Frontiers in Fungal Biology

Fungal genomics has expanded rapidly over the past 30 years, and recently the pace and breath has further quickened for many taxa, although many taxonomic gaps persist. With three decades of rapid growth, fungal genomics now merits a re-examination of its history, progress, and unresolved taxonomic gaps. Here, we review the development of fungal genomics from early efforts such as the Fungal Genome Initiative to current progress driven by third-generation long-read sequencing. We have compiled and summarised publicly available fungal genomes to highlight trends in assembly quality, adoption of long-read technologies, and taxonomic representation. Notably, substantial phylogenetic gaps remain, particularly outside Dikarya, and significant challenges persist for unculturable taxa. This review identifies priorities for the fungal community, including: (1) coordinated efforts to close major taxonomic gaps across the fungal tree of life; (2) improved repository metrics to facilitate identification of high-quality assemblies; and (3) improved and standardised genome annotation which is lacking for most assemblies. Together, these steps will support the development of reliable genomic resources that capture the full breadth of diversity across the fungal kingdom, generating foundational data for comparative genomics, evolutionary biology, functional studies, genetic studies and applied research.

Nakaseomyces glabratus is a globally distributed opportunistic fungal pathogen. An ongoing discussion in studies of N. glabratus population structure has been whether genetic clusters are best defined using multilocus sequence typing (MLST) or short-read whole-genome sequencing (WGS). To assess the concordance between MLST- and WGS-based phylogenies, we analyzed a dataset of 548 N. glabratus WGS sequences from 12 countries. Clusters identified from WGS largely recapitulated the MLST-defined sequence type (ST) groups: fourteen WGS clusters were composed of a single MLST ST, and the remaining contained STs with very closely related MLST profiles. We thus propose a pragmatic naming convention, consistent with the system used in other microbial species, which specifies WGS cluster labels based on the primary ST. From the large WGS isolate dataset, we determined the prevalence of admixture and genomic variants. Interestingly, seven of the nine singleton isolates were admixed, in addition to 58 isolates from six different clusters. Aneuploidy was detected in 4% of isolates, most commonly in chrE, which contains ERG11, the gene encoding the enzyme targeted by azole antifungals. Aneuploid chromosomes did not exhibit elevated heterozygosity relative to the sequencing error rate, consistent with instability of extra chromosome copies. Copy number variants were found in 3% of the isolates; some of the CNVs co-occurred with aneuploidies, and were primarily identified on chrD, chrE, chrI, and chrM. Our findings demonstrate that deep splits between clusters preserve the utility of MLST ST designations for clade-level designation, yet underscore the utility of WGS for high-resolution genomic analyses. Article SummaryThere is an ongoing debate in studies on Nakaseomyces glabratus about whether traditional MLST analysis is sufficient to determine population structure, or whether the precision of whole genome sequencing (WGS) is necessary. We analyzed WGS data from 548 isolates from around the world. We found a very strong agreement between the two methods. We propose a hybrid naming system, where cluster names are based on the dominant MLST group. We used the WGS data to show that admixed isolates, and those with extra chromosomes or CNVs are rare (<7% of isolates in each class) and are distributed throughout the phylogeny.

The fungal cell wall is populated by proteins (CWPs), mostly uncharacterized, that show an atypical evolutionary behavior. Most CWPs are glycosylphosphatidylinositol(GPI)-proteins, followed by proteins with internal repeats (PIR), and non-covalently attached proteins that harbor carbohydrate binding domains (CBM). Several structural CWPs are initially bound to the same wall carbohydrates, but either covalently or non-covalently. However, it is not clear whether they work in the same way and if they are subjected to the same evolutionary constraints. In Neurospora crassa, CWPs ACW-1 (NCU08936) and NCW-3 (NCU07817) bind to {beta}-1,3-glucans through a GPI anchor or a predicted CBM-52 domain, respectively. Here, the evolutionary trajectories and functional roles of both CWPs were analyzed. Both proteins localized primarily to distal septa and hyphal wall surfaces. Morphological characterization and stress cell wall assays suggested that both proteins contribute to cell wall integrity, but NCW-3 likely plays a more prominent role. ACW-1 and NCW-3 homologues were predominantly identified in Ascomycota. ACW-1 displayed a broader distribution than NCW-3, whose homologues were largely restricted to Sordariales. Despite these differences, both protein families exhibited similar moderate global conservation and signatures of purifying selection within shared taxa. Nevertheless, a divergence gradient was identified within ACW-1, related to its tandem leucine-rich repeat (LRR) regions. A similar local accumulation of evolutionary change was not observed within NCW-3. These findings suggested that distinct CWP architectures can accommodate different patterns of sequence diversification despite sharing similar global evolutionary change.

Fungi play central roles in terrestrial ecosystem functioning and are the major decomposers of dead wood in forest systems. Wood decay fungi are adapted to growth and decay under different environmental conditions, but we have limited insight into the intraspecific variability in fungal growth and decay. In Fennoscandia, there are two genetically and ecologically distinct ecotypes of the wood-decay fungus Meruliopsis taxicola: a northern Continental ecotype associated with Norway spruce (Picea abies) growing in moist old-growth forests, and a southern Coastal ecotype growing on Scots pine (Pinus sylvestris) in harsher habitats. The two ecotypes hybridize in a narrow contact zone running through Fennoscandia. Here, we investigate the level of adaptation the two ecotypes show in phenotypic traits, and how hybrid isolates perform as compared with the parental genotypes. We performed in vitro experiments to quantify mycelial growth rate under varying temperature and drought conditions, as well as decomposition of the two substrates, Scots pine and Norway spruce. Isolates of the Continental ecotype exhibited generally higher growth rates in all environments and caused higher mass loss of both substrates. This is consistent with a more competitive life history strategy in the Continental ecotype, whereas Coastal isolates showed adaptions indicative of greater stress tolerance. Hybrid isolates displayed largely intermediate growth responses relative to the parental ecotypes. Together, these results reveal clear phenotypic divergence between M. taxicola ecotypes consistent with contrasting life-history strategies.

Successful colonization of the wheat apoplast requires that Zymoseptoria tritici tolerate host-derived stresses, but the mechanisms underlying this adaptation remain poorly understood. We combined phenotypic assays, transcriptomics, and genome-wide association analyses to characterize fungal responses to acidic pH, salicylic acid, gibberellic acid, and oxidative stress. Exposure to salicylic acid inhibited in vitro growth across a global collection of 411 Z. tritici strains, whereas acidic pH promoted growth, illustrating contrasting effects on pathogen performance of environments simulating host-defense responses. At the transcriptional level, acidic pH and oxidative stress induced the strongest and most similar responses, while salicylic acid elicited a more distinct transcriptional program and gibberellic acid caused only limited transcriptional changes. Although the sets of differentially expressed genes were largely condition specific, overlapping enrichment of transport- and redox-related functions across conditions indicated shared transcriptional responses. K-mer based genome-wide association mapping identified five candidate loci associated with growth under acidic pH, gibberellic acid and salicylic acid, including four loci specific to a single growth condition. These loci colocalized with genes implicated in cell wall remodeling, nitrogen metabolite regulation, proteostasis, and ubiquitin-related processes. This study highlights the multifaceted strategies employed by Z. tritici to navigate environments simulating host-defense responses, involving shared and environment-specific adaptations. We provide new insights into the genetic and molecular basis of fungal resilience, with implications for understanding pathogen-host interactions.

Wheat take-all is a root disease which devastates crop yields, caused by the ascomycete fungus Gaeumannomyces tritici. The closely related root endophyte, G. hyphopodioides, has been found to induce local host defence responses which confer protection against take-all and reduce disease severity. Chancellor et al. (2024) investigated host transcriptional response to early colonisation by each of these two fungi. Using this RNA-seq dataset in conjunction with newly available Gaeumannomyces reference genomes, we have completed the picture by characterising the fungal transcriptional activity underpinning these different lifestyles. Even at early time points, their transcriptional profiles differ: G. hyphopodioides shows signs of transcriptional reprogramming between 4 and 5 days post inoculation (dpi), mirroring the wheat response, whereas G. tritici expression varied very little between these two time points despite progressing into the vasculature, instead exhibiting a stealthy expression profile dominated by gene downregulation at earlier time points. Moreover, GO term enrichment in this study identified a stress-response unique to G. hyphopodioides, which may explain the formation of its subepidermal vesicles (SEVs), putative resting structures that are a key difference between the pathogen and non-pathogen, alongside upregulation of many putative effectors and CAZymes. The enrichment of a key lignin-degrading CAZyme may contribute to the lack of stress-response identified in G. tritici, allowing fungal hyphae to overcome localised host lignification. These findings highlight the transcriptional basis of colonisation differences and are a step towards understanding how closely related fungi with different lifestyles modulate their interactions within a common host and tissue.

Cadophora luteo-olivacea is an ecologically versatile fungus associated with grapevine trunk diseases, yet the extent to which strains from different hosts and environments differ in genome composition, functional potential, and pathogenicity remains poorly understood. Here, we performed a comparative genomic analysis of 12 C. luteo-olivacea isolates recovered from grapevine, almond, apple, Crocus bulbs, soil, air, wastewater, and deep-sea sediment. Genome assemblies were highly complete (BUSCO >99%) and ranged from 46.94 to 50.70 Mbp. Pairwise average nucleotide identity (ANI) revealed a cohesive 11-strain group and one markedly divergent strain, CBS 266.93. Phylogenomic analysis based on 2,645 single-copy orthologs further showed that CBS 266.93 lies outside the main C. luteo-olivacea clade and forms a sister relationship with Cadophora malorum, indicating that its taxonomic placement warrants reassessment. Across the remaining strains, broad functional conservation was observed, including similar KOG profiles, extensive carbohydrate-active enzyme repertoires (798-849 genes per genome), and abundant biosynthetic gene clusters (26-35 per genome). Transposable element content varied substantially among strains (0.67-4.45% of genome), but this variation did not parallel overall functional profiles. All isolates colonized grapevine leaves in vitro, although lesion severity differed significantly among strains, indicating conserved plant-colonizing capacity with quantitative variation in aggressiveness. Small RNA profiling of inoculated grapevine leaves further revealed isolate-associated differences in host miRNA family expression, particularly for miR398, miR827, and miR156. Together, these results show that most C. luteo-olivacea strains share a conserved genomic framework compatible with plant colonization, while retaining lineage-and strain-level phenotypic and host-associated variation.

Botrytis cinerea is a necrotrophic fungal pathogen that infects thousands of plant species. During infection, these diverse plant hosts produce different specialized metabolites that can inhibit pathogen growth and shape pathogen fitness. However, the genetic architecture of pathogen resistance toward individual host defense metabolites remains poorly understood. To address this question, we exposed 83 B. cinerea isolates to the metabolite linalool and quantified metabolic and structural responses. Exposure revealed extensive phenotypic diversity across isolates. Genome-wide association identified 101 genes of interest associated with membrane transport and stress response regulation. Genetic associations were stronger for morphological traits than for metabolic traits, suggesting that hyphal architecture may have a complex genetic architecture contributing to linalool resistance. Together, these results establish natural variation in linalool response and provide candidate loci for understanding how generalist pathogens respond to host-derived chemical defenses. Article SummaryTo understand how a generalist pathogen responds to host defenses, we asked how Botrytis cinerea responds to linalool, a widespread monoterpene involved in plant defense. We exposed 83 B. cinerea isolates to 1000 {micro}M of linalool for 72 hours and quantified metabolic traits (growth curves and growth dynamics over time) and morphological traits (hyphal network features). Using GWA, we linked phenotypic variation to genetic variants. Results indicate substantial natural variation in linalool resistance and distinct genetic architectures across trait classes: metabolic responses are driven by a relatively small number of loci with larger effects, whereas structural/morphological responses appear more polygenic.

Epimutations are changes in chromatin modifications, such as DNA methylation or histone modifications. Some of these epigenetic changes can be inherited for several generations, and they potentially contribute to evolutionary processes. Estimates of epimutation rates now exists in a few species, but the presence and function of epigenetic marks are not conserved across different species. To understand the properties of epimutations in fungi, we performed a mutation accumulation experiment with the filamentous fungus Neurospora crassa and investigated spontaneous changes in DNA methylation and trimethylation of lysine 9 on histone H3 (H3K9me3) in the mutation accumulation lines. We observed that centromeric regions are hotspots of spontaneous DNA methylation changes in N. crassa. In these hotspot regions, DNA methylation changes were transmitted across mitoses, but changes occurring in euchromatin were not maintained. The rate of DNA methylation changes was around 30 000 fold faster than the genetic mutation rate. We did not observe spontaneous changes in H3K9me3 that were transmitted across mitoses. Our results show that while spontaneous epimutations occur in this species, they occur predominantly in gene poor heterochromatic regions, so their impact for evolutionary adaptation may be limited.

IntroductionThe use of Saccharomyces cerevisiae to ferment alcoholic beverages is an ancient tradition, with genetic evidence indicating origins in Neolithic Asia, although the domestication process of the species is not fully understood. Kveik is a group of traditional yeasts used in farmhouse brewing in western Norway preserved through generations of rural brewing practice. While recent studies have highlighted the distinctiveness of kveik, its precise phylogenetic position, genetic diversity, and domestication history remain unclear. ResultsWe performed whole-genome sequencing on 62 samples representing 25 unique Norwegian strains selected using cultural heritage criteria, and generated telomere-to-telomere (T2T) assemblies for representative isolates. Phylogenomic and population genetic analyses reveal that kveik forms a paraphyletic and early diverging group with respect to other domesticated S. cerevisiae strains. Most strains exhibit low within-strain diversity, strong geographic clustering, and little evidence of gene-flow or admixture. Mitochondrial genomes and Ty1 retrotransposon profiles corroborate this distinct lineage history. We further show that previously reported signals of gene flow between kveik and Asian fermentation strains are likely artifacts caused by population structure and selection. Divergence time estimates suggest that the common ancestor of beer, kveik, and other liquid-phase fermenting strains originated from ancestral populations 4,000 to 8,000 years ago. ConclusionKveik yeasts represent a relic of early S. cerevisiae domestication, shaped by ancient human practices, migrations, and the spread of agriculture. Our genomic resource sheds light on yeast evolution and domestication. They likely comprise some of the oldest domesticated lineages in continuous use until today, connecting endangered intangible cultural heritage to an early genetic origin.

Eukaryotic genomic DNA is packaged in the nucleus as chromatin - a DNA-protein aggregate regulating genome function, including transcription. Chromatin is classified as either active euchromatin or silent heterochromatin, with each marked by distinct histone post-translational modifications (PTMs). Chromatin composition also mediates genome organization, including how heterochromatin aggregates at the nuclear periphery while euchromatin localizes to the nucleus center. In fungi, heterochromatic loci cluster, including independent centromere and telomere clusters that form the Rabl chromosome conformation. However, it is unknown if chromatin composition and genome organization are conserved in closely related fungi, and how they are impacted by large-scale chromosomal rearrangements. Here, we examined differences in histone PTM deposition, gene expression, and genome organization in two yeast species from the order Pichiales, which diverged from the common ancestor shared with Saccharomyces cerevisiae more than 200 million years ago. We focused on Ogataea polymorpha, which is used for industrial protein production, and Ogataea haglerorum, an isolate of which harbors a translocation between chromosomes 1 and 6. We show that the enrichment of three activating PTMs - the trimethylation of lysine 4 of histone H3 (H3K4me3) and the acetylation of lysine 9 of histone H3 (H3K9ac) or lysine 16 of histone H4 (H4K16ac) - are similar genome-wide yet individual gene orthologs have distinct chromatin and expression patterns. While both Ogataea genomes organize into a Rabl conformation, the O. haglerorum translocation alters subtelomeric chromatin composition and expression of genes affected by the translocation. Our work highlights the genome function differences that occur on a microevolutionary scale.

Yeasts ability to invade surfaces has important implications for infections and food contamination. Invasive growth in yeast is influenced by genetic and environmental factors. In this exploratory study, we investigated the effects of sodium sulfide, gene deletions, and environmental conditions on the invasive behaviour of the wine yeast strain AWRI 796. Sodium sulfide enhanced invasion in the (parent) AWRI 796 strain under nitrogen-limiting conditions, although its effect was obscured by experimental variability and pre-culture conditions. Genetic factors had a major effect on the overall invasive phenotype, with deletion of key genes suppressing invasion. Most gene-deletion mutants did not significantly affect how the colony responded to sulfide. In addition to sulfide and genotype, environmental conditions also influenced invasive behaviour. The pre-2xSLAD pre-culture condition was best for detecting sulfide-induced growth, and later plate washing time and decreased nutrient levels enhanced invasiveness. Our experimental design and findings provide a framework for understanding the determinants of yeast invasiveness, which may inform future studies on filamentous yeast behaviour.

Gene-by-environment (GxE) interactions play a major role in shaping both phenotypic and molecular variation, with important implications for human health and disease. In this study, we used the Doxycycline (Dox) regulated, tetracycline-responsive (Tet-Off) promoter system to sequentially reduce or titrate gene expression levels of the essential yeast transcription factor Repressor Activator Protein 1 (RAP1) similar to a hypomorph allele series, across three distinct environments: Yeast Peptone Dextrose (YPD) media, YPD media with Heat Shock (HS), and Yeast Peptone Acetate (YPAC) media. We then performed RNA sequencing (RNA Seq) to assess global transcriptional responses to RAP1 reduction in these different growth environments. Our analysis first focused on the independent effects of varying RAP1 expression levels within and across environments. We then explored GxE interactions, revealing a subset of genes with significant consequences of reduced levels of RAP1 and environment-specific expression patterns. Notably, many genes exhibited opposite effects of RAP1 titration on gene expression when yeast were grown in YPAC media compared to YPD media and/or HS, suggesting environment-dependent regulatory architecture. This design reveals how cells integrate internal transcriptional and regulatory changes with external environmental cues, providing a deeper view of GxE architecture. Using Weighted Gene Co-expression Network Analysis (WGCNA), we identified co-regulated gene modules, and by combining this with transcription factor motif enrichment tests, our study identified candidate regulators driving their dynamics. Our findings demonstrate that gene regulatory networks can vary dramatically depending on the environmental context an organism experiences, which can then influence the specific phenotypes produced by a particular genetic perturbation. This illustrates the complexity of genotype-environment interactions and the importance of studying gene function in multiple environments to gain a truly comprehensive understanding of a genes sometimes numerous and diverse functions.

High-temperature stress is a major constraint on yeast growth and fermentation, and has traditionally been interpreted primarily in terms of intracellular molecular damage such as protein denaturation and aggregation. Despite this extensive focus on intracellular mechanisms, how physical factors within the extracellular environment influence yeast thermotolerance remains poorly understood. Here we demonstrate that increases in extracellular osmolarity markedly attenuate growth inhibition under high-temperature conditions in yeast. This protective effect was consistently observed in multiple laboratory and industrial strains of Saccharomyces cerevisiae, as well as in ascomycetous and basidiomycetous yeasts, indicating that osmotic pressure-dependent thermotolerance is a broadly conserved phenomenon. We also found that extracellular osmolarity dynamically increases during growth and then decreases in a diauxic shift-like pattern after growth arrest. At high temperature, the secretion of glucose-derived metabolites decreased, but that of other solutes increased, suggesting that heat stress alters the composition of extracellular solutes contributing to osmolarity. In addition, intracellular glycerol levels increased at high temperature, and this increase was further enhanced under high-osmolarity conditions. Notably, expression of a constitutively active Hog1 mutant exhibited raised intracellular glycerol levels, enhanced nuclear localization of Hog1, and improved growth under high-temperature conditions. Collectively, these findings support a model in which extracellular osmolarity is modulated to avoid excessive intracellular osmolarity under high-temperature conditions, while the intracellular accumulation of glycerol contributes to yeast adaptation at high-temperature. Our results highlight extracellular-intracellular osmotic coordination as an additional physiological layer of high-temperature stress adaptation in yeast.

The fungal crop pathogen Blumeria hordei, causal agent of powdery mildew on barley, presents life-history and epidemiological characteristics, as well as and selective pressures due to modern agriculture leading to expected sweepstakes reproduction, that is highly skewed offspring distributions. Using genome-wide polymorphism data and population genomics inferences, we aim to 1) infer the past demographic history and the strength of sweepstakes reproduction in B. hordei, and 2) quantify the contributions of these selective and neutral processes in the genome. An new inference method based on Neural Posterior Estimation and diversity and linkage disequilibrium statistics was developed and tested on simulated and B. hordei genomic data. We confirm that B. hordei exhibits a moderate sweepstakes reproduction (-parameter of 1.6). We highlight that the Site Frequency Spectrum (SFS) appears sensitive to the joint occurrence of sweepstakes and recent demographic changes, which may caution on the reliability of the SFS to infer sweepstakes reproduction. We then scan the genome for selective sweeps, adjusting the significance thresholds of the methods for demographic history and sweepstakes reproduction, thereby yielding a counterintuitive result. When conditioning the significance threshold for sweep detection on simulations under sweepstakes and demography, a very large number of putatively selected regions is found (11.6% of the genome). We suggest that sweepstakes reproduction in B. hordei is due to 1) neutrality (clonal/sexual phases and Boom-and-Bust cycles) generating a genome-wide level of background noise in the coalescent genealogies, and 2) selective sweepstakes due to pervasive positive selection. Our findings have important implications for both population genomic methodology and our understanding of pathogen evolution.

Changes in regulatory sequences controlling the timing and activity of gene products underlie much of natural phenotypic variation. Yet, what these changes are and how they impact gene expression remain largely unknown. To address this question, we investigated how transcriptional activity and homeostatic responsiveness of orthologous promoters of the metabolic gene TDH3 evolved among Saccharomyces yeast. We found that promoter expression level increased specifically in the S. cerevisiae lineage and that a substantial part of this increase was caused by genetic variants located between the well-characterized, conserved binding sites for two direct transcriptional regulators. These nucleotide changes altered the promoters expression levels while leaving the expression dynamics conserved. Further, the effects of these nucleotide changes were only seen in the presence of a third transcription factor, TYE7p, which is recruited by the other transcription factors through protein-protein interactions. These results suggest that the cis-regulatory changes act through their influence on the collective assembly/activation of the transcription factors, and that changes acting through such a mechanism can allow distinct parts of gene expression, such as expression level and dynamics, to be tuned separately.

Cell size in a proliferating cell population generally varies over a limited range ([~]2-4-fold). Within such populations, organelle content increases with cell size maintaining a relatively constant organelle density (amount per cell volume). However, cells of different types can differ greatly in cell size as well as in organelle composition. In such cases, it is often unclear to what degree, if any, the differences in organelle composition are due to the difference in cell size. In principle, this issue could be resolved by examining situations where a proliferating population of cells of the same cell type exhibit much greater size variation. Here we characterize how organelle content scales with cell volume in the polymorphic fungus, A. pullulans, whose proliferating cells span a [~]100-fold size range. We find that mitochondria and ER content increases in proportion to cell volume, while this is not the case for vacuoles and peroxisomes. Thus, organelle composition is affected by cell size in this system.

Plant tissues and surfaces are among the largest microbial habitats on Earth, and commensal yeasts are common members of these communities, where they can contribute to plant-microbe interactions including the biological control of plant diseases. Here, we describe a novel genus, Aimea, of unpigmented, plant-associated basidiomycete yeasts, in the class Microbotryomycetes, and name three new species (A. erigeronia, A. cardamina, and A. sorghi) represented by four isolates from leaves and roots of multiple hosts. We characterize these taxa through analyses of metabolic requirements, tolerance to differences in osmolarity, pH, and temperature, and enzymatic activities. In parallel, we generate near-chromosome-scale hybrid genomes annotated with transcriptome data. We employ whole-genome and multilocus phylogenetic approaches to infer the placement of these species within a monophyletic clade. We use comparative genomics to examine how the gene content of these yeasts differs from that of other members of the Microbotryomycetes, including an apparent proliferation of retrotransposons. We further demonstrate the genetic transformability of these taxa using Agrobacterium tumefaciens-mediated transformation. The description of these new species, together with high-quality genome resources and a genetic transformation protocol, establishes a foundation for experimental studies of these novel plant-associated yeasts and their interactions with hosts and other microbes.

DDI2 and DDI3 (DDI2/3) are duplicated genes in Saccharomyces cerevisiae that exhibit strong induction by a transcription factor Fzf1 in response to chemical treatments like cyanamide (CY) and methyl methanesulfonate (MMS). Although, like DDI2/3, SSU1, YHB1 and YNR064C also contain an Fzf1-binding consensus sequence CS2 and are coordinately regulated by Fzf1, these genes are only modestly induced by CY and MMS. To identify additional cis-acting elements in the DDI2/3 promoter, we made DDI2/3 promoter deletions in a reporter system and identified upstream repressing sequences (URS) spanning 480 nucleotides. To test a hypothesis that the chromatin structure constitutes the URS, we utilized a yeast strain capable of histone H3/H4 depletion by shifting carbon sources. Following histone depletion, DDI2/3 were strongly induced in an Fzf1 dependent manner, while YHB1 was repressed. Interestingly, under histone depletion conditions, CY or MMS treatment further increased expression of all Fzf1-regulated genes to comparable levels in an Fzf1 dependent manner. A genome-wide MNase-seq analysis showed that CY treatment reduced the nucleosome occupancy at the mapped DDI2/3 URS region in wild-type cells, but not in in fzf1{Delta} cells. These findings collectively indicate that Fzf1 plays dual roles in regulating the DDI2/3 response to CY. Firstly, it binds CS2 and serves as a transcription activator. Secondly, it is required for the chromatin remodeling at URS. This two-tier regulation at the DDI2/3 promoter helps to explain why DDI2/3 achieve much higher fold induction by CY and MMS than other Fzf1-regulated genes, suggesting Fzf1 to be a candidate pioneer transcription factor.

We often think of fungi as mysterious organisms that do not follow the general principles of other eukaryotes. Thus, when exciting results are found, these organisms do not always receive the rigorous level of scrutiny seen in other fields. For many fungal species, dispersal and reproduction relies on spores, either sexual or asexual. These spores can either have a single nucleus, or multiple nuclei, and the purpose of these presumably mitotic copies was unclear. Recently it was described that the multiple nuclei in these spores are not mitotic duplicates, but instead they share a single haploid set of chromosomes distributed across nuclei. Here, we provide fluorescent microscopy and UV mutagenesis data that is inconsistent with this hypothesis. Contrasting these previous results, we observe multiple sets of chromosomes in spores of both B. cinerea and N. crassa. We also observed a strong linear relationship between the number of nuclei in spores and the total acriflavine fluorescence, further supporting mitotic copies. Genome sequencing of colonies arising from UV-irradiated colonies also recovered variants at intermediate frequences, instead of the fixed 100% expected from the new model proposed. This evidence suggests that, as long suspected, these nuclei are indeed mitotic copies, and that a re-evaluation of fungal biology is not currently necessary.