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ABSTRACTHydrophobins are small, amphipathic proteins secreted by filamentous fungi. Hydrophobin RolA, which is produced by Aspergillus oryzae, attaches to solid surfaces, recruits the polyesterase CutL1, and consequently promotes hydrolysis of polyesters. Because this interaction requires the N-terminal, positively charged residue of RolA to be exposed on the solid surface, the orientation of RolA on the solid surface is important for recruitment. However, the process by which RolA forms the self-assembled structure at the interface remains unclear. Using the Langmuir–Blodgett technique, we analyzed the process by which RolA forms a self-assembled structure at the air–water interface and observed the structures on the hydrophobic or hydrophilic SiO2substrates via atomic force microscopy. We found that RolA formed self-assembled films in two steps during phase transitions. We observed different assembled structures of RolA on hydrophilic and hydrophobic SiO2substrates.

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 nidulansincreased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzaein 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 cutL1into A. oryzaewild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzaetripleΔ-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.

Disruption of the kexBencoding a subtilisin-like processing protease in Aspergillus oryzae(ΔkexB) leads to substantial morphological defects when the cells are grown on Czapek-Dox agar plates. We previously found that the disruption of kexBcauses a constitutive activation of the cell wall integrity pathway. To understand how the disruption of the kexBaffects cell wall organization and components, we analyzed the cell wall of ΔkexBgrown on the plates. The results revealed that both total N-acetylglucosamine content, which constitutes chitin, and chitin synthase activities were increased. Whereas total glucose content, which constitutes β-1,3-glucan and α-1,3-glucan, was decreased; this decrease was attributed to a remarkable decrease in α-1,3-glucan. Additionally, the β-1,3-glucan in the alkali-insoluble fraction of the ΔkexBshowed a high degree of polymerization. These results suggested that the loss of α-1,3-glucan in the ΔkexBwas compensated by increases in the chitin content and the average degree of β-1,3-glucan polymerization.

The transcriptional regulation of the MAPK mpkAand cell wall-related genes in Aspergillus nidulansdiffers from that of their counterparts in Saccharomyces cerevisiae. The A. nidulansMAPK MpkB is putatively orthologous to the yeast MAPKs Kss1p and Fus3p. To investigate MpkB and its contribution to cell wall integrity in A. nidulans, we constructed mpkB-disruptant (mpkB∆) strains. We previously showed that mpkA∆ strains exhibited reduced colony growth and increased sensitivity to the β-1,3-glucan synthase inhibitor micafungin. Like mpkA∆ strains, mpkB∆strains exhibited slight growth retardation and increased sensitivity to micafungin. Although MpkB-dependent signaling modulated the transcription of some cell wall-related genes, the sugar composition of cell wall fractions was similar among wild-type, mpkA∆, and mpkB∆ strains.To elucidate the relationship between MpkA and MpkB pathways, we compared conditional mutants of mpkBwith those with mpkAdeletion. Sensitivity testing suggested that MpkA and MpkB additively contribute to micafungin activity in A. nidulans.

The Aspergillus nidulanshigh-osmolarity glycerol response (AnHOG) pathway is involved in osmoadaptation. We found that fludioxonil, a fungicide, causes improper activation of HogA mitogen-activated protein kinase (MAPK) in A. nidulans. Here we present novel reporter systems for monitoring activation of the AnHOG pathway. The promoter region of gfdB(glycerol-3-phosphate dehydrogenase), whose expression depends on the presence of HogA, was fused to a β-glucuronidase uidAgene (GUS) to construct the reporter, which was introduced into A. nidulanswild type and hogAΔ. Increased GUS activity was detected in the wild type only when it was treated with high osmolarity or fludioxonil, while reporter activity was scarcely stimulated in the hogAΔ mutant. These results indicate that the reporter activity is controlled viaHogA activation. Furthermore, we present possible applications of the reporter systems in screening new antifungal compounds.