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7 recordsLinked to original sources
PubMed2025

Early germline sequestration in a basidiomycete fungus.

In sexual organisms, inheritance of new mutations is highly dependent on the timing of germline definition. Here, we used the fairy ring-forming fungus Marasmius oreades to challenge the general assumption of a late germline separation in the Fungi. We collected mushrooms from different parts of rings over a 7-year period and identified new mutations in different tissues by whole-genome sequencing. We found evidence that fertile and sterile tissues had accumulated different mutations, suggesting that the germ line, destined for spore production, is already defined in the mycelium in this species. Moreover, the germ line carried fewer mutations than sterile tissues, indicating a lower mutation rate. Our findings suggest that early germline sequestration is more widespread than previously considered across multicellular life.

Genome, Fungal↗
PubMed2026

The novel transcriptional activator Bhr1 combining NTPase and Zn(II)2Cys6 DNA-binding domains controls (hemi-)cellulase response to mannose-rich substrates in the white-rot fungus Dichomitus squalens.

The regulatory landscape responsible for lignocellulose degradation in white-rot basidiomycete fungi remains largely unexplored. In this study, we characterize a novel transcriptional activator, Bhr1, in the white-rot fungus Dichomitus squalens. Bhr1 exhibits an unusual domain architecture that combines a septin-like P-loop NTPase fold with Zn(II)2Cys6 DNA-binding domains and plays a critical role in activating (hemi-)cellulase enzyme production when D. squalens is exposed to mannose-rich substrates. Using CRISPR/Cas9-mediated gene editing, we generated a bhr1 disruption mutant that displayed distinct phenotypes and enzyme activity profiles on mannose and guar gum compared to the wild type. RNA sequencing data indicate that Bhr1 induces specific (hemi-)cellulase-encoding genes without altering the expression of genes encoding sugar transporters or sugar metabolic enzymes. Phylogenetic analyses show that Bhr1 is basidiomycete specific and largely restricted to saprotrophic and plant-associated Agaricomycetes fungi. Based on the domain architecture of Bhr1 and the effects of its disruption in D. squalens, our findings reveal a lineage-specific regulatory innovation in basidiomycetes that is distinct from those described in ascomycetes. Elucidating the function and evolutionary conservation of Bhr1 advances our understanding of lignocellulose degradation at the molecular level in basidiomycete fungi and may inform studies of their ecological adaptation and the development of biotechnological applications.IMPORTANCEUnderstanding the transcriptional regulatory mechanisms in white-rot fungi, such as Dichomitus squalens, is crucial for advancing our knowledge of lignocellulose degradation. This study identifies D. squalens Bhr1 as a key regulator of (hemi-)cellulase production on mannose-rich substrates and further distinguishes basidiomycete transcription factors involved in plant biomass degradation from their ascomycete counterparts. Our findings highlight the significance of lineage-specific regulators in facilitating adaptive enzyme production for efficient biomass utilization, which is critical to carbon cycling in terrestrial ecosystems. This work establishes a foundation for exploring novel regulatory strategies among wood-degrading fungi, potentially enabling targeted strain engineering in biotechnological applications.

Mannose↗
PubMed2025

Tracing the evolution and genomic dynamics of mating-type loci in Cryptococcus pathogens and closely related species.

Sexual reproduction in basidiomycete fungi is governed by MAT loci (P/R and HD), which exhibit remarkable evolutionary plasticity, characterized by expansions, rearrangements, and gene losses often associated with mating system transitions. The sister genera Cryptococcus and Kwoniella provide a powerful framework for studying MAT loci evolution owing to their diverse reproductive strategies and distinct architectures, spanning bipolar and tetrapolar systems with either linked or unlinked MAT loci. Building on recent comparative genomic analyses, we generated additional chromosome-level assemblies, uncovering distinct trajectories shaping MAT loci organization. Contrasting with the small-scale expansions and gene acquisitions observed in Kwoniella, our analyses revealed independent expansions of the P/R locus in tetrapolar Cryptococcus, possibly driven by pheromone gene duplications. Notably, these expansions coincided with a pronounced GC-content reduction best explained by reduced GC-biased gene conversion following recombination suppression, rather than relaxed codon usage selection. Diverse modes of MAT locus linkage were also identified, including three previously unrecognized transitions: one resulting in a pseudobipolar arrangement and two leading to bipolarity. All three transitions involved translocations. In the pseudobipolar configuration, the P/R and HD loci remained on the same chromosome but genetically unlinked, whereas the bipolar transitions additionally featured rearrangements that fused the two loci into a nonrecombining region. Mating assays confirmed a sexual cycle in C. decagattii, demonstrating its ability to undergo mating and sporulation. Progeny analysis in K. mangrovensis revealed substantial ploidy variation and aneuploidy, likely stemming from haploid-diploid mating, yet evidence of recombination and loss of heterozygosity indicates that meiotic exchange occurs despite irregular chromosome segregation. Our findings underscore the importance of continued diversity sampling and provide further evidence for convergent evolution of fused MAT loci in basidiomycetes, offering new insights into the genetic and chromosomal changes driving reproductive transitions.

MAT genes↗
PubMed2025

The complex evolution and genomic dynamics of mating-type loci in Cryptococcus and Kwoniella.

Sexual reproduction in basidiomycete fungi is governed by MAT loci (P/R and HD), which exhibit remarkable evolutionary plasticity, characterized by expansions, rearrangements, and gene losses often associated with mating system transitions. The sister genera Cryptococcus and Kwoniella provide a powerful framework for studying MAT loci evolution owing to their diverse reproductive strategies and distinct architectures, spanning bipolar and tetrapolar systems with either linked or unlinked MAT loci. Building on recent comparative genomic analyses, we generated additional chromosome-level assemblies, uncovering distinct trajectories shaping MAT loci organization. Contrasting with the small-scale expansions and gene acquisitions observed in Kwoniella, our analyses revealed independent expansions of the P/R locus in tetrapolar Cryptococcus, possibly driven by pheromone gene duplications. Notably, these expansions coincided with a pronounced GC-content reduction best explained by reduced GC-biased gene conversion following recombination suppression, rather than relaxed codon usage selection. Diverse modes of MAT locus linkage were also identified, including three previously unrecognized transitions: one resulting in a pseudobipolar arrangement and two leading to bipolarity. All three transitions involved translocations. In the pseudobipolar configuration, the P/R and HD loci remained on the same chromosome but genetically unlinked, whereas the bipolar transitions additionally featured rearrangements that fused the two loci into a nonrecombining region. Mating assays confirmed a sexual cycle in Cryptococcus decagattii, demonstrating its ability to undergo mating and sporulation. Progeny analysis in Kwoniella mangrovensis revealed substantial ploidy variation and aneuploidy, likely stemming from haploid-diploid mating, yet evidence of recombination and loss of heterozygosity indicates that meiotic exchange occurs despite irregular chromosome segregation. Our findings underscore the importance of continued diversity sampling and provide further evidence for convergent evolution of fused MAT loci in basidiomycetes, offering new insights into the genetic and chromosomal changes driving reproductive transitions.

Genes, Mating Type, Fungal↗
PubMed2026

Liquid fermentation and an adapted cetyltrimethylammonium bromide (CTAB) method enable sequencing-grade DNA extraction from Phanerodontia australis.

High-quality genomic DNA extraction from basidiomycete fungi remains challenging due to polysaccharide co-purification and nuclease-mediated DNA degradation. We systematically compared 22 experimental conditions combining five DNA extraction methods (one Dellaporta-based method, three CTAB-based variants, and one commercial kit) with five mycelium preparation techniques (scalpel scraping, fungal suspension, silica bead homogenization, liquid fermentation, and liquid nitrogen treatment) for Phanerodontia australis (BRM62389). DNA quality was assessed by 1% agarose gel electrophoresis, NanoDrop spectrophotometry, and Qubit fluorimetry. The optimal protocol combined liquid-fermentation mycelium with an adapted CTAB method, yielding Qubit-quantified concentrations of 34.2 and 62.6 ng/µL (samples S21 and S22), 260/280 ratios of 2.06 and 2.05, 260/230 ratios of 1.88 and 1.85, and no detectable DNA degradation by agarose gel electrophoresis. This protocol enabled whole-genome sequencing, yielding a 37.62 Mb assembly with 98.5% completeness as assessed by BUSCO v5 (basidiomycota_odb10 lineage dataset, n = 1,764). Liquid fermentation is associated with the production of younger, actively growing hyphae with reduced cell wall thickness, and the adapted CTAB method effectively removed residual polysaccharides. This protocol provides a reliable, cost-effective approach for obtaining sequencing-grade DNA from P. australis.

Basidiomycota↗
PubMed2025

CRISPR/Cas9-compatible plasmids enabling seven dominant genetic selection methods for the human fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is the most common cause of human fungal meningitis and an important model system for studying fundamental eukaryotic biology. Genetic manipulation of this organism relies on three dominant drug resistance markers (nourseothricin acetyltransferase [NAT], neomycin phosphotransferase II [NEO], and hygromycin B phosphotransferase [HYG]) and the recyclable dominant prototrophic marker amdS. With ongoing technological advances that are expanding our ability to explore cryptococcal gene function, contemporary studies often require multiple genetic manipulations in the same strain. Additional dominant selection methods would maximize the utility of these tools by facilitating their combinatorial use. Here, we identify blasticidin S resistance via the blasticidin S deaminase (BSD) or blasticidin S resistance (BSR) markers as a novel dominant selection method for C. neoformans. We further validate phleomycin resistance via the bleomycin resistance gene (BLE) marker as an additional selection method, confirming a study that first established this marker 25 years ago (J. Hua, J. D. Meyer, and J. K. Lodge, Clin Diagn Lab Immunol 7:125-128, 2000, https://doi.org/10.1128/cdli.7.1.125-128.2000). To enable highly efficient CRISPR/Cas9-mediated genome modification, we incorporated these markers, as well as the newly established dominant prototrophic marker ptxD (M. Khongthongdam, T. Phetruen, and S. Chanarat, Microbiol Spectr 13:e01618-24, 2025, https://doi.org/10.1128/spectrum.01618-24), into a vector series that enables the construction of fused marker-sgRNA products via PCR. Altogether, this work expands the number of dominant genetic selection methods for C. neoformans to seven, including five drug selection regimes and two prototrophic methods. The vector series has been deposited at Addgene. IMPORTANCE Cryptococcus neoformans is the top-ranked World Health Organization priority fungal pathogen due to its widespread distribution and inadequate treatment options. Additionally, as a basidiomycete yeast occupying an underexplored branch of the fungal kingdom, this organism is a powerful system for deciphering core eukaryotic biology that is absent in classic model fungi. Defining functions for novel cryptococcal genes is a crucial priority, and the availability of additional genetic selection methods would facilitate these efforts. In this study, we establish blasticidin S resistance as a novel genetic selection method for C. neoformans, and we validate a previous report using phleomycin resistance as such. This work expands the number of reliable dominant selection methods to seven, providing flexibility for the introduction of sequential genetic modifications into single strains.

Cryptococcus neoformans↗
PubMed2025

CRISPR/Cas9-directed disruption of wc-2 leads to the absence of fruiting body development in Pleurotus ostreatus.

Light, particularly blue light, is a key environmental factor that induces fruiting in certain agaricomycetes. In this study, we characterized mutant strains of Pleurotus ostreatus with disrupted wc-2, which encodes one of the white-collar proteins, Wc-2, to investigate the role of light in fruiting in P. ostreatus. We introduced two different plasmids containing expression cassettes for Cas9 and two different gRNAs targeting wc-2 separately into the dikaryotic P. ostreatus strain PC9×#64. Among the 11 dikaryotic hygromycin-resistant transformants, six strains did not form fruiting bodies. Genomic PCR followed by sequencing analysis suggested that all six fruitless strains were dikaryotic wc-2 disruptants. Small aggregate structures were not observed in the dikaryotic wc-2 disruptants grown under light conditions, as in PC9×#64 grown in a red box. These results suggest that Wc-2 is essential for the initiation of blue light-induced fruiting in P. ostreatus.

Pleurotus↗