Identification of Various Recombinants in a Patient Coinfected With the Different SARS-CoV-2 Variants.

BACKGROUND: Viral recombination that occurs by exchanging genetic materials between two viral genomes coinfecting the same host cells is associated with the emergence of new viruses with different virulence. Herein, we detected a patient coinfected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta and Omicron variants and identified various recombinants in the SARS-CoV-2 full-length spike gene using long-read and Sanger sequencing. METHODS: Samples from five patients in Japan with household transmission of coronavirus disease 2019 (COVID-19) were analyzed using molecular assays for detection and identification of SARS-CoV-2. Whole-genome sequencing was conducted using multiplex PCR with short-read sequencing. RESULTS: Among the five SARS-CoV-2-positive patients, the mutation-specific assay identified the Delta variant in three, the Omicron variant in one, and an undetermined in one. The undermined patient was identified as Delta using whole-genome sequencing, but samples showed a mixed population of Delta and Omicron variants. This patient was analyzed for viral quasispecies by long-read and Sanger sequencing using a full-length spike gene amplicon. In addition to the Delta and Omicron sequences, the viral quasispecies analysis identified nine different genetic recombinant sequences with various breakpoints between Delta and Omicron sequences. The nine detected recombinant sequences in the spike gene showed over 99% identity with viruses that were detected during the Delta and Omicron cocirculation period from the United States and Europe. CONCLUSIONS: This study demonstrates that patients coinfected with different SARS-CoV-2 variants can generate various viral recombinants and that various recombinant viruses may be produced during the cocirculation of different variants.

The Inhibition of TREK-1 K+ Channels via Multiple Compounds Contained in the Six Kamikihito Components, Potentially Stimulating Oxytocin Neuron Pathways.

Oxytocin, a significant pleiotropic neuropeptide, regulates psychological stress adaptation and social communication, as well as peripheral actions, such as uterine contraction and milk ejection. Recently, a Japanese Kampo medicine called Kamikihito (KKT) has been reported to stimulate oxytocin neurons to induce oxytocin secretion. Two-pore-domain potassium channels (K2P) regulate the resting potential of excitable cells, and their inhibition results in accelerated depolarization that elicits neuronal and endocrine cell activation. We assessed the effects of KKT and 14 of its components on a specific K2P, the potassium channel subfamily K member 2 (TREK-1), which is predominantly expressed in oxytocin neurons in the central nervous system (CNS). KKT inhibited the activity of TREK-1 induced via the channel activator ML335. Six of the 14 components of KKT inhibited TREK-1 activity. Additionally, we identified that 22 of the 41 compounds in the six components exhibited TREK-1 inhibitory effects. In summary, several compounds included in KKT partially activated oxytocin neurons by inhibiting TREK-1. The pharmacological effects of KKT, including antistress effects, may be partially mediated through the oxytocin pathway.

Identification of two genes essential for basidiospore formation during the postmeiotic stages in Pleurotus ostreatus.

A sporeless strain is an important breeding target in the mushroom industry. However, basidiospore production in the oyster mushroom Pleurotus ostreatus has been shown to be impaired by single-gene mutations in only two meiosis-related genes, mer3 and msh4. This study proposed a strategy for identifying the genes essential for basidiospore formation after meiotic division to determine new targets for molecular breeding. RNA-seq analysis was performed to identify P. ostreatus genes that are specifically expressed in the gill tissue of fruiting bodies, where basidiospore formation occurs. Transcriptome data during fruiting development of Coprinopsis cinerea, in which the meiotic steps progress synchronously, were then used to identify genes that are active in the postmeiotic stages. Based on these comparative analyses, five P. ostreatus genes were identified. Plasmids containing expression cassettes for hygromycin B-resistance screening, Cas9, and single-guide RNA targeting each gene were introduced into the protoplasts of dikaryotic strain, PC9×#64, to generate dikaryotic gene disruptants. Among the obtained transformants, three dikaryotic pcl1 disruptants and two cro6c disruptants did not produce basidiospores. Microscopic analyses indicated that spore formation was arrested at particular stages in these gene disruptants. These results indicate that these two genes are essential for mature spore formation in this fungus.

Visualizing organelles with recombinant fluorescent proteins in the white-rot fungus Pleurotus ostreatus.

White-rot fungi secrete numerous enzymes involved in lignocellulose degradation. However, the secretory mechanisms or pathways, including protein synthesis, folding, modification, and traffic, have not been well studied. In the first place, few experimental tools for molecular cell biological studies have been developed. As the first step toward investigating the mechanisms underlying protein secretion, this study visualized organelles and transport vesicles involved in secretory mechanisms with fluorescent proteins in living cells of the white-rot fungus Pleurotus ostreatus (agaricomycete). To this end, each plasmid containing the expression cassette for fluorescent protein [enhanced green fluorescent protein (EGFP) or mCherry] fused with each protein that may be localized in the endoplasmic reticulum (ER), Golgi, or secretory vesicles (SVs) was introduced into P. ostreatus strain PC9. Fluorescent microscopic analyses of the obtained hygromycin-resistant transformants suggested that Sec13-EGFP and Sec24-EGFP visualize the ER; Sec24-EGFP, mCherry-Sed5, and mCherry-Rer1 visualize the compartment likely corresponding to early Golgi and/or the ER-Golgi intermediate compartment; EGFP/mCherry-pleckstrin homology (PH) visualizes possible late Golgi; and EGFP-Seg1 and mCherry-Rab11 visualize SVs. This study successfully visualized mitochondria and nuclei, thus providing useful tools for future molecular cell biological studies on lignocellulose degradation by P. ostreatus. Furthermore, some differences in the Golgi compartment or apparatus and the ER-Golgi intermediate of P. ostreatus compared to other fungi were also suggested.

Experimental evidence that lignin-modifying enzymes are essential for degrading plant cell wall lignin by Pleurotus ostreatus using CRISPR/Cas9.

Lignin-modifying enzymes (LMEs), which include laccases (Lacs), manganese peroxidases (MnPs), versatile peroxidases (VPs), and lignin peroxidases (LiPs), have been considered key factors in lignin degradation by white-rot fungi because they oxidize lignin model compounds and depolymerize synthetic lignin in vitro. However, it remains unclear whether these enzymes are essential/important in the actual degradation of natural lignin in plant cell walls. To address this long-standing issue, we examined the lignin-degrading abilities of multiple mnp/vp/lac mutants of Pleurotus ostreatus. One vp2/vp3/mnp3/mnp6 quadruple-gene mutant was generated from a monokaryotic wild-type strain PC9 using plasmid-based CRISPR/Cas9. Also, two vp2/vp3/mnp2/mnp3/mnp6, two vp2/vp3/mnp3/mnp6/lac2 quintuple-gene mutants, and two vp2/vp3/mnp2/mnp3/mnp6/lac2 sextuple-gene mutants were generated. The lignin-degrading abilities of the sextuple and vp2/vp3/mnp2/mnp3/mnp6 quintuple-gene mutants on the Beech wood sawdust medium reduced drastically, but not so much for those of the vp2/vp3/mnp3/mnp6/lac2 mutants and the quadruple mutant strain. The sextuple-gene mutants also barely degraded lignin in Japanese Cedar wood sawdust and milled rice straw. Thus, this study presented evidence that the LMEs, especially MnPs and VPs, play a crucial role in the degradation of natural lignin by P. ostreatus for the first time.

The lignin-degrading abilities of Gelatoporia subvermispora gat1 and pex1 mutants generated via CRISPR/Cas9.

White-rot fungi efficiently degrade wood lignin; however, the mechanisms involved remain largely unknown. Recently, a forward genetics approach to identify several genes in Pleurotus ostreatus (Agaricales) in which mutations cause defects in wood lignin degradation was used. For example, pex1 encodes a peroxisome biogenesis factor and gat1 encodes a putative Agaricomycetes-specific DNA-binding transcription factor. In this study, we examined the effects of single-gene mutations in pex1 or gat1 on wood lignin degradation in another white-rot fungus, Gelatoporia (Ceriporiopsis) subvermispora (Polyporales), to investigate conserved and derived degradation mechanisms in white-rot fungi. G. subvermispora pex1 and gat1 single-gene mutant strains were generated from a monokaryotic wild-type strain, FP-90031-Sp/1, using plasmid-based CRISPR/Cas9. As in P. ostreatus, Gsgat1 mutants were nearly unable to degrade lignin sourced from beech wood sawdust medium (BWS), while Gspex1 mutants exhibited a delay in lignin degradation. We also found that the transcripts of lignin-modifying enzyme-encoding genes, mnp4, mnp5, mnp6, mnp7, and mnp11, which predominantly accumulate in FP-90031-Sp/1 cultured with BWS, were greatly downregulated in Gsgat1 mutants. Taken together, the results suggest that Gat1 may be a conserved regulator of the ligninolytic system of white-rot fungi and that the contribution of peroxisomes to the ligninolytic system may differ among species.

Double-gene targeting with preassembled Cas9 ribonucleoprotein for safe genome editing in the edible mushroom Pleurotus ostreatus.

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.

Lignocellulose molecular assembly and deconstruction properties of lignin-altered rice mutants.

Bioengineering approaches to modify lignin content and structure in plant cell walls have shown promise for facilitating biochemical conversions of lignocellulosic biomass into valuable chemicals. Despite numerous research efforts, however, the effect of altered lignin chemistry on the supramolecular assembly of lignocellulose and consequently its deconstruction in lignin-modified transgenic and mutant plants is not fully understood. In this study, we aimed to close this gap by analyzing lignin-modified rice (Oryza sativa L.) mutants deficient in 5-HYDROXYCONIFERALDEHYDE O-METHYLTRANSFERASE (CAldOMT) and CINNAMYL ALCOHOL DEHYDROGENASE (CAD). A set of rice mutants harboring knockout mutations in either or both OsCAldOMT1 and OsCAD2 was generated in part by genome editing and subjected to comparative cell wall chemical and supramolecular structure analyses. In line with the proposed functions of CAldOMT and CAD in grass lignin biosynthesis, OsCAldOMT1-deficient mutant lines produced altered lignins depleted of syringyl and tricin units and incorporating noncanonical 5-hydroxyguaiacyl units, whereas OsCAD2-deficient mutant lines produced lignins incorporating noncanonical hydroxycinnamaldehyde-derived units. All tested OsCAldOMT1- and OsCAD2-deficient mutants, especially OsCAldOMT1-deficient lines, displayed enhanced cell wall saccharification efficiency. Solid-state nuclear magnetic resonance (NMR) and X-ray diffraction analyses of rice cell walls revealed that both OsCAldOMT1- and OsCAD2 deficiencies contributed to the disruptions of the cellulose crystalline network. Further, OsCAldOMT1 deficiency contributed to the increase of the cellulose molecular mobility more prominently than OsCAD2 deficiency, resulting in apparently more loosened lignocellulose molecular assembly. Such alterations in cell wall chemical and supramolecular structures may in part account for the variations of saccharification performance of the OsCAldOMT1- and OsCAD2-deficient rice mutants.

Introducing multiple-gene mutations in Pleurotus ostreatus using a polycistronic tRNA and CRISPR guide RNA strategy.

The white-rot fungus Pleurotus ostreatus is an agaricomycete that is frequently used in molecular genetics studies as many useful tools are applicable to the fungus. In particular, efficient gene targeting using homologous recombination and CRISPR/Cas9 enables the introduction of a mutation in the gene of interest for functional analysis. Multiple genes encoding various lignocellulose-degrading enzymes are predicted to be present in the genome; therefore, analyses of multiple-gene mutants are required to elucidate the mechanisms underlying lignocellulose degradation by P. ostreatus. Conventional tools for generating multiple-gene mutations in P. ostreatus are laborious and time-consuming. Therefore, more efficient and practical methods are needed. In this study, we introduced CRISPR/Cas9-assisted multiple-gene mutations using a polycistronic tRNA and CRISPR guide RNA approach. The frequency (triple-gene mutation in fcy1, vp2, and 62347) was only 3.3% when a tetracistronic tRNA-sgRNA containing four different sgRNAs targeting fcy1, vp2, vp3, or 62347 was expressed. It increased to 20% (triple-gene mutation in vp1, vp2, and vp3) after a tricistronic tRNA-sgRNA was expressed with replaced/modulated promoter and tRNA sequences. This study demonstrated, for the first time, the applicability of a strategy to induce multiple-gene mutations in P. ostreatus in a transformation experiment.

Gene targeting of dikaryotic Pleurotus ostreatus nuclei using the CRISPR/Cas9 system.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted gene targeting is a promising method used in molecular breeding. We recently reported the successful introduction of this method in the monokaryotic Pleurotus ostreatus (oyster mushroom), PC9. However, considering their application in mushroom breeding, dikaryotic strains (with targeted gene mutations in both nuclei) need to be generated. This is laborious and time-consuming because a classical crossing technique is used. Herein, we report a technique that targets both nuclei of dikaryotic P. ostreatus, PC9×#64 in a transformation experiment using plasmid-based CRISPR/Cas9, with the aim of developing a method for efficient and rapid molecular breeding. As an example, we targeted strains with low basidiospore production ability through the meiosis-related genes mer3 or msh4. Four different plasmids containing expression cassettes for Cas9 and two different gRNAs targeting mer3 or msh4 were constructed and separately introduced into PC9×#64. Eight of the 38 dikaryotic transformants analyzed produced no basidiospores. Genomic PCR suggested that msh4 or mer3 mutations were introduced into both nuclei of seven out of eight strains. Thus, in this study, we demonstrated simultaneous gene targeting using our CRISPR/Cas9 system, which may be useful for the molecular breeding of cultivated agaricomycetes.

CRISPR/Cas9 using a transient transformation system in Ceriporiopsis subvermispora.

Ceriporiopsis subvermispora is a white-rot fungus with great potential for industrial and biotechnological applications, such as the pretreatment of lignocellulose in biorefineries, as it decomposes the lignin in the plant cell wall without causing severe cellulose degradation. A genetic transformation system was recently developed; however, gene-targeting experiments to disrupt or modify the gene(s) of interest remain challenging, and this is a bottleneck for further molecular genetic studies and breeding of C. subvermispora. Herein, we report efficient clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted gene mutagenesis in this fungus. Two plasmids expressing Cas9 together with a different pyrG-targeting single-guide RNA were separately introduced into the monokaryotic C. subvermispora strain FP-90031-Sp/1, which frequently generated strains that exhibited resistance to 5-fluoroorotic acid and uridine/uracil auxotrophy. Southern blot analyses and genomic polymerase chain reaction followed by DNA sequencing of some mutants revealed that they were pyrG mutants. We also observed that hygromycin resistance of the pyrG mutants was frequently lost after repeated subcultivations, indicating that a maker-free genome editing occurred successfully. It is also suggested that a gene mutation(s) can be introduced via a transient expression of Cas9 and a single-guide RNA; this feature, together with high-frequency gene targeting using the CRISPR/Cas9 system, would be helpful for studies on lignocellulose-degrading systems in C. subvermispora. KEY POINTS: • Efficient plasmid-based CRISPR/Cas9 was established in C. subvermispora. • The mutations can be introduced via a transient expression of Cas9 and sgRNA. • A maker-free CRISPR/Cas9 is established in this fungus.

Overexpressing Pleurotus ostreatus rho1b results in transcriptional upregulation of the putative cellulolytic enzyme-encoding genes observed in ccl1 disruptants.

The transcriptional expression pattern of lignocellulolytic enzyme-encoding genes in white-rot fungi differs depending on the culture conditions. Recently, it was shown that 13 putative cellulolytic enzyme-encoding genes were significantly upregulated in most Pleurotus ostreatus ligninolysis-deficient mutant strains on beech wood sawdust medium. However, the mechanisms by which this transcriptional shift is triggered remain unknown. In this study, we identified one mechanism. Our previous study implied that histone H3 N-dimethylation at lysine 4 level possibly affects the shift; therefore, we analysed the expression pattern in the disruptants of P. ostreatus ccl1, which encodes a putative component of the COMPASS complex mediating the methylation. The results showed upregulation of 5 of the 13 cellulolytic enzyme-encoding genes. We also found that rho1b, encoding a putative GTPase regulating signal transduction pathways, was upregulated in the ccl1 disruptants and ligninolysis-deficient strains. Upregulation of at least three of the five cellulolytic enzyme-encoding genes was observed in rho1b-overexpressing strains but not in ccl1/rho1b double-gene disruptants, during the 20-day culture period. These results suggest that Rho1b may be involved in the upregulation of cellulolytic enzyme-encoding genes observed in the ccl1 disruptants. Furthermore, we suggest that Mpk1b, a putative Agaricomycetes-specific mitogen-activated protein kinase, functions downstream of Rho1b.

Complete Genome Sequences of Enterovirus D68 Clade A and D Strains in the Philippines.

Complete genome sequences were determined for 4 clade A and 12 clade D enterovirus D68 strains detected in nasopharyngeal swabs from children with acute respiratory illness in the Philippines. These sequence data will be useful for future epidemiological monitoring, including watching for viral evolution.

Gene targeting using pre-assembled Cas9 ribonucleoprotein and split-marker recombination in Pleurotus ostreatus.

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.

Functional analyses of Pleurotus ostreatus pcc1 and clp1 using CRISPR/Cas9.

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).

Efficient genome editing with CRISPR/Cas9 in Pleurotus ostreatus.

Pleurotus ostreatus is one of the most commercially produced edible mushrooms worldwide. Improved cultivated strains with more useful traits have been obtained using classical breeding, which is laborious and time-consuming. Here, we attempted efficient gene mutagenesis using plasmid-based CRISPR/Cas9 as the first step for non-genetically modified (non-GM) P. ostreatus generation. Plasmids harboring expression cassettes of Cas9 and different single guide RNAs targeting fcy1 and pyrG were individually transferred into fungal protoplasts of the PC9 strain, which generated some strains exhibiting resistance to 5-fluorocytosine and 5-fluoroorotic acid, respectively. Genomic PCR followed by sequencing revealed small insertions/deletions or insertion of a fragment from the plasmid at the target site in some of the drug-resistant strains. The results demonstrated efficient CRISPR/Cas9-assisted genome editing in P. ostreatus, which could contribute to the molecular breeding of non-GM cultivated strains in the future. Furthermore, a mutation in fcy1 via homology-directed repair using this CRISPR/Cas9 system was also efficiently introduced, which could be applied not only for precise gene disruption, but also for insertions leading to heterologous gene expression in this fungus.

Comparative transcriptional analyses of Pleurotus ostreatus mutants on beech wood and rice straw shed light on substrate-biased gene regulation.

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.

Targeted disruption of hir1 alters the transcriptional expression pattern of putative lignocellulolytic genes in the white-rot fungus Pleurotus ostreatus.

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 promoter assay system using gene targeting in agaricomycetes Pleurotus ostreatus and Coprinopsis cinerea.

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.