Yingying Wu

Hypsizygus marmoreus (Peck) H.E. Bigelow is a commercial edible mushroom includes two primary commercial varieties: brown and white. To reveal the genetic and metabolic differences between these two varieties, genomic and metabolomic comparisons of the white strain F4 and the brown strain B5-15 were performed. The assembled genome sizes were 40,851,948 bp for F4 and 41,902,673 bp for B5-15. Molecular clock analysis estimated that H. marmoreus diverged from Termitomyces sp. approximately 59.4 million years ago during the Paleocene based on the genomic information. The two genomes showed little difference in the gene compositions related to β-Glucosidase and certain lignin degrading auxiliary enzymes. In contrast, the structures of the mating-type loci, including gene copy numbers and the transcriptional orientation of open reading frames, differed between the varieties, and it exhibited higher mating-type locus diversity. Comparative genomic analysis further indicated that the brown strain can biosynthesize melanin-like compounds using chorismate as the starting molecule, with tyrosinase acting as a key enzyme. Moreover, metabolomic profiling based on principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) revealed distinct metabolic profiles between the two varieties. Collectively, these findings improve our understanding of the genetic basis underlying the phenotypic differences between the two H. marmoreus varieties.

Following the elucidation of the fungal bioluminescence pathway (FBP), it was quickly adopted as a reporter system in plants; however, no such application has been documented in fungi to date. In this study, we established for the first time a luminescent reporter in the commercially important mushroom Hypsizygus marmoreus by expressing the luciferase gene from the luminous fungus Neonothopanus nambi. Using an established Agrobacterium-mediated transformation method, we separately introduced the wild-type luciferase gene nnLuz and the previously reported optimized variant nnLuz-v4 that can enhance bioluminescence expression into H. marmoreus arthroconidia. Both genes were stably integrated into the genome and expressed under the control of the H. marmoreus Glycerol 3-phosphate dehydrogenase (GPD) gene promoter. Upon addition of exogenous luciferin, transformants carrying the wild-type nnLuz produced clear, readily detectable bioluminescence signals, whereas no luminescence was observed in untransformed controls. Unexpectedly, the wild-type luciferase consistently exhibited substantially higher luminescence intensity than the optimized nnLuz-v4 variant. This finding suggests that codon optimization may be unnecessary or even detrimental when the donor and host are phylogenetically close basidiomycetes. The successful deployment of the fungal luciferase gene in H. marmoreus provides a sensitive and non-invasive genetic tool that does not require external excitation. This system opens new avenues for promoter characterization, real-time gene expression monitoring during mushroom development, and molecular breeding efforts aimed at improving agronomically important traits.

Sparassis latifolia is an edible and medicinal mushroom with significant economic value, now commercially cultivated on a large scale in China. However, current cultivars face challenges, including an extended mycelial growth period and unstable fruiting body yields. Advances in molecular breeding and functional genomics for this species are hindered by the absence of a reliable genetic transformation system. In this study, we first determined that S. latifolia is highly sensitive to carboxin and hygromycin, two selective agents commonly used in fungal genetics. We subsequently constructed a novel binary vector, pCbxHyg, harboring a carboxin resistance cassette driven by its native Pleurotus eryngii promoter and a hygromycin resistance cassette under the control of the P. eryngii Glycerol 3-phosphate dehydrogenase (GPD) gene promoter. Initial transformation attempts using Agrobacterium-mediated transformation of liquid-cultured mycelial pellets were unsuccessful. During microscopic examination, we discovered that S. latifolia mycelia produce abundant asexual chlamydospores. Using these chlamydospores as recipient material, we efficiently and reproducibly obtained transformants with the pCbxHyg vector under both carboxin and hygromycin selection. This method highlights the advantage of using asexual spores of Basidiomycetes as recipients for genetic transformation. PCR analysis confirmed the stable integration of the exogenous resistance genes into the fungal genome. The functionality of the system was further validated by transforming chlamydospores with a vector carrying a β-glucuronidase (GUS) reporter gene, whose expression was confirmed via histochemical staining of the resulting transformant mycelia. This work establishes the first successful Agrobacterium-mediated genetic transformation system for S. latifolia, providing a foundational platform for future gene function studies and molecular breeding efforts.

Mitochondria regulate nuclear genomes and their own genetic material, primarily to provide energy in eukaryotes. Currently, high-throughput sequencing technologies are being used to resolve the mitochondrial genomes of various edible fungi. However, the application of pan-genomes for the analysis of edible mushroom mitochondrial genomes remains unexplored. In this study, we conducted a comparative mitochondrial genome analysis of 31 Hypsizygus marmoreus strains (four newly sequenced monotypes and 27 public datasets), ranging from 98,284 to 111,087 bp. This variation was determined to be primarily driven by dynamic changes in non-coding regions, particularly intronic polymorphisms in the cox1 gene. Further, transfer RNA (tRNA) secondary structures exhibited atypical globular and elongated conformations alongside copy number variations. Additionally, codon usage showed a pronounced A/T bias, whereas core respiratory chain genes demonstrated an evolutionary pattern of strong purifying selection. Furthermore, the 31 mitochondrial genomes of H. marmoreus were found to harbor eight gene rearrangement patterns and five genetic clusters, and the pan-genome analysis (220,364 bp, 217 nodes) captured abundant single-nucleotide polymorphisms (SNPs), insertions/deletions (InDels), and structural variations. This study provides breeding-relevant genetic markers and a genomic framework for H. marmoreus germplasm classification, genetic improvements, and the molecular breeding of stress-resilient varieties.

As semi-autonomous organelles, mitochondria function through the coordinated regulation of nuclear genomes and their own genetic material, primarily providing energy for eukaryotic organisms. Currently, high-throughput sequencing technologies have been used to resolve the mitochondrial genomes of various edible fungi. With advances in sequencing technology, species genome characterization has evolved from single genomes to pan-genomes. However, the application of pan-genomes for the analysis of edible mushroom mitochondrial genomes remains unexplored. In this study, we conducted a comparative mitochondrial genome analysis of 31 Hypsizygus marmoreus strains (4 newly sequenced monotypes and 27 public datasets). The results revealed that the mitochondrial genome sizes ranged from 98,284 to 111,087 bp, exhibiting significant structural diversity. This variation is primarily driven by dynamic changes in non-coding regions, particularly intronic polymorphisms in the cox1 gene. This study revealed that tRNA secondary structures exhibit atypical globular and elongated conformations alongside copy number variations. Additionally, codon usage showed a pronounced A/T bias, whereas core respiratory chain genes demonstrated an evolutionary pattern of strong purifying selection. Furthermore, the 31 mitochondrial genomes of H. marmoreus were identified 8 gene rearrangement patterns and 5 genetic clusters, and the pan-genome (220,364 bp, 217 nodes) captured abundant SNPs, InDels and structural variations. This study provides breeding-relevant genetic markers and a genomic framework for germplasm classification, genetic improvement and stress-resilient variety molecular breeding of Hypsizygus marmoreus.

Core germplasm, a strategically selected subset of the original germplasm, aims to maximize the representation of genetic diversity within the entire collection. Establishing a germplasm resource bank is essential for the effective management and sustainable utilization of genetic resources. This study developed a core germplasm repository for Hypsizygus marmoreus, a commercially important mushroom species, to capture the genetic diversity of the original collection with a minimal sample size. Genetic diversity and cluster analyses were conducted on 57 representative strains of H. marmoreus, including both cultivated and wild accessions from different regions, using 15 pairs of simple sequence repeat (SSR) markers. DNA molecular identity cards were generated for all germplasms, and cultivation trials with agronomic trait assessments were performed on 24 core accessions. A total of 115 distinct alleles were identified, with genetic similarity coefficients ranging from 0.70 to 1.00. Clustering at a similarity threshold of 0.76 classified the strains into five groups. The core germplasm panel, comprising 24 accessions (42.11% of the total collection), retained full allelic diversity and preserved the genetic and phenotypic variability of the original population, confirming its suitability for parental selection in breeding programs. unique molecular identity codes were developed for each H. marmoreus germplasm by integrating SSR marker profiles with data on geographical origin, fruiting body color, and cultivation traits. These were converted into DNA molecular ID codes, providing a reliable system for rapid identification and traceability of germplasm resources. The findings offer a valuable reference for breeding improvement and the protection of edible fungal varieties with independent intellectual property rights.

In the original publication [...].

Ganoderma leucocontextum, a newly identified species from the Tibetan Plateau, has been mainly studied for its polysaccharides and triterpenoids, with no prior reports on fungal immunomodulatory proteins (FIPs). This study explores the biological activity of FIP-gle2, cloned from G. leucocontextum and expressed in Pichia pastoris. The effects and mechanisms of recombinant FIP-gle2 (rFIP-gle2) on cell activity and melanin synthesis in mouse melanoma B16-F10 cells were investigated in vitro. The results showed that the FIP-gle2 gene, with an open reading frame (ORF) of 333 bp, encodes a 111-amino acid polypeptide with a molecular weight of 12.60 kDa and an isoelectric point of 4.48. We achieved a yield of 184.18 mg/L of rFIP-gle2. In vitro functional experiments showed that rFIP-gle2 significantly inhibited the proliferation of B16-F10 melanoma cells and induced apoptosis in a dose-dependent manner, particularly at concentrations above 1 μg/mL. At 3 μg/mL, rFIP-gle2 effectively inhibited tyrosinase activity and reduced melanin content, downregulating microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), and tyrosinase-related proteins (TRP-1 and TRP-2). Furthermore, RNA-seq analysis indicated that differentially expressed genes in treated cells were enriched in the mitogen-activated protein kinase (MAPK) signaling pathway, with Western blotting confirming enhanced phosphorylation of JNK, ERK, and p38 proteins. Thus, P. pastoris is an effective host for rFIP-gle2 production, which shows potential for applications in pharmaceuticals, cosmeceuticals, and food fields.

Pleurotus eryngii is a tasty and low-calorie mushroom containing abundant high-quality protein. This study aims to improve the digestibility of P. eryngii protein (PEP) and hence to facilitate its development as a healthy alternative protein. The extracted PEP was pretreated with 1000–5000 U of papain, neutral protease and alkaline protease. The Chyme collected from in vitro simulated gastrointestinal digestion was analyzed by fluorescence microscopy and protein particle analyzer, and the endpoint profiles of peptides and amino acids were determined by UHPLC-MS/MS and NanoLC-MS/MS. The particle size curve and fluorescence microscopy images jointly supported that protease hydrolysis improved decomposition and dispersion of PEP during digestion, particularly in the gastric phase. The impact on Zeta potential was minimal. Proteases effectively increased the abundance of amino acids after digestion, particularly L-isomer Lys and Arg Maximum release was achieved when pretreated with 5000 U of alkaline protease, reaching 7.54 times that of control. Pretreatments by proteases also notably increased digestive yields of 16,736–19,870 peptides, with the maximum reaching 1.70 times that of the control, which mainly consisted of small peptides composed of 7–15 amino acids with molecular weight below 800 Da. The findings indicated that protease hydrolysis, especially pretreatment with 5000 U of alkaline protease, effectively enhanced the digestibility of PEP, which shed light on providing enzymatic approaches for improving bioavailability and developing healthy fungal proteins.

Flammulina filiformis is one of the widely produced edible fungi worldwide. It is rich in γ-aminobutyric acid (GABA), a non-protein amino acid with important physiological functions in humans. To investigate the functions of key genes in the GABA metabolic pathway of F. filiformis, we isolated the monokaryon Fv-HL23-1 from the factory-cultivated F. filiformis strain Fv-HL23 and then sequenced and assembled the genome using the PacBio Sequel and Illumina NovaSeq sequencing platforms. The results showed that the genome comprised 140 scaffolds with a total length of 40.96 Mb, a GC content of 49.62%, an N50 of 917,125 bp, and 14,256 protein-coding genes. Phylogenetic analysis based on the whole genome revealed a close evolutionary relationship of Fv-HL23-1 with Armillaria mellea, Lentinula edodes, and Schizophyllum commune. A total of 589 carbohydrate-active enzymes were identified in the genome of Fv-HL23-1, suggesting its strong lignocellulose degradation ability, and 108 CYP450 gene family members were identified, suggesting important functions such as resistance to stress, secondary metabolite synthesis, and growth and development. The F. filiformis proteins glutamate decarboxylase 1 (Ff-GAD1) and glutamate decarboxylase 2 (Ff-GAD2), which may be responsible for GABA synthesis, were identified by protein alignment. Molecular docking analysis showed that Ff-GAD2 may have better catalytic activity than Ff-GAD1. To verify the function of Ff-gad2, its heterologous expression in the mycelia of the mononuclear Hypsizigus marmoreus was analyzed. Compared with wild type, the GABA content of mycelia was increased by 85.40–283.90%, the growth rate was increased by 9.39 ± 2.35%, and the fresh weight was increased by 18.44 ± 7.57%. Ff-GAD2 may play a catalytic role in GABA synthesis. In addition, the expression of the full-length Ff-gad2 gene was increased by 7.96 ± 1.39 times compared with the exon expression level in H. marmoreus mycelia, suggesting that the intron may contribute to the heterologous expression of Ff-GAD2. Based on whole-genome sequencing, we analyzed the enzyme system related to the important life activities of F. filiformis, focusing on the function of Ff-GAD, a key enzyme in the GABA synthesis pathway. The results lay a foundation for elucidating the GABA metabolism pathway of edible fungi and developing targeted breeding strategies for GABA-producing edible fungi.