G3

The diploid genome of the fungal pathogen Candida albicans is highly heterozygous, with most allele pairs diverging at either the coding or regulatory level. When faced with selection pressure like antifungal exposure, this hidden genetic diversity can provide a reservoir of adaptive mutations through loss of heterozygosity (LOH) events. Validating the potential phenotypic impact of LOH events observed in clinical or experimentally evolved strains can be difficult due to the challenge of precisely targeting one allele over the other. Here, we show that a CRISPR-Cas9 system can be used to overcome this challenge. By designing allele-specific guide RNA sequences, we can induce targeted, directed LOH events, which we validate by whole-genome long-read sequencing. Using this approach, we efficiently recapitulate a recently described LOH event that increases resistance to the antifungal fluconazole. Additionally, we find that the recombination tracts of these induced LOH events have similar lengths to those observed naturally. To facilitate future use of this method, we provide a database of allele-specific sgRNA sequences for Cas9 that provide near genome-wide coverage of heterozygous sites through either direct or indirect targeting. This approach will be useful in probing the adaptive role of LOH events in this important human pathogen.

Fusarium head blight (FHB), a major disease of wheat, is primarily managed through applications of demethylation inhibitor (DMI) fungicides during anthesis. However, repeated use of DMIs has led to the emergence of Fusarium graminearum isolates with reduced sensitivity and, in some cases, resistance. In this study, we evaluated the sensitivity of 152 F. graminearum isolates to propiconazole and tebuconazole. While sensitivity varied among isolates, no resistant strains were detected. We also conducted a genome-wide association study (GWAS) to investigate the genetic basis of DMI sensitivity. GWAS identified 48 and 39 quantitative trait nucleotides (QTNs) associated with propiconazole and tebuconazole sensitivity, respectively, with 12 QTNs common to both fungicides--supporting their common mode of action. Candidate gene analysis highlighted genes encoding transporters, secondary metabolite synthesis enzymes, transcription factors, and a heat shock protein as potential candidates for DMI fungicide response. We propose that tolerance to DMIs in F. graminearum is linked to active fungicide efflux out of fungal cells, mediated by transporters, including those associated with secondary metabolite pathways. Data summaryInformation on all strains used in the experiments have been included in a supplemental file. All sequencing data used in this study are publicly available and were described in our previous publication, which has been cited. Additional supporting data (isolate genotype and phenotype files) and code written for the analyses described here are made available on GitHub: https://github.com/Toomajian-laboratory/files_fungicide_manuscript/ Impact statementIsolates from field populations of fungal plant pathogens vary in their sensitivity to DMI fungicides, though in most cases the genetic determinants of this variation are poorly understood. In this study, we measure the sensitivity of isolates from the main US population of Fusarium graminearum to two DMI fungicides. We used GWAS to identify genomic loci and candidate genes that might underlie variation in DMI sensitivity. Our work contributes to the broader effort to understand the evolution of fungicide tolerance and resistance in populations of plant pathogens. The candidate genes identified, most of which are novel, provide good targets for functional studies of the tools fungi use to survive fungicides. The identified variants can also be screened to monitor potential increases in fungicide tolerance. The high-throughput, rapid, and large-scale monitoring of fungicide sensitivity described here advances both fundamental research and resistance management efforts.

In this study, we introduce a novel approach for analysing long, repetitive genomic sequences. Our methods significantly advance research on rDNA polymorphism. First, we describe a technique for isolating high-molecular-weight DNA from individual chromosomes, enabling selective enrichment of sequencing libraries for extensive genomic regions of interest. Second, we present rDNAmine, a bioinformatic toolkit for capturing and examining large repetitive arrays in Oxford Nanopore sequencing data. This approach facilitates the study of polymorphisms within long repeats, bypassing traditional alignment-based methods and providing a more efficient and scalable solution for investigating repetitive regions. We demonstrate the effectiveness of our approach through the analysis of rDNA arrays in two yeast species, Saccharomyces cerevisiae and Candida albicans. In S. cerevisiae, rDNA arrays show limited polymorphism, while in C. albicans, we observe substantial variation in rDNA module size, with two distinct repeat populations within the array. These findings reveal species-specific differences in the structural organisation of rDNA loci, highlighting the diverse nature of tandem repeat architecture. The rDNAmine toolkit is broadly applicable to various organisms and repetitive genomic contexts, offering a versatile platform for studying repetitive sequences. Take AwayO_LIYeast rDNA serves as a benchmark to validate tools for analysing long repetitive sequences. C_LIO_LIA chromosome-specific DNA extraction method has been introduced to enable targeted enrichment of repetitive loci. C_LIO_LIThe rDNAmine pipeline was designed to analyse long tandem repeats from noisy long-read data without requiring global alignment. C_LI

The fission yeast species Schizosaccharomyces japonicus is currently divided into two varieties - S. japonicus var. japonicus and S. japonicus var. versatilis. Here we examine the var. versatilis isolate CBS5679. The CBS5679 genome shows 88% coding sequence identity to the reference genome of S. japonicus var. japonicus at the coding sequence level, with phylogenetic analyses suggesting that it has split from the S. japonicus lineage 25 million years ago. The CBS5679 genome contains a reciprocal translocation between chromosomes 1 and 2, together with several large inversions. The products of genes linked to the major translocation are associated with "metabolism" and "cellular assembly" ontology terms. We further show that CBS5679 does not generate viable progeny with the reference strain of S. japonicus. Although CBS5679 shares closer similarity to the "type" strain of var. versatilis as compared to S. japonicus, it is not identical to the type strain, suggesting population structure within var. versatilis. We recommend that the taxonomic status of S. japonicus var. versatilis is raised, with it being treated as a separate species, Schizosaccharomyces versatilis. Take-awayO_LIThe taxonomic status of Schizosaccharomyces versatilis is addressed. C_LIO_LIS. versatilis diverged from S. japonicus around 25 million years ago. C_LIO_LIS. versatilis does not produce viable progeny in crosses with S. japonicus. C_LIO_LIS. versatilis has a reciprocal translocation between chromosomes 1 and 2. C_LIO_LIThe Gene Ontology terms for genes in the translocations are enriched for terms connected to "metabolism" and "cellular assembly". C_LI

The overall process of meiosis is conserved in many species, including some lineages that have lost various ancestrally present meiosis genes. The extent to which individual meiosis gene losses are independent from or dependent on one another is largely unknown. Various Eurotiomycete fungi were investigated as a case system of recent meiosis gene losses after BLAST and synteny comparisons found Msh4, Msh5, Pch2, and Zip3 to be either pseudogenized or undetected in Aspergillus nidulans yet intact in congeners such as A. fumigatus. Flanking gene-targeted degenerate PCR primers applied to 9 additional Aspergillus species found (i) Msh4, Msh5, and Zip3 pseudogenized in A. rugulosus (sister taxon to A. nidulans) but intact in all other amplified sequences; and (ii) Pch2 not present at the syntenic locus in most of the 9 species. Topology tests suggested two independent Pch2 losses in genus Aspergillus, neither directly coinciding with pseudogenization of the other three genes. The A. nidulans-A. conjunctus clade Pch2 loss was not associated with significant Ka/Ks changes for Msh4, Msh5, or Zip3; this suggests against prior Pch2 loss directly altering sequence evolution constraints on these three genes. By contrast, Zip3 Ka/Ks tended to be elevated in several other Eurotiomycete fungi with independently pseudogenized Msh4 and Msh5 (Talaromyces stipitatus, Eurotium herbariorum). The coinciding Ka/Ks elevation and/or clear pseudogenization of Zip3 in taxa with pseudogenized Msh4 and Msh5 is consistent with some degree of molecular coevolution. Possible molecular, environmental, and life history variables (e.g., homothallism) that may be associated with these numerous independent meiosis gene losses (Msh4: 3, Msh5: 3, Zip3: [≥] 1, Pch2: 4) are discussed.

Multicopy nuclear ribosomal (rDNA) genes have been used as markers for fungal identification for three decades. The rDNA sequences in a genome are thought to be homogeneous due to concerted evolution. However, intragenomic variation of rDNA sequences has recently been observed in many fungi, which cause problems in fungal identification and species abundance estimation. Various sequence-based methods have been used to demonstrate rDNA sequence heterogeneity, but there is no technical assessment of the comparability of results from these methods. In this article, we sampled smut fungi representing all major lineages of subphylum Ustilaginomycotina as a system to examine sequence heterogeneity in the rDNA repeats. Three methods were used: PCR-cloning-Sanger sequencing, targeted amplicon high-throughput sequencing, and WGS high-throughput sequencing. Based on our analyses, Ceraceosorus is the only sampled fungal genus in Ustilaginomycotina showing intragenomic variation, with up to 27 nucleotide variant sites in the ITS1-5.8S-ITS2 region and 2.6% divergence among analyzed ITS haplotypes. We found many conflicting patterns across the three detection methods, with up to 28 conflicting variant sites in one sample. Surprisingly, at least 40% of these conflicts are due to PCR-cloning-sequencing errors, as the corresponding variant sites were not observed in the other methods. Based on our data and the literature, we evaluated the characteristics and advantages/disadvantages of each detection method. A model for how intragenomic variation may arise in the rDNA region is presented. Finally, we describe the fourth known species of Ceraceosorus, C. americanus, isolated from an asymptomatic rosemary leaf collected in Louisiana, USA. We anticipate that our study will provide a framework for future research in rDNA regions as well as other similar multicopy genes. Author SummaryRibosomal DNA (rDNA) genes are one of the most ancient multicopy genes in cellular organisms. They function as a part of the protein synthesis machinery in a cell. The rDNA sequences have also been used in species identification and microbial community profiling. Despite these utilities, little is known how the rDNA genes have evolved. Biologists initially thought the sequences among rDNA copies are homogeneous, but many recent cases illustrated rDNA sequence heterogeneity. In this article, we utilized the fungal genus Ceraceosorus together with allied smut fungi as a system to study sequence heterogeneity in the rDNA genes using various detection methods. Our system found rDNA sequence homogeneity as a common form, while sequence heterogeneity is taxon-specific. Based on our data and literature review, we explained possible sources for sequence heterogeneity in the rDNA genes. Our study also noticed result discrepancies across variant detection methods. These include artefactual variants from the PCR-cloning-sequencing method, inconsistent detected variants from the independent runs of high-throughput sequencing, and technical errors in bioinformatic analyses. We therefore emphasize the importance of methodological choices which have different pros and cons for studying intragenomic variation of rDNA genes, as well as other multicopy gene families.

Kveik is the common name of yeast that has been used in traditional farmhouse brewing of western Norway for generations. Its fast fermentation, increased flocculation, temperature tolerance, and rich flavor profile have led to growing interest in recent years. Previous genetic analyses have shown that kveik forms a distinct group within the Saccharomyces cerevisiae tree and placed its origins within the Beer I clade of industrial brewing yeasts, although with signs of mixed ancestry. In this study, we revisited the phylogenetic position of kveik within the S. cerevisiae tree. We searched for traditional farm breweries in western Norway and collected ten samples of potential kveik yeast. Using Illumina whole genome shotgun sequencing, we reconstructed the phylogenetic tree of kveik based on de novo genome assemblies and variant calls of our new kveik samples, along with published wild and domesticated S. cerevisiae strains. We calibrated and used sequential computational experiments at different thresholds to determine the most probable phylogenetic position of kveik yeast. Previously sequenced kveik genotypes form a clade with our new samples clustering partially by place of origin. Our results indicate that kveik is indeed a compact clade within S. cerevisiae with significantly reduced polymorphism compared to common brewing yeasts and wild strains. Contrary to what was previously thought, our analyses support a more ancient divergence of kveik and place it closer to the root of the S. cerevisiae tree. In conclusion, our genetic analyses suggest that kveik is a unique and ancient yeast group, distinct from other domesticated S. cerevisiae strains. Considering a possible far east origin of kveik yeast, the apparent endemism to western Norway remains as a big paradox These findings have important implications for the understanding of yeast domestication and the use of kveik in modern brewing practices.

Recent technological developments have made genome sequencing and assembly accessible to many groups. However, the presence in sequenced organisms of certain genomic features such as high heterozygosity, polyploidy, aneuploidy, or heterokaryosis can challenge current standard assembly procedures and result in highly fragmented assemblies. Hence, we hypothesized that genome databases must contain a non-negligible fraction of low-quality assemblies that result from such type of intrinsic genomic factors. Here we present Karyon, a Python-based toolkit that uses raw sequencing data and de novo genome assembly to assess several parameters and generate informative plots to assist in the identification of non-chanonical genomic traits. Karyon includes automated de novo genome assembly and variant calling pipelines. We tested Karyon by diagnosing 35 highly fragmented publicly available assemblies from 19 different Mucorales (Fungi) species. Our results show that 6 (17%) of the assemblies presented signs of unusual genomic configurations, suggesting that these are common, at least within the Fungi.

Zinc is a major soil contaminant and high zinc levels can disrupt growth, survival, and reproduction of fungi. Some fungal species have evolved zinc tolerance through cell processes mitigating zinc toxicity, though the genes and detailed mechanisms underlying fungal zinc tolerance remain unexplored. To fill this gap in knowledge, we investigated the gene expression of zinc tolerance in the mycorrhizal fungus Suillus luteus. We found that zinc tolerance in this species is both a constitutive and environmentally dependent trait. Highly differentially expressed genes were predicted to be involved in transmembrane transport, metal chelation, oxidoreductase activity, and signal transduction. Some of these genes were previously reported as candidates for S. luteus zinc tolerance, while others are reported here for the first time. Overall, we found S. luteus zinc tolerance is associated with differences in expression of genes involved in metal exclusion and immobilization, as well as recognition and mitigation of metal-induced oxidative stress. Our results contribute to understanding the mechanisms of fungal metal tolerance and pave the way for further research on the role of metal tolerance in mycorrhizal associations.

Climate change can alter interactions between plants and their pathogens, which could adversely affect crop production. To better understand the molecular mechanisms underlying the responses of pathogenic fungal to temperature stress, we conducted a quantitative trait loci (QTL) mapping study in the wheat pathogen Zymoseptoria tritici to identify genomic regions associated with colony growth and melanisation at three temperatures (10{degrees}C, 18{degrees}C, 27{degrees}C). We then identified likely candidate genes for thermal adaptation within these intervals by combining information regarding gene function, GO annotation enrichment, transcriptional profile, and results from previous genome wide association studies (GWAS) investigating responses to climate, temperature and thermal adaptation. The QTL mapping, conducted for two separate crosses involving four Swiss parents, found significant QTL uniquely associated with traits measured in high and low temperatures. These intervals contained many genes known to regulate responses to temperature stress, including heat shock proteins (HSPs) and proteins involved in the mitogen-activated protein kinase (MAPK) pathways, and were enriched for genes with a zinc ion binding GO annotation. We highlight the most promising candidate genes for thermal adaptation, including an ammonium transporter gene, a stress response factor (Whi1) and two MAPK pathway genes - SSk2 and Opy2. Future validation work on these candidate genes could provide novel insight into the molecular mechanisms underlying temperature adaptation in this important wheat pathogen.

Antifungal drug resistance across fungal and yeast pathogens presents one of the major concerns for global public health. Understanding the interactions between genetic background and environment is important for the development of new, effective treatments of infections. Allelic variation within populations of Ascomycota as well as hybridisation impacts the phenotype in response to stressful conditions, including to antifungal drugs. We exploited recent advances in multigenerational breeding of Saccharomyces interspecies hybrids to study the impact of hybridisation on antifungal resistance and identify quantitative trait loci (QTL) responsible for the phenotypes observed. A library of Saccharomyces cerevisiae x S. kudriavzevii hybrid offspring was screened in the presence of sub-lethal concentrations of six antifungal drugs and revealed a broad phenotypic diversity across the progeny. QTL analysis was carried out comparing alleles between the pools of high and low fitness offspring, identifying hybrid-specific genetic regions involved in resistance to fluconazole, micafungin and flucytosine. We found both drug specific and pleiotropic regions, and through gene ontology and SIFT analysis we identify potential causal genes, such as BCK2 and DNF1 that were validated via reciprocal hemizygosity analysis. We highlight 41 regions that contain genes not previously associated with resistance phenotypes in the literature. The results of this screening will help identify new pathways contributing to drug resistance, and lead to greater understanding of how allelic variation, hybridisation and evolution affect antifungal drug resistance in yeast and fungi.

BackgroundOne way single-celled eukaryotes respond to DNA damage stress is by modifying their gene expression, facilitating genomic repair. Gene expression responses to DNA damage induced by methyl methanesulfonate (MMS) have been studied in the model organism Saccharomyces cerevisiae. However, lacking are investigations of the MMS stress responses in evolutionarily-related sensu stricto Saccharomyces species, including Saccharomyces cerevisiae. MethodsNext-generation Illumina RNA-sequencing was to characterize the entire transcriptomes of four evolutionarily-related species of yeast, S. cerevisiae, S. paradoxus, S. mikatae, and S. bayanus, under control and experimental (MMS) conditions. Subsequent genomic studies included gene set enrichment analysis, promoter analysis, and concentration gradient studies. ResultsS. mikitae and S. paradoxus grew well in light of MMS while S. bayanus showed no growth. While there was fair overlap in induced and repressed genes, overall each species had unique expression responses. S. paradoxus and S. bayanus showed the most distinct changes with the former greatly inhibiting a large segment of its genome while the latter induced such segments. Gene set enrichment analysis revealed significantly modulated biologic, cellular, and molecular processes in each species. Promoter analysis revealed sets of induced/repressed transcription factors for genes highly modulated in the stress response. Concentration gradient studies of S. cerevisiae showed linear increase in gene expression of RAD54, DIN7, and IRC19 in response to increasing concentrations of MMS. ConclusionOverall, we depict the transcriptome changes of four evolutionarily-related sensu stricto yeast species and several functional genomic analyses to provide a novel understanding of their responses to MMS.

How genomic differences contribute to phenotypic differences across species is a major question in biology. The recently characterized genomes, isolation environments, and qualitative patterns of growth on 122 sources and conditions of 1,154 strains from 1,049 fungal species (nearly all known) in the subphylum Saccharomycotina provide a powerful, yet complex, dataset for addressing this question. In recent years, machine learning has been successfully used in diverse analyses of biological big data. Using a random forest classification algorithm trained on these genomic, metabolic, and/or environmental data, we predicted growth on several carbon sources and conditions with high accuracy from presence/absence patterns of genes and of growth in other conditions. Known structural genes involved in assimilation of these sources were important features contributing to prediction accuracy, whereas isolation environmental data were poor predictors. By further examining growth on galactose, we found that it can be predicted with high accuracy from either genomic (92.6%) or growth data in 120 other conditions (83.3%) but not from isolation environment data (65.7%). When we combined genomic and growth data, we noted that prediction accuracy was even higher (93.4%) and that, after the GALactose utilization genes, the most important feature for predicting growth on galactose was growth on galactitol. These data raised the hypothesis that several species in two orders, Serinales and Pichiales (containing Candida auris and the genus Ogataea, respectively), have an alternative galactose utilization pathway because they lack the GAL genes. Growth and biochemical assays of several of these species confirmed that they utilize galactose through an oxidoreductive D-galactose pathway, rather than the canonical GAL pathway. We conclude that machine learning is a powerful tool for investigating the evolution of the yeast genotype-phenotype map and that it can help uncover novel biology, even in well-studied traits.

Exophiala dermatitidis is a polyextremotolerant black yeast species. E. dermatitidis produces 1,8 dihydroxynaphthalene (DHN) melanin via the Polyketide Synthase 1 (PKS1) pathway enabling it to survive harmful conditions. This study focused on random mutagenesis to obtain albino (alb) and hyper-pigmented (hyp) mutants. Notably, all 17 alb mutants possessed mutations in PKS1 whereas the 133 hyper-pigmented (hyp) mutants harbored mutations impacting a range of functions. Cell morphology and phenotypic assays showed additional differences between the alb and hyp mutants. Strikingly, three of the albino mutants (alb1, alb2, and alb3) were conditional in that despite the presence of mutations in PKS1 they were able to produce melanin upon exposure to different carbon sources. These mutants otherwise shared similar cell morphology and growth patterns with the obligate albinos. No additional shared mutations were found among the conditional albinos. Temperature and UV irradiation assays demonstrated reduced growth of albino mutants at higher temperatures (i.e., 42{degrees}C) and a greater sensitivity to higher doses of UV. Single nucleotide variant (SNVs) calling showed that hyper-pigmented mutants had a greater number of SNVs compared to albino strains. To date this is the first study to generate and characterize conditional albino mutants in E. dermatitidis with the ability to recover melanin production.

Basidiobolus is a globally distributed genus of early-diverging fungi within Zoopagomycota, known for its presence in diverse ecological niches ranging from soil and decaying organic matter to vertebrate gastrointestinal tracts. Despite its ecological and medical relevance, the taxonomy and evolutionary relationships within the genus remain poorly resolved due to limited genomic resources. In this study, we present nineteen newly sequenced Basidiobolus genomes, expanding the available genomic data. Using short-read Illumina sequencing, assembly, and annotation pipelines, we characterize genic content, assess completeness, and explore biosynthetic gene content across isolates. Phylogenomic analysis reveals two major clades corresponding to B. meristosporus and B. ranarum, while B. heterosporus forms a distinct lineage. Several isolate clusters exhibit deep divergence suggestive of cryptic species, underscoring the need for expanded sampling and taxonomic revision. Functional annotations reveal a rich repertoire of biosynthetic gene clusters, including non-ribosomal peptide synthetases, polyketide synthases, and hybrid clusters, pointing to an underexplored reservoir of secondary metabolite diversity. These findings position Basidiobolus as a compelling model for investigating fungal evolution, ecological adaptation, and natural product biosynthesis.

Jian et al. (2024) describe de novo genome assemblies for two strains of Prymnesium parvum (sensu lato, s.l.), a cryptic species complex of toxic, unicellular algae responsible for harmful algal blooms around the world. Here, we present evidence that the labels for UTEX 2797 and CCMP 3037 were inadvertently swapped by Jian et al. (2024). This resulted in sequence data labeled "UTEX 2797" but derived from strain CCMP 3037, and vice versa. Strain misidentification is a major risk with cryptic species like P. parvum s.l., and our reanalysis of the data in Jian et al. (2024) underscores the urgent need for clade-specific markers to ensure accurate and efficient strain identification.

The TOR (Target of Rapamycin) signalling pathway is found in all eukaryotes and integrates nutrient and stress signals to control cell growth. It is well-studied in yeast and mammals but is less well understood in filamentous fungi. We previously identified TOR pathway components VTA (homologous to the vacuole-bound EGO complex of yeast) and GTR2 (homologous to Rag GTPases) as essential to maintaining circadian rhythms in the filamentous fungus Neurospora crassa. Therefore we are interested in defining the TOR pathway and its regulation in N. crassa. In yeast and mammals, TOR kinase is activated by carbon sources and amino acids. We report here that on high glucose medium, TOR is insensitive to added amino acids. On low glucose, TOR is activated by added glucose and amino acids. VTA and GTR2 knockouts block the activation of TOR by amino acids but not by glucose, identifying their function in an amino acid-sensing pathway. Live cell microscopy of KOG1 (a component of TOR complex 1) and GTR2 localizes them to punctate bodies near the vacuole. This localization is lost and the proteins are largely cytoplasmic under starvation conditions, in the presence of TOR inhibitor Torin II, and in the VTA knockout. This indicates that VTA acts as the vacuolar anchor for activated TOR complex. Co-immunoprecipitation of KOG1-FLAG and GTR2-FLAG confirms their cytoplasmic localization in VTA knockout and identifies TORC1 complex components TCO89 and LST8. These results focus attention on amino acid sensing through VTA and GTR2 as potentially regulating circadian rhythmicity in N. crassa.

During cultivation, mixing of different heterokaryotic individuals of the button mushroom, Agaricus bisporus, generally reduces yield. This phenomenon could be caused by direct antagonistic responses and/or reduced synchronization by not forming a chimeric hyphal network. In other fungi, highly divergent alleles for a set of genes affect successful network formation between individuals either by preventing fusion or, more commonly, triggering cell death post-fusion. To understand this process in A. bisporus, it is important to identify the allelic variants allowing these fungi to discriminate self from nonself. We leverage a recently described cell death staining method utilizing Evans Blue to visualize mycelial compatibility. Here, we provide results of a first genetic mapping of incompatibility alleles in A. bisporus. Crossing strains between A. bisporus var. bisporus and A. bisporus var. burnetti we find segregation ratios of compatible progeny generally consistent with three nuclear loci. To identify these regions, we first use a set of single Chromosome Substitution Lines (CSLs), produced by genotyping progeny with recombination skewed to the very chromosome ends. We localize the main effect to be between two and three chromosomes, depending on the common nucleus of interacting heterokaryons. Using genome-wide markers for 167 sexual progeny, we identify loci controlling mycelial compatibility on chromosomes 4, 6 and 7, the same chromosomes as indicated by chromosome substitution lines. Notably, while the choice of a common nucleus seemed to affect the compatibility of CSLs, it did not seem to affect the loci identified in the sexual progeny. The ability to mix different strains of this mushroom-forming fungus could allow additional cultivation approaches, combining strains with complementary characteristics. These results provide a starting point towards understanding the molecular mechanisms underlying this fundamental property of hyphal networks in basidiomycetes.

A. fumigatus is a deadly fungal pathogen, responsible for >400,000 infections/year and high mortality rates. A. fumigatus strains exhibit variation in infection-relevant traits, including in their virulence. However, most A. fumigatus protein-coding genes, including those that modulate its virulence, are shared between A. fumigatus strains and closely related non-pathogenic relatives. We hypothesized that A. fumigatus genes exhibit substantial genetic variation in the non-coding regions immediately upstream to the start codons of genes, which could reflect differences in gene regulation between strains. To begin testing this hypothesis, we identified 5,812 single-copy orthologs across the genomes of 263 A. fumigatus strains. A. fumigatus non-coding regions showed higher levels of sequence variation compared to their corresponding protein-coding regions. Specifically, we found that 1,274 non-coding regions exhibited <75% nucleotide sequence similarity (compared to 928 protein-coding regions) and 3,721 non-coding regions exhibited between 75% and 99% similarity (compared to 2,482 protein-coding regions) across strains. Only 817 non-coding regions exhibited [&ge;]99% sequence similarity compared to 2,402 protein-coding regions. By examining 2,482 genes whose protein-coding sequence identity scores ranged between 75% and 99%, we identified 478 total genes with signatures of positive selection only in their non-coding regions and 65 total genes with signatures only in their protein-coding regions. 28 of the 478 non-coding regions and 5 of the 65 protein-coding regions under selection are associated with genes known to modulate A. fumigatus virulence. Non-coding region variation between A. fumigatus strains included single nucleotide polymorphisms and insertions or deletions of at least a few nucleotides. These results show that non-coding regions of A. fumigatus genes harbor greater sequence variation than protein-coding regions, raising the hypothesis that this variation may contribute to A. fumigatus phenotypic heterogeneity.

Histoplasma spp. is a dimorphic fungal primary pathogen that infects people worldwide and frequently affects immunosuppressed patients. Previous studies have identified the AMY1 gene product, the -amylase Amy1p, as essential for -glucan production and virulence in Histoplasma capsulatum. We identified two new genes (AMY2 and AMY3) in the Histoplasma genome that encode putative -amylases and made mutants using CRISPR/Cas9 technology, followed by evaluation of their role in -glucan biosynthesis and virulence. We also searched for AMY gene copies in 19 fungal genomes with the goals of identifying orthologs for AMY2 and AMY3, and establishing how many AMY copies existed across different fungi. We found that the number and type of -amylases vary depending on the fungal species; that all -amylases related to Histoplasma Amy1p belong to the GH13_5 subfamily, and all orthologs related to Histoplasmas Amy2p and Amy3p belong to the GH13_1 subfamily. We performed phylogenetic analyses of the three paralogs and revealed that the Histoplasma AMY duplications are ancient. We further established Amy2 is an ortholog of Aspergillus niger AgtA, and Aspergillus nidulans AmyD, and that it is partially involved in Histoplasma -glucan biosynthesis and virulence, while Amy3p is an ortholog of Aspergillus flavus Amy1, and it is dispensable for -glucan biosynthesis and virulence.