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Currently, heavy metal pollution has emerged as a global issue. Compared with green plants, edible fungi, significant crops cultivated worldwide, present a greater capacity to accumulate heavy metals (HMs). However, the enrichment characteristics and functions of heavy metal transporters (HMATs) in the accumulation of HMs in edible fungi are still unclear. Cadmium (Cd) and mercury (Hg) are the primary HMs enriched in edible fungi. This study focused on Pleurotus ostreatus, the second largest edible mushroom worldwide, to examine the enrichment process. In this study, a series of different concentrations of CdCl 2 and HgCl 2 (0, 0.01, 0.05, 0.5, 2, 5, 10, and 20 mg/L) were used to mimic HMs pollution. HMs in the experimental concentration range did not affect the mycelial growth rate or fruiting body yield of P. ostreatus. However, in the 20 mg/L treatment group, the HMs were mainly concentrated in the cap, with about 4.4 mg/kg Cd and 2.7 mg/kg Hg, and were predominantly present in the most toxic ion exchange state. Thirteen HMATs were identified in the genome database of P. ostreatus. Using RT-qPCR, seven HMATs (24093, 1066001, 1106787, 1066344, 1079972, 1095088, and 1104877) whose expression levels were more than twice that of the control under most concentrations of HMs were selected for further investigation of their transport functions and their involvement in signal regulation. Among them, gene 24093 was involved in the absorption of Cd and Hg. These transporters are regulated by ROS, Ca 2+ , and NO signals under HM stress. This study provides target genes for reducing the risk of HM accumulation through molecular means, and serves as a reference for HM remediation using edible fungi., Competing Interests: Declaration of competing interest There is no conflict of interest between the authors., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pleurotus ostreatus is a popular edible mushroom cultivated worldwide. However, the mechanism of P. ostreatus primordia formation is unclear. Pofst3 is a MHR superfamily transcription factor, which has the function of regulating primordia formation. In this study, the target genes of Pofst3 in P. ostreatus were identified by DAP-Seq approach at the genome level, 1481 peaks were obtained and the Pofst3 binding motif sequence was GARGRVGARGAR. The interaction between transcription factor Pofst3 and this motif was confirmed in vitro and in vivo through electrophoretic mobility shift (EMSA) and yeast one-hybrid screening (Y1H) assays. Among the top 20 GO enrichment results, most were related to transcriptional regulation, and some transcription factor encoding genes, such as HMG-box (gene_5346), MADS-box (gene_86), FOG (gene_6211) and RFX (gene_3496) were obtained. Besides basic metabolism, MAPK signaling pathway, Inositol phosphate metabolism, Glycosylphosphatidylinositol (GPI)-anchor biosynthesis and Pentose phosphate pathway were significantly enriched in the KEGG pathway analysis. The expression levels of randomly selected 11 genes, some transcription factor genes, and genes involved in metabolic pathways in wild and Pofst3 transgenic P. ostreatus strains indicated that target genes likely involved in the development of the P. ostreatus primordia. These results indicated that transcription factor Pofst3 ultimately negatively regulated the development of P. ostreatus primordia very likely through regulating a series of biological pathways., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest that could have appeared to influence the work reported in this paper., (Copyright © 2024 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

High-temperature or heat stress (HS) represents a significant environmental challenge that adversely affects crop growth and poses a substantial threat to agricultural production. Pleurotus ostreatus , recognized as the second most widely cultivated edible fungus worldwide, is particularly susceptible to the detrimental effects of HS. Enhancing the HS resistance of P. ostreatus is crucial for increasing its yield. In a prior investigation, we discovered that salicylic acid (SA) enhanced the resistance of P. ostreatus mycelia to HS through a metabolic rearrangement. The present study further investigated the effects of SA on P. ostreatus under HS. Cultivation experiments revealed that exogenous SA improved the mycelium recovery growth rate, yield, and fruiting body quality after HS. Further experiments revealed that exogenous SA mitigated the damage to the MAPK-Slt2 signal produced by HS while maintaining cell wall integrity. Furthermore, we hypothesized that the phenylalanine ammonia-lyase pathway might serve as a source for SA. In this context, we identified two salicylic hydroxylases, Po1102164 and Po1104438. Both HS and exogenous SA were found to elevate intracellular SA levels, thereby enhancing the resistance of P. ostreatus to HS.

Pleurotus ostreatus is one of the most cultivated edible fungi worldwide, but its lignocellulose utilization efficiency is relatively low (<50 %), which eventually affects the biological efficiency of P. ostreatus. Improving cellulase production and activity will contribute to enhancing the lignocellulose-degrading capacity of P. ostreatus. AMP-activated/Snf1 protein kinase plays important roles in regulating carbon and energy metabolism. The Snf1 homolog (PoSnf1) in P. ostreatus was obtained and analyzed using bioinformatics. The cellulose response of PoSnf1, the effect of the phosphorylation level of PoSnf1 on the expression of cellulose degradation-related genes, the putative proteins that interact with the phosphorylated PoSnf1 (P-PoSnf1), the cellobiose transport function of two sugar transporters (STP1 and STP2), and the interactions between PoSnf1 and STP1/STP2 were studied in this research. We found that cellulose treatment improved the phosphorylation level of PoSnf1, which further affected cellulase activity and the expression of most cellulose degradation-related genes. A total of 1, 024 proteins putatively interacting with P-PoSnf1 were identified, and they were enriched mainly in the substances transport and metabolism. Most of the putative cellulose degradation-related protein-coding genes could respond to cellulose. Among the P-PoSnf1-interacting proteins, the functions of two sugar transporters (STP1 and STP2) were further studied, and the results showed that both could transport cellobiose and were indirectly regulated by P-PoSnf1, and that STP2 could directly interact with PoSnf1. The results of this study indicated that PoSnf1 plays an important role in regulating the expression of cellulose degradation genes possibly by affecting cellobiose transport., Competing Interests: Declaration of competing interest There is no conflict of interest between the authors., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

GABA is a health-promoting bioactive substance. Here, the GABA biosynthetic pathways were investigated, and then the dynamic quantitative changes in GABA and the expression levels of genes related to GABA metabolism under heat stress or at different developmental stages of fruiting bodies in Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm were determined. We found that the polyamine degradation pathway was the main route of GABA production under growth normal condition. The accumulation of GABA and the expression of most genes related to GABA biosynthesis, including genes encoding glutamate decarboxylase (PoGAD-2), polyamine oxidase (PoPAO-1), diamine oxidase (PoDAO) and aminoaldehyde dehydrogenase (PoAMADH-1 and PoAMADH-2), were significantly suppressed by heat stress and the excessive maturity of fruiting bodies. Finally, the effects of GABA on the mycelial growth, heat tolerance and the morphogenesis and development of fruiting bodies were studied, the results showed that the deficiency of endogenous GABA inhibited the mycelial growth and primordial formation and aggravated heat damage, whereas exogenous application of GABA could improve thermotolerance and promote the development of fruiting bodies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Primordia formation is the first and most critical step in the development of fruiting bodies of edible fungi. In this study, the effects of exogenous ascorbic acid (ASA) on the Pleurotus ostreatus mycelia growth and primordia formation were researched and the results showed that the growth rate of P. ostreatus mycelia was accelerated and the time of primordia formation was advanced. The protein content and ascorbate oxidase (AAO) activity analysis showed that with the increase of ASA concentration, the protein content of mycelia first decreased and then increased, and in a certain concentration range, exogenous ASA could significantly promote the activity of AAO. Further expression analysis of the development regulating genes (Pofst3 and Pofst4) as well as blue light receptor coding genes (PoWC-1 and PoWC-2) showed the expression levels of those four genes all changed after the exogenous ASA addition, which indicated that the expression changes of PoWC-1 and PoWC-2, two key genes in the light morphogenesis, might affect the expression levels of development regulating genes Pofst3 and Pofst4, so as to lead to the formation of primordia in advance., (© 2021 Wiley-VCH GmbH.)

Blue light is necessary for initiation of mushroom formation of Pleurotus ostreatus. In this study, we isolated homologues of the blue light receptor genes wc-1 and wc-2 from P. ostreatus, PoWC-1 and PoWC-2. The PoWC-1 contained three typical PAS domains and one PAS domain exhibited significant similarity to the LOV domain of known blue light receptors. The PoWC-2 had one typical PAS domain and one ZnF domain. The qRT-PCR analysis showed that PoWC-1 and PoWC-2 expression increased in a short time, and the final level tended to be stable along with the light illumination. The PoWC-1 and PoWC-2 expression levels of the primordium period was higher than that of mature fruiting-body period; and in the pileus were the highest, followed by the stipe and the gills. The expression of PoWC-1 and PoWC-2 in pre-primordial mycelia was induced by light exposure. In vivo analysis through yeast two-hybrid experiment disclosed that PoWC-1 and PoWC-2 could form heterologous complex to activate the reporter genes and the complex perform the transcription factor function requiring the addition of FAD., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Only a few transcription factors involved in mushroom development have been reported till now, Therefore, identification of transcription factors in common edible mushroom has commercial and scientific importance. In this study, the Pofst3 gene from Pleurotus ostreatus was cloned and characterized. Bioinformatics analysis showed that Pofst3 protein had 71% sequence similarity with fst3 of model mushroom Schizophyllum commune. Furthermore, the function of Pofst3 gene was analyzed by overexpression and antisense silencing in P. ostreatus via Agrobacterium-mediated transformation. Expression verification of Pofst3 in transformants through qRT-PCR showed that compared with the wild type strains, the transcription level was about 1.26 ∼ 9.59 and 0.01 ∼ 0.30 fold in Pofst3 overexpressing and silencing strains, respectively. Petri dish and bag cultivation tests of transgenic strains showed that the number of primordia and the type of fruiting bodies of Pofst3 overexpressing strains were consistent with the wild type strains, i.e. fewer primordia and larger fruiting bodies; the number of primordia formed by Pofst3 silencing strains were more than those of wild type strains, but fruiting bodies were smaller. It was very likely that Pofst3 was involved in the regulation of P. ostreatus development through inhibiting the formation of clusters of primordia., (Copyright © 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

In order to isolate the differentially expressed genes in the primordium stage of Pleurotus ostreatus, the SSH cDNA library was constructed using the cDNA from dikaryotic mycelium stage as a driver and the cDNA from primordium stage as a tester. There were 423 significantly differently expressed clones among 2055 positive clones after three times of reverse Northern blot differential screening. After the repeated sequences being removed, 46 genes were identified which were putatively involved in cell rescue and defense, energy metabolism, transcription and protein regulation, membrane proteins, and signal transduction; 18 genes encoding hypothetical proteins with unknown function; 5 genes without any homology. PoALDH1 and its full-length cDNA sequence were cloned using the Aldehyde dehydrogenase EST isolated from the library. The amino acid sequence of PoALDH1 contains conservative glutamic acid and cysteine residues active sites of aldehyde dehydrogenase family. When exposed to different concentrations of sodium chloride, the mycelium growth was inhibited and the expression level of PoALDH1 was significantly higher than that of the control one, which indicated that PoALDH1 may have the ability to relieve salt stress. The results of this study will provide useful information for isolating growth and development related genes of P. ostreatus., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pleurotus ostreatus laccase gene poxc is transcriptionally induced by copper, and several putative MREs have been found and confirmed in its promoter region. However, the related transcription factors mediating copper response via MREs have not been reported. To isolate MRE binding proteins, we carried out yeast one-hybrid screens. Reporter genes containing two trimers of the cMRE2 and cMRE3 element were prepared and introduced into a yeast strain. The yeast was transformed with library cDNA that represents RNA isolated from CuSO 4 -treated fungi of P. ostreatus. From the screen of yeast transformants, we isolated ltf4 which encoded protein with HTH DNA binding domain. Electrophoretic mobility shift assay suggested direct and specific interaction of ltf4 with the MRE2 of poxc. Quantitative RT-PCR showed that the transcription of ltf4 was significantly up-regulated after the copper addition, and the expression trend was consistent with the poxc after copper introduction. The results indicated that ltf4 could interact with cMRE2 and thus participated in the regulation of copper mediated poxc transcription in P. ostreatus., (Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Dikarya is a subkingdom of fungi that includes Ascomycota and Basidiomycota. The gene expression patterns of dikaryon are poorly understood. In this study, we bred a dikaryon DK13×3 by mating monokaryons MK13 and MK3, which were from the basidiospores of Pleurotus ostreatus TD300. Using RNA-Seq, we obtained the transcriptomes of the three strains. We found that the total transcript numbers in the transcriptomes of the three strains were all more than ten thousand, and the expression profile in DK13×3 was more similar to MK13 than MK3. However, the genes involved in macromolecule utilization, cellular material synthesis, stress-resistance and signal transduction were much more up-regulated in the dikaryon than its constituent monokaryons. All possible modes of differential gene expression, when compared to constituent monokaryons, including the presence/absence variation, and additivity/nonadditivity gene expression in the dikaryon may contribute to heterosis. By sequencing the urease gene poure sequences and mRNA sequences, we identified the monoallelic expression of the poure gene in the dikaryon, and its transcript was from the parental monokaryon MK13. Furthermore, we discovered RNA editing in the poure gene mRNA of the three strains. These results suggest that the gene expression patterns in dikaryons should be similar to that of diploids during vegetative growth., (Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ostreatus. The promoter for β-1,3-glucan synthase gene (GLS) was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR), and was introduced into P. ostreatus through PEG/CaCl2-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of β-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of β-glucans. This is the first report of successful swapping of promoters in filamentous fungi.

Three hygromycin B phosphotransferase (hph) gene expression systems for culinary-medicinal Oyster mushroom, Pleurotus ostreatus, plasmid pSHC, pAN7-1, and pBHt1 were evaluated through PEG/CaCl(2)-mediated protoplast transformation. Plasmid pSHC is a newly constructed hph gene expression system, composed of Escherichia coli hph gene, the P. ostreatus sdi promoter, and the CaMV35S terminator. The vector pAN7-1 was commonly used for integrative transformation in filamentous fungi. Plasmid pBHtl is a T-DNA binary vector, usually introduced into fungi by Agrobacterium-mediated transformation. The results showed that plasmids pSHC, pAN7-1, and pBHt1 were all integrated into the host chromosomes and expressed hygromycin B resistance in P. ostreatus. pAN7-1 had the highest transformation efficiency and hph gene expression level, pSHC the second, and pBHt1 the lowest. Growth rates of the transformants on plates containing hygromycin B were in correspondence with their hph gene expression levels. To our knowledge, this is the first report on integrated transformation of plasmid pAN7-1 and pBHt1 in P. ostreatus.

Many cultivated mushroom strains, such as Pleurotus ostreatus TD300, displayed symptoms of degeneration. A spherical virus POSV and four dsRNA segments were extracted from mycelium of P. ostreatus TD300. POSV had a diameter of 23 nm and encapsidated a 2.5kb dsRNA segment with coat proteins whose molecular weights were 39 kDa and 30 kDa. Four dsRNA segments were 8.2 kb, 2.5 kb, 2.0 kb, and 1.1 kb in size, respectively. The 1.1 kb dsRNA segment often escaped detection. The cDNA and the amino acid sequences of the 8.2 kb dsRNA were homologous to those of RNA-dependent RNA polymerases (RDRP) of ssRNA oyster mushroom spherical virus (OMSV), and contained conserved motifs A to D which were almost identical to those in RDRP of OMSV. The cDNA and amino acid sequences of the 2.5 kb and 2.0 kb dsRNA segments were homologous to that of RDRP and capsid protein of dsRNA virus P. ostreatus virus 1 (PoV1), respectively. In particular, the amino acid sequence of 2.5 kb dsRNA segment had high identity with the conserved motifs A to C in RDRP of PoV1, a Partiviridae virus. After eliminating the viruses in P. ostreatus TD300, the symptoms of degeneration completely disappeared. The results reveal that P. ostreatus TD300 was at least infected by a particle virus POSV, and two naked viruses, one was a dsRNA virus with a 2.0 kb dsRNA segment, the other was an ssRNA virus whose replicating form of genome was an 8.2 kb dsRNA segment. Mycoviruses infection is a causative agent of mushroom strain degeneration., (Copyright © 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)