-1,3-glucan synthase

Under liquid culture conditions, the hyphae of filamentous fungi aggregate to form pellets, which reduces cell density and fermentation productivity. Previously, we found that loss of α-1,3-glucan in the cell wall of the fungus Aspergillus nidulans increased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzae in which the three genes encoding α-1,3-glucan synthase were disrupted (tripleΔ). Although the hyphae of the tripleΔ mutant were not fully dispersed, the mutant strain did form smaller pellets than the wild-type strain. We next examined enzyme productivity under liquid culture conditions by transforming the cutinase-encoding gene cutL1 into A. oryzae wild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzae tripleΔ-cutL1 formed smaller hyphal pellets and showed both greater biomass and increased CutL1 productivity compared with wild-type-cutL1, which might be attributable to a decrease in the number of tripleΔ-cutL1 cells under anaerobic conditions.     

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     

Comparative Analysis of Aspergillus oryzae with Normal and Abnormal Color Conidia

This study focuses on the characteristic of strains with anomalous color conidium and compares with normal color conidium. Comparative analysis of enzymes activity and extracellular proteins revealed that A. oryzae with anomalous color conidium was not different from the strain with normal color conidium. In addition, A. oryzae with anomalous color conidium could not influence the palatability and quality of the soy sauce. These findings provide an insight into A. oryzae with anomalous color conidium.     

Magnaporthe oryzae nucleoside diphosphate kinase is required for metabolic homeostasis and redox-mediated host innate immunity suppression

The fungus Magnaporthe oryzae causes blast, the most devastating disease of cultivated rice. After penetrating the leaf cuticle, M. oryzae grows as a biotroph in intimate contact with living rice epidermal cells before necrotic lesions develop. Biotrophic growth requires maintaining metabolic homeostasis while suppressing plant defenses, but the metabolic connections and requirements involved are largely unknown. Here, we characterized the M. oryzae nucleoside diphosphate kinase-encoding gene NDK1 and discovered it was essential for facilitating biotrophic growth by suppressing the host oxidative burst—the first line of plant defense. NDK enzymes reversibly transfer phosphate groups from tri- to diphosphate nucleosides. Correspondingly, intracellular nucleotide pools were perturbed in M. oryzae strains lacking NDK1 through targeted gene deletion, compared to WT. This affected metabolic homeostasis: TCA, purine and pyrimidine intermediates, and oxidized NADP⁺, accumulated in Δndk1. cAMP and glutathione were depleted. ROS accumulated in Δndk1 hyphae. Functional appressoria developed on rice leaf sheath surfaces, but Δndk1 invasive hyphal growth was restricted and redox homeostasis was perturbed, resulting in unsuppressed host oxidative bursts that triggered immunity. We conclude Ndk1 modulates intracellular nucleotide pools to maintain redox balance via metabolic homeostasis, thus quenching the host oxidative burst and suppressing rice innate immunity during biotrophy.