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Hydrophobins are small-secreted proteins comprising both hydrophobic and hydrophilic parts, that can self-assemble into an amphiphilic film at the air-liquid interface. More than 20 hydrophobin genes have been estimated in the white-rot fungus Pleurotus ostreatus. In our previous studies, three hydrophobin genes were shown to be predominantly expressed under ligninolytic conditions, and only vmh3 was downregulated in both the delignification-deficient mutant Δgat1 and Δhir1 strains. Here, we focused on the function of the hydrophobin Vmh3 to clarify its physiological role in lignin degradation. When the hyphae were observed by transmission electron microscopy, deletion of vmh3 resulted in the disappearance of black aggregates at the interface between the cell wall and outer environment. Deletion of vmh3 resulted in reduced hydrophobicity when 0.2% sodium dodecyl sulfate was dropped onto the mycelial surface. These results suggest that Vmh3 functions on the cell surface and plays a major role in mycelial hydrophobization. Furthermore, the Δvmh3 strain showed a marked delay in lignin degradation on beech wood sawdust medium, while the production of lignin-modifying enzymes was not reduced. This study demonstrated, for the first time, the possible effect of hydrophobin on lignin degradation by a white-rot fungus.

Hydrophobins, which are small-secreted proteins with both hydrophobic and hydrophilic parts, can self-assemble into an amphiphilic film at the air-water interface, helping the fungus to form aerial hyphae. In the agaricomycete Pleurotus ostreatus, more than 20 putative hydrophobin genes have been predicted. Of these, two hydrophobin genes, vmh2 and vmh3, are predominantly expressed in the vegetative mycelium. In this study, we focused on the functions of Vmh2 and Vmh3 in vegetative mycelia. Based on the observation of the mycelial cross-section by transmission electron microscopy and the disappearance time of water droplets on the mycelial surface, Vmh2 and Vmh3 were considered essential for the maintenance of the surface hydrophobicity of the mycelium. The Δvmh3 and Δvmh2Δvmh3 strains exhibited relatively slower aerial mycelia formation on a liquid medium, and no significant alteration was observed in Δvmh2 strains. Only the Δvmh3 and Δvmh2Δvmh3 strains grew slower than the wild-type strain under stress conditions involving SDS and H2O2 on agar plates. This study revealed possible distinct roles for these hydrophobins in stress resistance. These results suggest that Agaricomycetes, including P. ostreatus, have evolved to possess multiple different hydrophobins as a means of adapting to various environments.

CRISPR/Cas9 has potential for efficient molecular breeding. Recently, a foreign-DNA-free gene-targeting technology was established by introducing a preassembled Cas9 ribonucleoprotein (RNP) complex into the oyster mushroom Pleurotus ostreatus. However, the target gene was restricted to such a gene like pyrG, since screening of a genome-edited strain was indispensable and could be performed via examination of 5-fluoroorotic acid (5-FOA) resistance caused by the disruption of the target gene. In this study, we simultaneously introduced the Cas9 RNP complex targeting fcy1, a mutation that conferred P. ostreatus resistance to 5-fluorocytosine (5-FC), together with that targeting pyrG. A total of 76 5-FOA resistant strains were isolated during the first screening. Subsequently, a 5-FC resistance examination was conducted, and three strains exhibited resistance. Genomic PCR experiments followed by DNA sequencing revealed that mutations were successfully introduced into fcy1 and pyrG in the three strains. The results indicated that double gene-edited mutants could be obtained in one experiment employing 5-FOA resistance screening for strains with Cas9 RNP incorporation. This work may pave the way for safe CRISPR/Cas9 technology to isolate mutant strains in any gene of interest without an ectopic marker gene.

Quantitative reverse transcription PCR experiments were done to analyze the effects of light and a temperature downshift on transcriptional expressions of a variety of developmentally regulated genes in the initial stages of fruiting-body formation of Lentinula edodes wild-type strain FMC2. It was revealed that both proper light conditions and a temperature downshift are required for high levels of transcriptions of the genes and the formation of fruiting bodies. In L. edodes MIL-LEW-M13-1 strain, which has been reported previously to be capable of forming fruiting bodies without a low-temperature treatment, the expressions of the developmentally regulated genes were enhanced by light exposure.