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Until recently, classical breeding has been used to generate improved commercial mushroom strains; however, classical breeding remains to be laborious and time-consuming. In this study, we performed gene mutagenesis using Cas9 ribonucleoprotein (Cas9 RNP) as a plasmid-free genome editing in Pleurotus ostreatus, which is one of the most economically important cultivated mushrooms. The pre-assembled Cas9/sgRNA targeting pyrG was introduced into protoplasts of a wild-type monokaryotic P. ostreatus strain PC9, which resulted in a generation of strains exhibiting resistance to 5-fluoroorotic acid. Small insertions/deletions at the target site were identified using genomic PCR followed by sequencing. The results showed Cas9 RNP-assisted gene mutagenesis could be applied for the molecular breeding in P. ostreatus and in other edible mushroom strains. Furthermore, gene disruption via split-marker recombination using the Cas9 RNP system was also successfully demonstrated in wild-type P. ostreatus PC9. This method could overcome the disadvantages of NHEJ-deficiency in conventional studies with gene targeting, and also difficulty in gene targeting in various non-model agaricomycetes.

Use of the ku70-deficient strain of Coprinopsis cinerea enabled confirmation within the native context of the central role the sesquiterpene synthase Cop6 plays in lagopodin biosynthesis. Furthermore, yeast in vivo bioconversion and in vitro assays of two cytochrome P450 monooxygenases Cox1 and Cox2 allowed elucidation of the network of oxidation steps that build structural complexity onto the α-cuprenene framework during the biosynthesis of lagopodins. Three new compounds were identified as intermediates formed by the redox enzymes.

Pleurotus ostreatus is frequently used in molecular genetics and genomic studies on white-rot fungi because various molecular genetic tools and relatively well-annotated genome databases are available. To explore the molecular mechanisms underlying wood lignin degradation by P. ostreatus, we performed mutational analysis of a newly isolated mutant UVRM28 that exhibits decreased lignin-degrading ability on the beech wood sawdust medium. We identified that a mutation in the hir1 gene encoding a putative histone chaperone, which probably plays an important role in DNA replication-independent nucleosome assembly, is responsible for the mutant phenotype. The expression pattern of ligninolytic genes was altered in hir1 disruptants. The most highly expressed gene vp2 was significantly inactivated, whereas the expression of vp1 was remarkably upregulated (300-400 fold) at the transcription level. Conversely, many cellulolytic and xylanolytic genes were upregulated in hir1 disruptants. Chromatin immunoprecipitation analysis suggested that the histone modification status was altered in the 5'-upstream regions of some of the up- and down-regulated lignocellulolytic genes in hir1 disruptants compared with that in the 20b strain. Hence, our data provide new insights into the regulatory mechanisms of lignocellulolytic genes in P. ostreatus.

Terminators and introns are vital regulators of gene expression in many eukaryotes; however, the functional importance of these elements for controlling gene expression in Agaricomycetes remains unclear. In this study, the effects of Ceriporiopsis subvermispora terminators and introns on the expression of a recombinant hygromycin B phosphotransferase gene (hph) were characterized. Using a transient transformation system, we proved that a highly active terminator (e.g., the gpd terminator) is required for the efficient expression of the hph gene. Mutational analyses of the C. subvermispora gpd terminator revealed that hph expression was dictated by an A-rich region, which included a putative positioning element, and polyadenylation sites. In contrast, our results indicated that introns are not required for the expression of hph directed by the Csβ1-tub and Csgpd promoters in C. subvermispora. This study provides insights into the functions and cis-element requirements of transcriptional terminators in Agaricomycetes, which may be relevant for designing recombinant genes for this important fungal class.

Understanding the molecular mechanisms controlling dikaryon formation in Agaricomycetes, which is basically controlled by A and B mating-type loci, contributes to improving mushroom cultivation and breeding. In Coprinopsis cinerea, various mutations in the SRY-type high mobility group protein-encoding gene, pcc1, were shown to activate the A-regulated pathway to induce pseudoclamp (clamp cells without clamp connection) and fruiting body formation in monokaryons. The formation of clamp cells was blocked in AmutBmut strain 326 with clp1-1 mutation in C. cinerea. However, considering the diverse mechanisms of sexual development among Agaricomycetes, it remains unclear whether similar phenotypes are also observed in clp1 or pcc1 mutants in cultivated mushrooms. Therefore, phenotypic analyses of Pleurotus ostreatus pcc1 or clp1 (Popcc1 or Poclp1) mutants generated using CRISPR/Cas9 were performed in this study. Plasmids with Cas9 expression cassette and different single guide RNAs targeting Popcc1 or Poclp1 were individually introduced into a monokaryotic P. ostreatus strain PC9 to obtain the mutants. Unlike in C. cinerea, the pseudoclamp cell was not observed in monokaryotic Popcc1 mutants, but it was observed after crossing two compatible strains with Popcc1 mutations. In Poclp1 mutants, dikaryosis was impaired as clamp cells were not observed after crossing, suggesting that Poclp1 functions may be essential for clamp cell formation, like in C. cinerea. These results provided a clue with respect to conserved and diverse mechanisms underlying sexual development in Agaricomycetes (at least between C. cinerea and P. ostreatus).

We introduced a site-directed mutation in the sdi1 gene and used it as a selective marker for the polyethylene glycol-mediated transformation of Pleurotus eryngii monokaryon protoplasts. The transformants displayed obvious and stable resistance to the fungicide carboxin indicating that the mutant Pesdi1 gene is an efficient selective marker.

Of all of the natural polymers, lignin, an aromatic heteropolymer in plant secondary cell walls, is the most resistant to biological degradation. White-rot fungi are the only known organisms that can depolymerize or modify wood lignin. Investigating the mechanisms underlying lignin biodegradation by white-rot fungi would contribute to the ecofriendly utilization of woody biomass as renewable resources in the future. Efficient gene disruption, which is generally very challenging in the white-rot fungi, was established in Pleurotus ostreatus (the oyster mushroom). Some of the genes encoding manganese peroxidases, enzymes that are considered to be involved in lignin biodegradation, were disrupted separately, and the phenotype of each single-gene disruptant was analysed. However, it remains difficult to generate multi-gene disruptants in this fungus. Here we developed a new genetic transformation marker in P. ostreatus and demonstrated two marker recycling methods that use counter-selection to generate a multigene disruptant. This study will enable future genetic studies of white-rot fungi, and it will increase our understanding of the complicated mechanisms, which involve various enzymes, including lignin-degrading enzymes, underlying lignin biodegradation by these fungi.

Agaricomycetes exhibit a remarkable morphological differentiation from vegetative mycelia to huge fruiting bodies. To investigate the molecular mechanism underlying the fruiting body development, we have isolated and characterized many Coprinopsis cinerea mutant strains defective in fruiting initiation to date. Dikaryon formation in agaricomycetes, which is followed by fruiting development, is governed by the mating type loci, A and B. Recently, mutations in the Cc.snf5 gene, which encodes a putative component of the chromatin remodeling complex switch/sucrose non-fermentable (SWI/SNF), were shown to cause defects in A-regulated clamp cell morphogenesis, as well as in fruiting initiation. Here, we demonstrate that Cc.arp9, which encodes a putative actin-related protein associated with two chromatin remodeling complexes, SWI/SNF and remodels the structure of chromatin (RSC), is also essential for fruiting initiation. In contrast to Cc.snf5 mutants, Cc.arp9 mutants were not defective in clamp cell formation. The effects of mutations in Cc.arp9 and Cc.snf5 on oidia production and the transcriptional expression levels of clp1 and pcc1, which are under the control of the A gene, were also examined. These indicated that Cc.Snf5 is involved in A-regulated pathways, whereas Cc.Arp9 is not apparently. Cc.arp9/Cc.snf5 double-gene disruptants were generated and their phenotypes were analyzed, which suggested a complicated developmental regulation mechanism mediated by chromatin remodeling.

Peroxisomes are well-known organelles that are present in most eukaryotic organisms. Mutant phenotypes caused by the malfunction of peroxisomes have been shown in many fungi. However, these have never been investigated in Agaricomycetes, which include white-rot fungi that degrade wood lignin in nature almost exclusively and play an important role in the global carbon cycle. Based on the results of a forward genetics study to identify mutations causing defects in the ligninolytic activity of the white-rot Agaricomycete Pleurotus ostreatus, we report phenotypes of pex1 disruptants in P. ostreatus, which are defective in two major features of white-rot Agaricomycetes: lignin biodegradation and mushroom formation. Pex1 disruption was also shown to cause defects in the hyphal growth of P. ostreatus on certain sawdust and minimum media. We also demonstrated that pex1 is essential for fruiting initiation in the non-wood decaying Agaricomycete Coprinopsis cinerea. However, unlike P. ostreatus, significant defects in hyphal growth on the aforementioned agar medium were not observed in C. cinerea. This result, together with previous C. cinerea genetic studies, suggests that the regulation mechanisms for the utilization of carbon sources are altered during the evolution of Agaricomycetes or Agaricales.

We characterized a Coprinopsis cinerea mutant strain, Spe20, defective in fruiting initiation, which was isolated after restriction enzyme-mediated integration (REMI) mutagenesis of a homokaryotic fruiting strain, 326. A plasmid rescue followed by complementation experiments, RACE, and cDNA analyses revealed that the gene, a mutation of which is responsible for the phenotype, is predicted to encode a protein that exhibits a high similarity to yeast Snf5p, a key component of the chromatin remodeling complex SWI/SNF, and named Cc.snf5. Cc.Snf5 is, however, different from Snf5p in that the former has, in addition to an Snf5 domain comprising N-terminal repeat1 (rp1) and C-terminal repeat2 (rp2) subdomains in a middle region, a GATA Zn-finger domain in a C-terminal region. In strain Spe20, plasmid pPHT1 used for REMI is inserted in the ORF encoding rp2. This raised the possibility that in strain Spe20, the disrupted Cc.Snf5 is functionally active albeit incompletely because it retains rp1. Thus, we disrupted the whole SNF5 domain and its downstream peptide and found that the disruption results in inhibition of not only fruiting initiation but also dikaryon development, a prerequisite for fruiting. We also found that specific disruption of the Zn-finger domain results in inhibition of fruiting initiation. These results indicate that Cc.Snf5 plays an essential role in sexual development of C. cinerea.

White-rot fungi play an important role in the global carbon cycle because they are the species that almost exclusively biodegrade wood lignin in nature. Lignin peroxidases (LiPs), manganese peroxidases (MnPs) and versatile peroxidases (VPs) are considered key players in the ligninolytic system. Apart from LiPs, MnPs and VPs, however, only few other factors involved in the ligninolytic system have been investigated using molecular genetics, implying the existence of unidentified elements. By combining classical genetic techniques with next-generation sequencing technology, they successfully showed an efficient forward genetics approach to identify mutations causing defects in the ligninolytic system of the white-rot fungus Pleurotus ostreatus. In this study, they identified two genes - chd1 and wtr1 - mutations in which cause an almost complete loss of Mn2+ -dependent peroxidase activity. The chd1 gene encodes a putative chromatin modifier, and wtr1 encodes an agaricomycete-specific protein with a putative DNA-binding domain. The chd1-1 mutation and targeted disruption of wtr1 hamper the ability of P. ostreatus to biodegrade wood lignin. Examination of the effects of the aforementioned mutation and disruption on the expression of certain MnP/VP genes suggests that a complex mechanism underlies the ligninolytic system in P. ostreatus.

The agaricomycete (homobasidiomycete) Coprinopsis cinerea has been used as a model to study the molecular mechanism for photomorphogenesis. Molecular genetic analyses of mutants defective in fruiting body (mushroom) photomorphogenesis of C. cinerea identified two genes, dst1 and dst2. dst1 encodes a homolog of WC-1, a fungal blue-light photoreceptor first identified in Neurospora crassa, while dst2 encodes a novel protein with a putative flavin adenine dinucleotide (FAD)-binding-4 domain. In addition, reverse genetic analysis revealed that disruption of a C. cinerea gene encoding a WC-2 homolog, the partner of WC-1, causes the same blind phenotype. Searches on the genome data show that both WC-1 and WC-2 homologs are present in some agaricomycetes other than C. cinerea. Furthermore, in an agaricomycete, Lentinula edodes, it has been shown in vitro that the WC-1 and WC-2 homologs interact with each other. These findings suggest that the presumptive mechanism for blue-light sensing in agaricomycetes is fundamentally similar to that in Neurospora crassa, in which the WC-1/WC-2 complex plays a central role. Since the WC-1/WC-2 complex operates as a photoreceptor and a transcription factor, future studies will include identification of the targets of the WC-1/WC-2 complex that regulate photomorphogenesis in agaricomycetes. Another future challenge will be elucidation of the role of the newly identified photomorphogenetic protein, Dst2, in the blue-light-sensing mechanism.

The fruiting-body primordium of Coprinopsis cinerea exhibits remarkable photomorphogenesis. Under a 12-h light/12-h dark regime, the primordium proceeds to the fruiting-body maturation phase in which the primordium successively undergoes basidiospore formation, stipe elongation and pileus expansion, resulting in the mature fruiting-body. In continuous darkness, however, the primordium never proceeds to the maturation phase: the pileus and stipe tissues at the upper part of the primordium remain rudimentary while the basal part of the primordium elongates, producing the etiolated "dark stipe" phenotype. In our previous studies, blind mutants, which produce dark stipes under light conditions that promote fruiting-body maturation in the wild-type, have been isolated, and two genes, dst1 and dst2, responsible for the mutant phenotype have been identified. In this study we show that the dst2-1 mutant exhibits a blind phenotype during asexual spore production in addition to that in fruiting-body photomorphogenesis. We also reveal that dst2 is predicted to encode a protein with a putative flavin adenine dinucleotide (FAD)-binding-4 domain. The two blind phenotypes, together with the existence of an FAD-binding domain in Dst2, suggest that Dst2 may play a role in perceiving blue light.

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.

The formation and proliferation of the dikaryon in the agaricomycete Coprinopsis cinerea is controlled by the mating type genes, A and B. The B genes, which encode pheromones and pheromone receptors, control nuclear migration for dikaryosis as well as the fusion of the clamp cell with the subterminal cell while the A genes, which encode two classes of homeodomain proteins, control conjugate nuclear division associated with clamp connection development. We characterized the mutant, B28, which was newly isolated as a strain that fails to form a primary hyphal knot, the first visible sign toward fruiting, from a homokaryotic fruiting strain after REMI mutagenesis. Detailed phenotypic analysis revealed that strain B28 exhibits, in addition to the fruiting defect, a defect in A-regulated clamp cell morphogenesis as well as a defect in B-regulated nuclear migration for dikaryosis. The mutant clamp cells are unique in that they continue growing like branches without fusing with the subterminal cells, in contrast to the unfused pseudoclamp which are normally formed in A-on B-off strains, providing evidence for the existence of an as yet unidentified mechanism for the growth suppression of the clamp cell. Molecular analysis revealed that the gene responsible for the phenotypes, designated Cc.ubc2, encodes a protein similar to Ubc2, an adaptor protein for filamentous growth, pheromone response and virulence in the smut fungus Ustilago maydis. In addition, western blot analysis demonstrated that the Cc.ubc2-1 mutation blocks phosphorylation of a presumptive MAP kinase.

We isolated a target gene for the Lentinula edodes putative transcription factor Le.CDC5 that contains a c-Myb-type DNA-binding domain. The gene, termed ctg1, encodes a novel protein (159 amino acid residues) with a leucine zipper-like sequence and contains a 7-bp Le.CDC5-binding sequence, 5'GCAATCT3', in its transcribed region downstream of the start codon. Chromatin immunoprecipitation analysis strongly suggested that intracellular Le.CDC5 binds to this 7-bp sequence on L. edodes chromatin. Binding was most efficient on chromatin from the stipes of mature fruiting bodies. Two Le.CDC5-interaction partners were identified in L. edodes and named CIPA and CIPB. The CIPB protein (127 amino acid residues) binds to a 6-bp sequence with the consensus sequence 5'CAACAC/T/G3'. The ctg1 gene contains nine 6-bp consensus (or consensus-like) sequences, six are in the 5'-upstream region and three in the transcribed region downstream of the start codon. At least two each of the upstream and downstream sequences appear to bind CIPB in vitro. We suggest that Le.CDC5 and CIPB can cooperatively regulate the expression of ctg1.

Immunoblot analysis of Le.CDC5 (842 amino acid residues), the expressed product of the cDNA of Le.cdc5 gene that has been previously reported to be most actively transcribed in primordia and small immature fruiting bodies of the basidiomycete Lentinula edodes, showed that the primordia, immature fruiting bodies and mature fruiting bodies contain similar amounts of Le.CDC5 protein. This indicates that the Le.CDC5 protein molecules synthesized in the beginning and early stage of fruiting-body formation remains in mycelial tissues even after small immature fruiting bodies developed and matured. Immunohistochemical analysis showed that Le.CDC5 is present everywhere in the mycelial tissues of immature fruiting body, but prehymenophore, the border between pileus and stipe, and the bottom of stipe seem likely to contain larger amounts of Le.CDC5. Within the hymenophore of mature fruiting body, the hymenium (in/on which a large number of basidia and basidiospores are formed) contains the Le.CDC5 most exclusively.