Biology subjects

Coelho, M. A.

Publications and source records attributed to Coelho, M. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Structure-function analysis of PI3K signalling cascade base editing screens in cancer cells

Knowledge of protein structure and function underpins rational drug discovery, yet many targets lack known selectively druggable sites. Furthermore, the identification of secondary druggable sites offers a strategy to overcome drug resistance. Fragment-based drug discovery (FBDD) can identify new ligandable binding pockets, though how to triage those with the ability to exert biologically relevant effects can be unclear. Systematic approaches to identify novel functionally-important protein sites for therapeutic intervention, such as allosteric pockets or protein-protein interaction (PPI) interfaces, has the potential to accelerate drug discovery, particularly when combined with structure-based hit-finding modalities. The phosphoinositide-3 kinase (PI3K) signalling pathway is frequently altered in human cancer and resistance to approved inhibitors is an ongoing challenge. Here, we performed large-scale CRISPR base editing mutagenesis screens across 30 PI3K pathway proteins in three disease-relevant cancer cell models to systematically map functional residues. Integration of base editing data with structural information identified residues corresponding to known catalytic sites, fragment-binding pockets and PPI interfaces, providing validation for the approach. Additionally, we identified putative allosteric pockets near regions of undefined function. Together, these findings establish high-throughput base editing mutagenesis combined with structural analysis as a scalable strategy to delineate structure-function relationships and inform drug development.

genomics↗

bioRxiv2026

Host skin lipids trigger MAT-dependent mating, pathogenic hyphal growth, and parasexual reproduction of Malassezia furfur

The lipophilic yeast genus Malassezia dominates the human skin mycobiome and, while typically commensal, is associated with skin disorders in which hyphal cells are frequently observed. However, the mechanisms underlying hyphal differentiation and its contribution to pathogenesis remain poorly understood. Here, we show that hyphal growth in Malassezia furfur requires a complete mating-type system and is promoted by noncanonical reproductive interactions. Interspecific mating generates stable diploid hybrids with evidence of recombination, whereas intraspecific mating produces cell-fusion products that undergo recombination and haploidization, consistent with a parasexual cycle. Functional analyses reveal that the pheromone/receptor (P/R) locus governs cell recognition and fusion, while the homeodomain (HD) locus is required for hyphal development despite having lost canonical mating-type determination. Leveraging in vitro and in vivo skin models, we show that hyphal cells invade the epidermis more efficiently and elicit stronger inflammatory responses than yeast cells. Together, these findings link parasexual-like reproduction, mating-type loci function, and morphogenesis in Malassezia to pathogenic interactions with the host.

microbiology↗

bioRxiv2026

Aimea gen. nov. defines a novel plant-associated yeast genus in Microbotryomycetes with three novel species

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.

genomics↗

bioRxiv2026

Fourier transform infrared spectroscopy reveals high intraspecies diversity of Malassezia pachydermatis in dogs with atopic dermatitis

1Canine atopic dermatitis (CAD) is a chronic inflammatory skin condition sometimes associated with microbial dysbiosis, including alterations in colonization by the lipophilic yeast Malassezia pachydermatis. This study investigated the population diversity of M. pachydermatis in the ear canals of healthy and CAD-affected dogs using Fourier-transform infrared (FTIR) spectroscopy and whole genome sequencing (WGS). Among 60 dogs, M. pachydermatis prevalence was significantly higher in CAD cases than in healthy controls. FTIR spectroscopy revealed greater strain heterogeneity in CAD-affected dogs, often with distinct genotypes in each ear, while healthy dogs exhibited more homogeneous populations. Using a previously developed FTIR-based artificial neural network classifier, we assigned strains to three phylogroups. Strains from phylogroups I and III were significantly enriched in CAD-affected dogs, while phylogroup II was most prevalent overall and the dominant phylogroup in healthy controls. This suggests that CAD-associated inflammation may favor specific M. pachydermatis phylogroups and sub-clusters within phylogroups, shaping colonization dynamics. FTIR-based typing showed full concordance with WGS across 35 sequenced isolates, recapitulating relationships among phylogenetically related isolates and their similar phenotypic profiles. Overall, our findings reveal strain-level shifts in M. pachydermatis populations associated with CAD and establish FTIR spectroscopy as a rapid, cost-effective tool for large-scale epidemiological studies.

microbiology↗

bioRxiv2025

Fourier transform infrared spectroscopy enables rapid species discrimination across Malassezia and strain-level typing in M. pachydermatis

1Malassezia pachydermatis is a zoophilic yeast found on the skin and in the outer ear canal of many mammals. It normally maintains a commensal lifestyle but can cause dermatitis and otitis in predisposed hosts, particularly in atopic dogs. M. pachydermatis is genetically diverse, with strains clustering into at least three phylogroups based on molecular typing, a pattern we now confirm through whole-genome sequencing (WGS). Accurate species and strain-level identification is essential for understanding its epidemiology, pathogenic potential, and response to treatment. In this study, we established Fourier Transform Infrared (FTIR) spectroscopy as a rapid, cost-effective method for distinguishing M. pachydermatis from other Malassezia species, including M. globosa, M. furfur, M. restricta, and M. sympodialis. Within M. pachydermatis, FTIR spectroscopy resolved even closely related strains with high accuracy producing clusters congruent with WGS-based phylogeny. The incorporation of an Artificial Neural Network classifier further enhanced the discriminatory power, enabling robust and automated strain assignment. These findings demonstrate the potential of FTIR spectroscopy as a practical tool for large-scale epidemiological surveillance of M. pachydermatis and for clinical and veterinary applications where strain-level identification could inform treatment and management of Malassezia-associated diseases. ImportanceMalassezia pachydermatis is a yeast that commonly inhabits the skin and ear canals of mammals but can cause dermatitis and otitis in predisposed hosts, especially dogs with allergies. This species displays substantial genetic diversity, with strains falling into distinct phylogroups that may differ in their biology and clinical significance. Determining these differences has typically required advanced molecular or genomic methods, which can be costly and time-consuming. In this study, we demonstrate that Fourier transform infrared spectroscopy can rapidly and accurately distinguish M. pachydermatis from other Malassezia species and resolve genetic groups within the species in a way that reflects whole-genome relationships. This capability offers a practical tool for investigating the epidemiology and inter-/intraspecies diversity of M. pachydermatis and for guiding targeted management of Malassezia-associated diseases in both veterinary and, potentially, human medicine.

microbiology↗

bioRxiv2025

Genomic rearrangements at the MAT locus suggest reproductive shifts in Rhodotorula yeasts

Transitions from canonical outcrossing to rare, cryptic, or noncanonical reproduction are widespread in fungi, but how mating-type (MAT) systems are reorganized at the genomic and population levels during these shifts remains unclear. The coexistence of sexually reproducing species and putatively asexual lineages makes Rhodotorula a natural comparative system for addressing this question. We analyzed 249 globally sampled strains using a combination of long-read and short-read sequencing. Phylogenomics resolved three major clades and several closely related species complexes. Chromosome-scale assemblies confirmed physical separation of the pheromone/receptor (P/R) and homeodomain (HD) loci, whereas non-random P/R-HD associations revealed constrained tetrapolar inheritance. The P/R locus showed conserved synteny within mating types but extensive structural divergence between A1 and A2 alleles, whereas the HD locus remained compact and conserved. Mating-type distributions varied across clades, with both A1 and A2 present in Clade C, lineage-structured partitioning in Clade B, and pronounced A2 dominance in Clade A. Genome-wide linkage disequilibrium (LD) decay in putatively asexual R. mucilaginosa, although slower than in sexual R. diobovata, was consistent with historical recombination in a species without an observed sexual cycle. Clade A further exhibited deep trans-specific polymorphism of STE3.A2 and relaxed purifying selection, mosaic A2 P/R configurations retaining RHA.A1, A1/A2 and A2/A2 hybrid genomes, and A2-associated gene content beyond MAT loci. In conclusion, our findings underscore the dynamic nature of MAT-locus remodeling in Rhodotorula and establish this genus as a valuable model for investigating transitions from heterothallism toward reproductive strategies less dependent on opposite-type partners.

evolutionary biology↗

bioRxiv2025

Genomic and phenotypic insights into the expanding phylogenetic landscape of the Cryptococcus genus

The fungal genus Cryptococcus includes several life-threatening human pathogens as well as diverse saprobic species whose genome architecture, ecology, and evolutionary history remain less well characterized. Understanding how some lineages evolved into major pathogens remains a central challenge and may be advanced by comparisons with their nonpathogenic counterparts. Integrative approaches have become essential for delimiting species and reconstructing evolutionary relationships, particularly in lineages with cryptic diversity or extensive chromosomal rearrangements. Here, we formally characterize six Cryptococcus species representing distinct evolutionary lineages, comprising both newly discovered and previously recognized but unnamed taxa, through a combination of phylogenomic analyses, divergence metrics, chromosomal comparisons, mating assays, and phenotypic profiling. Among pathogenic taxa, we formally name Cryptococcus hyracis sp. nov., corresponding to the previously characterized VGV lineage within the C. gattii complex. In parallel, we describe five saprobic, nonpathogenic species isolated from fruit, soil, and bark beetle galleries, spanning four phylogenetic clades. We identify a strong ecological association with bark beetles for Cryptococcus porticicola sp. nov., the only newly described nonpathogenic species with multiple sequenced strains from diverse sites. In this species, we detect strain-level chromosomal variation and evidence of sexual reproduction, along with population-level signatures of recombination. Across the genus, chromosome-level comparisons reveal extensive structural variation, including species- and strain-specific rearrangements that may restrict gene flow. We also identify multiple instances of chromosome number reduction, often accompanied by genomic signatures consistent with centromere inactivation or loss of centromeric identity. Comparative metabolic profiling with Biolog phenotype microarrays reveals clade-level differentiation and distinct substrate preferences, which may reflect metabolic divergence and habitat-specific diversification. Notably, we confirm that thermotolerance is restricted to clinically relevant taxa. These findings refine the species-level taxonomy of Cryptococcus, broaden its known genomic and ecological diversity, and strengthen the framework for investigating speciation, adaptation, and the emergence of pathogenicity within the genus. Author SummaryCryptococcus is a genus of fungi that includes both pathogenic species capable of causing life-threatening infections in humans and many environmental species that inhabit soil, fruit, decaying wood, and insect-associated environments with no known link to disease. Here, we formally describe six previously unnamed species based on genome-wide comparisons, genetic divergence metrics, chromosome structure, reproductive behavior, and metabolic profiling. One of these species belongs to the pathogenic lineage, while the others are distinct environmental taxa, including several recovered from bark beetles or their galleries, indicating repeated occurrence in this ecological niche. For one of these insect-associated species, represented by multiple isolates, we detected signatures of sexual reproduction and genetic exchange in natural populations. This study expands the framework for investigating how certain Cryptococcus lineages adapt to new environments or evolve traits relevant to pathogenicity. It also emphasizes that continuous environmental sampling remains essential for capturing fungal diversity and illustrates the power of combining genomic and phenotypic data to define species boundaries and uncover lineages that may otherwise remain undetected.

genomics↗

bioRxiv2025

BEstimate: a computational tool for the design and interpretation of CRISPR base editing experiments

CRISPR base editors enable scalable targeted DNA mutagenesis and are a powerful tool for the interpretation of gene function, the study of variants of unknown significance, and disease modelling. Existing guide RNA (gRNA) design tools focus on engineering a small number of targeted variants in a given gene or lack comprehensive functional annotation of target sequences. Here we developed BEstimate, a flexible computational pipeline that systematically specifies base editor gRNA target sites, generates gRNA off-target predictions, and rich functional, structural and clinical annotations of targeted edits. BEstimate accommodates custom gRNA design against variant alleles, improving editing accuracy and facilitating the programmed reversion of disease variants. BEstimate is a freely available, versatile tool for designing gRNA libraries and analysing base editor screens.

bioinformatics↗

bioRxiv2025

CRISPR screens in the context of immune selection identify CHD1 and MAP3K7 as mediators of cancer immunotherapy resistance

Cancer immunotherapy is only effective in a subset of patients, highlighting the need for effective biomarkers and combination therapies. Here we systematically identify genetic determinants of cancer cell sensitivity to anti-tumor immunity by performing whole-genome CRISPR/Cas9 knock-out screens in autologous tumoroid-T cell co-cultures, isogenic cancer cell models deficient in interferon signaling, and in the context of four cytokines. We discover that loss of CHD1 and MAP3K7 potentiates the transcriptional response to IFN-{gamma}, thereby creating an acquired vulnerability through sensitizing cancer cells to tumor-reactive T cells. Immune checkpoint blockade was more effective in a syngeneic mouse model of melanoma deficient in Chd1 and Map3k7 and was associated with elevated intra-tumoral CD8+ T cell numbers and activation. CHD1 and MAP3K7 are recurrently mutated in cancer and reduced expression in tumors correlates with response to immune checkpoint inhibitors in patients, nominating these genes as potential biomarkers of immunotherapy response.

cancer biology↗

bioRxiv2025

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.

genetics↗

bioRxiv2023

Comparative genomics of Cryptococcus and Kwoniella reveals pathogenesis evolution and contrasting modes of karyotype evolution via chromosome fusion or intercentromeric recombination

A large-scale comparative genomic analysis was conducted for the global human fungal pathogens within the Cryptococcus genus, compared to non-pathogenic Cryptococcus species, and related species from the sister genus Kwoniella. Chromosome-level genome assemblies were generated for multiple species of both genera, resulting in a dataset encompassing virtually all of their known diversity. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at pre-adaptive pathogenic potential, our analysis found evidence in pathogenic Cryptococcus species of specific examples of gene gain (via horizontal gene transfer) and gene loss, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the two genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5 or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion-fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus. Instead, Cryptococcus species with less than 14 chromosomes, underwent chromosome reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Taken together, our findings advance our understanding of genomic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.

genomics↗

bioRxiv2023

High-throughput phenotyping of single nucleotide variants by linking transcriptomes to genotypes in single cells

CRISPR screens with single-cell transcriptomic readouts are a valuable tool to understand the effect of genetic perturbations, but are currently limited because genotypes are inferred from the guide RNA identity. We have developed a technique that couples single-cell genotyping to transcriptomics of the same cells to enable screening for the effects of single nucleotide variants. Analysis of variants tiling across the JAK1 gene demonstrates the importance of determining the precise genetic perturbation and classifies missense variants into three functional categories.

genetics↗

bioRxiv2023

Genetic and genomic analysis of Malassezia reveals transitions in mating-type locus chromosomal organization and early steps in sexual reproduction

Fungi in the basidiomycete genus Malassezia are the most prevalent eukaryotic microbes resident on the skin of human and other warm-blooded animals and have been implicated in skin diseases and systemic disorders. Analysis of Malassezia genomes revealed that key adaptations to the skin microenvironment have a direct genomic basis, and the identification of mating/meiotic genes suggests a capacity to reproduce sexually, even though no sexual cycle has yet been observed. In contrast to other bipolar or tetrapolar basidiomycetes that have either two linked mating-type-determining (MAT) loci or two MAT loci on separate chromosomes, in Malassezia species studied thus far the two MAT loci are arranged in a pseudobipolar configuration (linked on the same chromosome but capable of recombining). By incorporating newly generated chromosome-level genome assemblies, and an improved Malassezia phylogeny, we infer that the pseudobipolar arrangement was the ancestral state of this group and revealed six independent transitions to tetrapolarity, seemingly driven by centromere fission or translocations in centromere- flanking regions. Additionally, in an approach to uncover a sexual cycle, Malassezia furfur strains were engineered to express different MAT alleles in the same cell. The resulting strains produce hyphae reminiscent of early steps in sexual development and display upregulation of genes associated with sexual development as well as others encoding lipases and a protease potentially relevant for pathogenesis of the fungus. Our study reveals a previously unseen genomic relocation of mating-type loci in fungi and provides insight towards the discovery of a sexual cycle in Malassezia, with possible implications for pathogenicity. Significance StatementMalassezia, the dominant fungal group of the mammalian skin microbiome, is associated with numerous skin disorders. Sexual development and yeast-to-hyphae transitions, governed by genes at two mating-type (MAT) loci, are thought to be important for fungal pathogenicity. However, Malassezia sexual reproduction has never been observed. Here, we used chromosome-level assemblies and comparative genomics to uncover unforeseen transitions in MAT loci organization within Malassezia, possibly related with fragility of centromeric-associated regions. Additionally, by expressing different MAT alleles in the same cell, we show that Malassezia can undergo hyphal development and this phenotype is associated with increased expression of key mating genes along with other genes known to be virulence factors, providing a possible connection between hyphal development, sexual reproduction, and pathogenicity.

genetics↗

bioRxiv2022

Obligate sexual reproduction of a homothallic fungus closely related to the Cryptococcus pathogenic species complex

Sexual reproduction is a ubiquitous and ancient trait of eukaryotic life. While sexual organisms are usually faced with the challenge of finding a compatible mating partner, species as diverse as animals, plants, and fungi have repeatedly evolved the ability to reproduce sexually without an obligate requirement for another individual. Here, we uncovered the underlying mechanism of self-compatibility (homothallism) in Cryptococcus depauperatus, a fungal species sister to the clinically relevant human fungal pathogens Cryptococcus neoformans and Cryptococcus gattii species complexes. In contrast to C. neoformans or C. gattii, which grow as a yeast in the asexual stage, and produce hyphae, basidia, and infectious spores during the sexual stage, C. depauperatus grows exclusively as hyphae decorated with basidia and abundant spores and appears to be continuously engaged in sexual reproduction. By combining the insights from comparative genomics and genetic analyses of mutants defective in key mating and meiosis genes, we demonstrate the sexual cycle of C. depauperatus involves meiosis, and reveal that self-compatibility is orchestrated by the expression, in the same cell, of an unlinked mating receptor (Ste3a) and pheromone ligand (MF) pair seemingly derived from opposite mating types of a heterothallic (self-sterile) ancestor. We identified a putative mating-type (MAT) determining region containing genes phylogenetically aligned with MATa alleles of other species, and a few MAT gene alleles scattered and unlinked throughout the genome, but no homologs of the mating-type homeodomain genes SXI1 (HD1) and SXI2 (HD2). Comparative genomic analyses suggested a dramatic remodeling of the MAT locus possibly owing to reduced selective constraints to maintain mating-type genes in tight linkage, associated with a transition to self-fertility. Our findings support C. depauperatus as an obligately sexual, homothallic fungal species and provide additional insight into the repeated transitions between modes of sexual reproduction that have occurred throughout the fungal kingdom.

microbiology↗

bioRxiv2022

Base editing screens map mutations affecting IFNγ signalling in cancer

IFN{gamma}signalling underpins host responses to infection, inflammation and anti-tumour immunity. Mutations in the IFN{gamma}signalling pathway cause immunological disorders, haematological malignancies, and resistance to immune checkpoint blockade (ICB) in cancer, however the function of most clinically observed variants remain unknown. Here, we systematically investigate the genetic determinants of IFN{gamma}response in colorectal cancer cells using CRISPR-Cas9 screens and base editing mutagenesis. Deep mutagenesis of JAK1 with cytidine and adenine base editors, combined with pathway-wide screens, reveal loss-of-function and gain-of-function mutations with clinical precedence, including causal variants in haematological malignancies and mutations detected in patients refractory to ICB. We functionally validate variants of uncertain significance in primary tumour organoids, where engineering missense mutations in JAK1 enhanced or reduced sensitivity to autologous tumour-reactive T cells. By classifying > 300 missense variants altering IFN{gamma} pathway activity, we demonstrate the utility of base editing for mutagenesis at scale, and generate a resource to inform genetic diagnosis.

cancer biology↗

bioRxiv2021

Dynamic genome plasticity during unisexual reproduction in the human fungal pathogen Cryptococcus deneoformans

Genome copy number variation occurs during each mitotic and meiotic cycle and it is crucial for organisms to maintain their natural ploidy. Defects in ploidy transitions can lead to chromosome instability, which is a hallmark of cancer. Ploidy in the haploid human fungal pathogen Cryptococcus neoformans is exquisitely orchestrated and ranges from haploid to polyploid during sexual development and under various environmental and host conditions. However, the mechanisms controlling these ploidy transitions are largely unknown. During C. deneoformans (formerly C. neoformans var. neoformans, serotype D) unisexual reproduction, ploidy increases prior to the onset of meiosis, can be independent from cell-cell fusion and nuclear fusion, and likely occurs through an endoreplication pathway. To elucidate the molecular mechanisms underlying this ploidy transition, we identified twenty cell cycle-regulating genes encoding cyclins, cyclin-dependent kinases (CDK), and CDK regulators. We characterized four cyclin genes and two CDK regulator genes that were differentially expressed during unisexual reproduction and contributed to diploidization. To detect ploidy transition events, we generated a ploidy reporter, called NURAT, which can detect copy number increases via double selection for nourseothricin-resistant, uracil-prototrophic cells. Utilizing this ploidy reporter, we showed that ploidy transition from haploid to diploid can be detected during the early phases of unisexual reproduction. Interestingly, selection for the NURAT reporter revealed several instances of segmental aneuploidy of multiple chromosomes, which conferred azole resistance in some isolates. These findings provide further evidence of ploidy plasticity in fungi with significant biological and public health implications. Author SummaryPloidy is an intrinsic fundamental feature of all eukaryotic organisms, and ploidy variation and maintenance are critical to the organism survival and evolution. Fungi exhibit exquisite plasticity in ploidy variation in adaptation to various environmental stresses. For example, the haploid opportunistic human fungal pathogen C. deneoformans can generate diploid blastospores during unisexual reproduction and also forms polyploid titan cells during host infection, however, the mechanisms underlying these ploidy transitions are largely unknown. In this study, we elucidated the genetic regulatory circuitry governing ploidy duplication during C. deneoformans unisexual reproduction through the identification and characterization of cell cycle regulators that are differentially expressed during unisexual reproduction. We showed that four cyclin and two cyclin-dependent kinase regulator genes function in concert to orchestrate ploidy transition during unisexual reproduction. To trace and track ploidy transition events, we also generated a ploidy reporter and revealed the formation of segmental aneuploidy in addition to diploidization, illustrating the diverse mechanisms of genome plasticity in C. deneoformans.

microbiology↗

bioRxiv2020

Multiple pathways to homothallism in closely related yeast lineages in the Basidiomycota

Sexual reproduction in fungi relies on proteins with well-known functions encoded by the mating-type (MAT) loci. In the Basidiomycota, MAT loci are often bipartite, with the P/R locus encoding pheromone precursors and pheromone receptors and the HD locus encoding heterodimerizing homeodomain transcription factors (Hd1/Hd2). The interplay between different alleles of these genes within a single species usually generates at least two compatible mating types. However, a minority of species are homothallic, reproducing sexually without an obligate need for a compatible partner. Here we examine the organization and function of the MAT loci of Cystofilobasidium capitatum, a species in the order Cystofilobasidiales, which is unusually rich in homothallic species. We determined MAT gene content and organization in C. capitatum and found that it resembles a mating type of the closely related heterothallic species Cystofilobasidium ferigula. To explain the homothallic sexual reproduction observed in C. capitatum we examined HD-protein interactions in the two Cystofilobasidium species and determined C. capitatum MAT gene expression both in a natural setting and upon heterologous expression in Phaffia rhodozyma, a homothallic species belonging to a clade sister to Cystofilobasidium. We conclude that the molecular basis for homothallism in C. capitatum appears to be distinct from that previously established for P. rhodozyma. Unlike the latter species, homothallism in C. capitatum may involve constitutive activation or dispensability of the pheromone receptor and the functional replacement of the usual Hd1/Hd2 heterodimer by an Hd2 homodimer. Overall, our results suggest that homothallism evolved multiple times within the Cystofilobasidiales. ImportanceSexual reproduction is important for the biology of eukaryotes because it strongly impacts the dynamics of genetic variation. In fungi, although sexual reproduction is usually associated with the fusion between cells belonging to different individuals (heterothallism), sometimes a single individual is capable of completing the sexual cycle alone (homothallism). Homothallic species are unusually common in a fungal lineage named Cystofilobasidiales. Here we studied the genetic bases of homothallism in one species in this lineage, Cystofilobasidium capitatum, and found it to be different in several aspects when compared to another homothallic species, Phaffia rhodozyma, belonging to the genus most closely related to Cystofilobasidium. Our results strongly suggest that homothallism evolved independently in Phaffia and Cystofilobasidium, lending support to the idea that transitions between heterothallism and homothallism are not as infrequent as previously thought. Our work also helps to establish the Cystofilobasidiales as a model lineage in which to study these transitions.

microbiology↗

bioRxiv2020

Factors enforcing the species boundary between the human pathogens Cryptococcus neoformans and Cryptococcus deneoformans

Hybridization has resulted in the origin and variation in extant species, and hybrids continue to arise despite pre- and post-zygotic barriers that limit their formation and evolutionary success. One important system that maintains species boundaries in prokaryotes and eukaryotes is the mismatch repair pathway, which blocks recombination between divergent DNA sequences. Previous studies illuminated the role of the mismatch repair component Msh2 in blocking genetic recombination between divergent DNA during meiosis. Loss of Msh2 results in increased interspecific genetic recombination in bacterial and yeast models, and increased viability of progeny derived from yeast hybrid crosses. Hybrid isolates of two pathogenic fungal Cryptococcus species, Cryptococcus neoformans and Cryptococcus deneoformans, are isolated regularly from both clinical and environmental sources. In the present study, we sought to determine if loss of Msh2 would relax the species boundary between C. neoformans and C. deneoformans. We found that crosses between these two species in which both parents lack Msh2 produced hybrid progeny with increased viability and high levels of aneuploidy. Whole-genome sequencing revealed few instances of recombination among hybrid progeny and did not identify increased levels of recombination in progeny derived from parents lacking Msh2. Several hybrid progeny produced structures associated with sexual reproduction when incubated alone on nutrient-rich medium in light, a novel phenotype in Cryptococcus. These findings represent a unique, unexpected case where rendering the mismatch repair system defective did not result in increased meiotic recombination across a species boundary. This suggests that alternative pathways or other mismatch repair components limit meiotic recombination between homeologous DNA and enforce species boundaries in the basidiomycete Cryptococcus species. Author summarySeveral mechanisms enforce species boundaries by either preventing the formation of hybrids, known as pre-zygotic barriers, or preventing the viability and fecundity of hybrids, known as post-zygotic barriers. Despite these barriers, interspecific hybrids form at an appreciable frequency, such as hybrid isolates of the human fungal pathogenic species, Cryptococcus neoformans and Cryptococcus deneoformans, which are regularly isolated from both clinical and environmental sources. C. neoformans x C. deneoformans hybrids are typically highly aneuploid, sterile, and display phenotypes intermediate to those of either parent, although self-fertile isolates and transgressive phenotypes have been observed. One important mechanism known to enforce species boundaries or lead to incipient speciation is the DNA mismatch repair system, which blocks recombination between divergent DNA sequences during meiosis. The aim of this study was to determine if genetically deleting the DNA mismatch repair component Msh2 would relax the species boundary between C. neoformans and C. deneoformans. Progeny derived from C. neoformans x C. deneoformans crosses in which both parental strains lacked Msh2 had higher viability, and unlike previous studies in Saccharomyces, these Cryptococcus hybrid progeny had higher levels of aneuploidy and no observable increase in meiotic recombination at the whole-genome level.

genetics↗