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Gene targeting is useful to isolate strains with mutations in a gene of interest for efficient breeding. In this study, we generated msh4 or mer3 single-gene disruptant monokaryons using a Pleurotus ostreatus Δku80 strain for efficient gene targeting. Dikaryons of P. ostreatus Δmsh4×Δmsh4 or Δmer3×Δmer3 were isolated via backcrosses, and the number of basidiospores produced was measured. The number of basidiospores fell by an average 1/13.7 in the P. ostreatus Δmsh4×Δmsh4 dikaryons versus the P. ostreatus msh4+×Δmsh4 dikaryons, and 1/82.6 in the P. ostreatus Δmer3×Δmer3 dikaryons versus the P. ostreatus mer3+×Δmer3 dikaryons. To demonstrate the effects of ku80 disruption, P. ostreatus Δku80×Δku80 dikaryon strains were isolated and no significant effects on basidiospore production were observed. Fluorescence microscopy showed meiotic progression was arrested during prophase I in the msh4 or mer3 disruptants. To our knowledge, this is the first report on molecular breeding of sporeless strains in cultivated mushrooms using an efficient method for targeted gene disruption.

White-rot fungi efficiently degrade lignin and, thus, play a pivotal role in the global carbon cycle. However, the mechanisms of lignin degradation are largely unknown. Recently, mutations in four genes, namely wtr1, chd1, pex1, and gat1, were shown to abrogate the wood lignin-degrading ability of Pleurotus ostreatus. In this study, we conducted a comparative transcriptome analysis to identify genes that are differentially expressed in ligninolysis-deficient mutant strains. Putative ligninolytic genes that are highly expressed in parental strains are significantly downregulated in the mutant strains. On the contrary, many putative cellulolytic and xylanolytic genes are upregulated in the chd1-1, Δpex1, and Δgat1 strains. Identifying transcriptional alterations in mutant strains could provide new insights into the regulatory mechanisms of lignocellulolytic genes in P. ostreatus.

A new endophytic fungus, described herein as Cunninghamella saisamornae, was isolated from soil collected from Chiang Mai Province, Thailand. This species was distinguished from the previously described Cunninghamella species by the presence of a chlamydospore formation, the lack of a zygospore stage and a maximum growth temperature of 45°C. Multi-gene phylogenetic analysis of internal transcribed spacers (ITS) and large subunit (LSU) of the nuclear ribosomal DNA (rDNA), along with the translation elongation factor 1-alpha (tef-1) genes, support the finding that C. saisamornae is distinct from other species within the genus Cunninghamella. A full description, color photographs, illustrations and a phylogenetic tree showing the position of C. saisamornae are provided.

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.

Cis-acting elements play a vital role in regulation of transcription initiation. Several cis-acting elements have been identified in filamentous fungi; however, the fundamental requirements for basic promoter function in basidiomycetes are obscure. In this study, core elements in β1-tubulin promoters of basidiomycetes were functionally characterized. Using transient transformation in Ceriporiopsis subvermispora as a promoter assay, we found that a 14-bp region (β1-tubulin core promoter element, BCE), as well as CT-rich stretch, in the β1-tubulin promoter of the species played a critical role in the expression of a recombinant hph as a reporter gene. In addition, in silico analysis revealed other members of basidiomycetes also harboured the BCE motif as well as CT-rich stretch in the β1-tubulin promoter region, suggesting their functional conservation among the species of basidiomycetes. To confirm the function of BCE, we investigated the effects of BCE motif deletion in the Pleurotus ostreatus β1-tubulin promoter on expression levels of a recombinant luminous shrimp luciferase reporter gene, which was targeted into the Pofcy1 locus. Intriguingly, luciferase activity was abolished when the BCE motif was deleted in the β1-tubulin promoter, strongly demonstrating its essential function in transcription from this promoter on the chromosome. This study clearly demonstrates the crucial role of the BCE as well as the CT-rich stretch regions in the β1-tubulin promoter among basidiomycetes and provides new insights into the fundamental mechanism of transcription initiation in this group.

In nature, white-rot fungi efficiently degrade lignin present in wood biomass. Elucidation of molecular mechanisms underlying wood lignin biodegradation by white-rot fungi would contribute to the development of efficient and ecofriendly methods of producing valuable chemical products from wood biomass. Here, using forward genetics approach, we demonstrate that the mutant of a putative transcription factor gene, gat1-1, significantly decreases the ligninolytic activity of the white-rot fungus Pleurotus ostreatus, when grown on beech wood sawdust medium. We also show that this phenotype is dominant. In Schizophyllum commune, Gat1 was previously shown to be involved in fruiting body development. In this study, we reveal that the mutations in gat1 gene cause defects in fruiting body development in P. ostreatus. Unlike the previously reported recessive gene mutations that decrease the ligninolytic activity of P. ostreatus, the gat1-1 mutation and Δgat1 are dominant and would thus be useful for future studies on the functional role of the orthologs in other white-rot fungi.

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.

Distinct wood degraders occupying their preferred habitats have biased enzyme repertoires that are well fitted to their colonized substrates. Pleurotus ostreatus, commonly found on wood, has evolved its own enzyme-producing traits. In our previous study, transcriptional shifts in several P. ostreatus delignification-defective mutants, including Δhir1 and Δgat1 strains, were analyzed, which revealed the downregulation of ligninolytic genes and the upregulation of cellulolytic and xylanolytic genes when compared to their parental strain 20b on beech wood sawdust medium (BWS). In this study, rice straw (RS) was used as an alternative substrate to examine the transcriptional responses of P. ostreatus to distinct substrates. The vp1 gene and a cupredoxin-encoding gene were significantly upregulated in the 20b strain on RS compared with that on BWS, reflecting their distinct regulation patterns. The overall expression level of genes encoding glucuronidases was also higher on RS than on BWS, showing a good correlation with the substrate composition. Transcriptional alterations in the mutants (Δhir1 or Δgat1 versus 20b strain) on RS were similar to those on BWS, and the extracellular lignocellulose-degrading enzyme activities and lignin-degrading ability of the mutants on RS were consistent with the transcriptional alterations of the corresponding enzyme-encoding genes. However, transcripts of specific genes encoding enzymes belonging to the same CAZyme family exhibited distinct alteration patterns in the mutant strains grown on RS compared to those grown on BWS. These findings provide new insights into the molecular mechanisms underlying the transcriptional regulation of lignocellulolytic genes in P. ostreatus.Key Points• P. ostreatus expressed variable enzymatic repertoire-related genes in response to distinct substrates.• A demand to upregulate the cellulolytic genes seems to be present in ligninolysis-deficient mutants.• The regulation of some specific genes probably driven by the demand is dependent on the substrate.

The contamination of paraquat (1,1'-dimethyl-4,4'-bipyridylium dichloride) herbicide from the farming area has become a public concern in many countries. This herbicide harms to human health and negatively effects the soil fertility. Several methods have been introduced for the remediation of paraquat. In this study, 20 isolates of the paraquat-tolerant fungi were isolated from the contaminated soil samples in northern Thailand. We found that isolate PRPY-2 and PFCM-1 exhibited the highest degradation activity of paraquat on synthetic liquid medium. About 80 and 68% of paraquat were removed by PRPY-2 and PFCM-1, respectively, after 15 days of cultivation. Based on the morphological characteristic and molecular analysis, the fungal isolate PRPY-2 and PFCM-1 were identified as Aspergillus tamarii and Cunninghamella sp., respectively. The biosorption of paraquat on these fungal mycelia was also investigated. It was found that only 8-10% of paraquat could be detected on their mycelia, while 24-46% of paraquat was degraded by fungal mycelia. This is the first report on paraquat degrading ability by A. tamarii and Cunninghamella sp. It is demonstrated that these filamentous fungi are promising microorganisms available for remediation of paraquat contaminated environment.

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.

A novel promoter assay was developed for Agaricomycetes, using a gene-targeting approach, with or without the CRISPR/Cas9 technique. It enables precise evaluation of promoter activity at the original site of the chromosome without random and multiple integrations in conventional transformation experiments.

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.

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.