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Asset Tag: Fungus

April 21, 2020  |  

Genome Sequencing of Cladobotryum protrusum Provides Insights into the Evolution and Pathogenic Mechanisms of the Cobweb Disease Pathogen on Cultivated Mushroom.

Cladobotryum protrusum is one of the mycoparasites that cause cobweb disease on cultivated edible mushrooms. However, the molecular mechanisms of evolution and pathogenesis of C. protrusum on mushrooms are largely unknown. Here, we report a high-quality genome sequence of C. protrusum using the single-molecule, real-time sequencing platform of PacBio and perform a comparative analysis with closely related fungi in the family Hypocreaceae. The C. protrusum genome, the first complete genome to be sequenced in the genus Cladobotryum, is 39.09 Mb long, with an N50 of 4.97 Mb, encoding 11,003 proteins. The phylogenomic analysis confirmed its inclusion in Hypocreaceae, with its evolutionary divergence time estimated to be ~170.1 million years ago. The genome encodes a large and diverse set of genes involved in secreted peptidases, carbohydrate-active enzymes, cytochrome P450 enzymes, pathogen?host interactions, mycotoxins, and pigments. Moreover, C. protrusum harbors arrays of genes with the potential to produce bioactive secondary metabolites and stress response-related proteins that are significant for adaptation to hostile environments. Knowledge of the genome will foster a better understanding of the biology of C. protrusum and mycoparasitism in general, as well as help with the development of effective disease control strategies to minimize economic losses from cobweb disease in cultivated edible mushrooms.

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April 21, 2020  |  

Effector gene reshuffling involves dispensable mini-chromosomes in the wheat blast fungus.

Newly emerged wheat blast disease is a serious threat to global wheat production. Wheat blast is caused by a distinct, exceptionally diverse lineage of the fungus causing rice blast disease. Through sequencing a recent field isolate, we report a reference genome that includes seven core chromosomes and mini-chromosome sequences that harbor effector genes normally found on ends of core chromosomes in other strains. No mini-chromosomes were observed in an early field strain, and at least two from another isolate each contain different effector genes and core chromosome end sequences. The mini-chromosome is enriched in transposons occurring most frequently at core chromosome ends. Additionally, transposons in mini-chromosomes lack the characteristic signature for inactivation by repeat-induced point (RIP) mutation genome defenses. Our results, collectively, indicate that dispensable mini-chromosomes and core chromosomes undergo divergent evolutionary trajectories, and mini-chromosomes and core chromosome ends are coupled as a mobile, fast-evolving effector compartment in the wheat pathogen genome.

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April 21, 2020  |  

Infection mechanisms and putative effector repertoire of the mosquito pathogenic oomycete Pythium guiyangense uncovered by genomic analysis.

Pythium guiyangense, an oomycete from a genus of mostly plant pathogens, is an effective biological control agent that has wide potential to manage diverse mosquitoes. However, its mosquito-killing mechanisms are almost unknown. In this study, we observed that P. guiyangense could utilize cuticle penetration and ingestion of mycelia into the digestive system to infect mosquito larvae. To explore pathogenic mechanisms, a high-quality genome sequence with 239 contigs and an N50 contig length of 1,009 kb was generated. The genome assembly is approximately 110 Mb, which is almost twice the size of other sequenced Pythium genomes. Further genome analysis suggests that P. guiyangense may arise from a hybridization of two related but distinct parental species. Phylogenetic analysis demonstrated that P. guiyangense likely evolved from common ancestors shared with plant pathogens. Comparative genome analysis coupled with transcriptome sequencing data suggested that P. guiyangense may employ multiple virulence mechanisms to infect mosquitoes, including secreted proteases and kazal-type protease inhibitors. It also shares intracellular Crinkler (CRN) effectors used by plant pathogenic oomycetes to facilitate the colonization of plant hosts. Our experimental evidence demonstrates that CRN effectors of P. guiyangense can be toxic to insect cells. The infection mechanisms and putative virulence effectors of P. guiyangense uncovered by this study provide the basis to develop improved mosquito control strategies. These data also provide useful knowledge on host adaptation and evolution of the entomopathogenic lifestyle within the oomycete lineage. A deeper understanding of the biology of P. guiyangense effectors might also be useful for management of other important agricultural pests.

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April 21, 2020  |  

Intercellular communication is required for trap formation in the nematode-trapping fungus Duddingtonia flagrans.

Nematode-trapping fungi (NTF) are a large and diverse group of fungi, which may switch from a saprotrophic to a predatory lifestyle if nematodes are present. Different fungi have developed different trapping devices, ranging from adhesive cells to constricting rings. After trapping, fungal hyphae penetrate the worm, secrete lytic enzymes and form a hyphal network inside the body. We sequenced the genome of Duddingtonia flagrans, a biotechnologically important NTF used to control nematode populations in fields. The 36.64 Mb genome encodes 9,927 putative proteins, among which are more than 638 predicted secreted proteins. Most secreted proteins are lytic enzymes, but more than 200 were classified as small secreted proteins (< 300 amino acids). 117 putative effector proteins were predicted, suggesting interkingdom communication during the colonization. As a first step to analyze the function of such proteins or other phenomena at the molecular level, we developed a transformation system, established the fluorescent proteins GFP and mCherry, adapted an assay to monitor protein secretion, and established gene-deletion protocols using homologous recombination or CRISPR/Cas9. One putative virulence effector protein, PefB, was transcriptionally induced during the interaction. We show that the mature protein is able to be imported into nuclei in Caenorhabditis elegans cells. In addition, we studied trap formation and show that cell-to-cell communication is required for ring closure. The availability of the genome sequence and the establishment of many molecular tools will open new avenues to studying this biotechnologically relevant nematode-trapping fungus.

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April 21, 2020  |  

Multi-omics characterization of the necrotrophic mycoparasite Saccharomycopsis schoenii.

Pathogenic yeasts and fungi are an increasing global healthcare burden, but discovery of novel antifungal agents is slow. The mycoparasitic yeast Saccharomycopsis schoenii was recently demonstrated to be able to kill the emerging multi-drug resistant yeast pathogen Candida auris. However, the molecular mechanisms involved in the predatory activity of S. schoenii have not been explored. To this end, we de novo sequenced, assembled and annotated a draft genome of S. schoenii. Using proteomics, we confirmed that Saccharomycopsis yeasts have reassigned the CTG codon and translate CTG into serine instead of leucine. Further, we confirmed an absence of all genes from the sulfate assimilation pathway in the genome of S. schoenii, and detected the expansion of several gene families, including aspartic proteases. Using Saccharomyces cerevisiae as a model prey cell, we honed in on the timing and nutritional conditions under which S. schoenii kills prey cells. We found that a general nutrition limitation, not a specific methionine deficiency, triggered predatory activity. Nevertheless, by means of genome-wide transcriptome analysis we observed dramatic responses to methionine deprivation, which were alleviated when S. cerevisiae was available as prey, and therefore postulate that S. schoenii acquired methionine from its prey cells. During predation, both proteomic and transcriptomic analyses revealed that S. schoenii highly upregulated and translated aspartic protease genes, probably used to break down prey cell walls. With these fundamental insights into the predatory behavior of S. schoenii, we open up for further exploitation of this yeast as a biocontrol yeast and/or source for novel antifungal agents.

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April 21, 2020  |  

Finding the needle in a haystack: Mapping antifungal drug resistance in fungal pathogen by genomic approaches.

Fungi are ubiquitous on earth and are essential for the maintenance of the global ecological equilibrium. Despite providing benefits to living organisms, they can also target specific hosts and inflict damage. These fungal pathogens are known to affect, for example, plants and mam- mals and thus reduce crop production necessary to sustain food supply and cause mortality in humans and animals. Designing defenses against these fungi is essential for the control of food resources and human health. As far as fungal pathogens are concerned, the principal option has been the use of antifungal agents, also called fungicides when they are used in the environment.

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April 21, 2020  |  

Anthropogenic N deposition, fungal gene expression, and an increasing soil carbon sink in the Northern Hemisphere.

Terrestrial ecosystems in the Northern Hemisphere are a globally important sink for anthropogenic CO2 in the Earth’s atmosphere, slowing its accumulation as well as the pace of climate warming. With the use of a long-term field experiment (ca. 20 yr), we show that the expression of fungal class II peroxidase genes, which encode enzymes mediating the rate-limiting step of organic matter decay, are significantly downregulated (-60 to -80%) because of increases in anthropogenic N deposition; this response was consistent with a decline in extracellular peroxidase enzyme activity in soil, the slowing of organic-matter decay, and greater soil C storage. The reduction in peroxidase expression we document here occurred in the absence of a compositional shift in metabolically active fungi, indicating that an overall reduction in peroxidase expression underlies the slowing of decay and increases in soil C storage. This molecular mechanism has global implications for soil C storage and should be represented in coupled climate-biogeochemical models simulating the influence of enhanced terrestrial C storage on atmospheric CO2 and the future climate of an N-enriched Earth. © 2019 by the Ecological Society of America.

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April 21, 2020  |  

A whole genome scan of SNP data suggests a lack of abundant hard selective sweeps in the genome of the broad host range plant pathogenic fungus Sclerotinia sclerotiorum.

The pathogenic fungus Sclerotinia sclerotiorum infects over 600 species of plant. It is present in numerous environments throughout the world and causes significant damage to many agricultural crops. Fragmentation and lack of gene flow between populations may lead to population sub-structure. Within discrete recombining populations, positive selection may lead to a ‘selective sweep’. This is characterised by an increase in frequency of a favourable allele leading to reduction in genotypic diversity in a localised genomic region due to the phenomenon of genetic hitchhiking. We aimed to assess whether isolates of S. sclerotiorum from around the world formed genotypic clusters associated with geographical origin and to determine whether signatures of population-specific positive selection could be detected. To do this, we sequenced the genomes of 25 isolates of S. sclerotiorum collected from four different continents-Australia, Africa (north and south), Europe and North America (Canada and the northen United States) and conducted SNP based analyses of population structure and selective sweeps. Among the 25 isolates, there was evidence for two major population clusters. One of these consisted of 11 isolates from Canada, the USA and France (population 1), and the other consisted of nine isolates from Australia and one from Morocco (population 2). The rest of the isolates were genotypic outliers. We found that there was evidence of outcrossing in these two populations based on linkage disequilibrium decay. However, only a single candidate selective sweep was observed, and it was present in population 2. This sweep was close to a Major Facilitator Superfamily transporter gene, and we speculate that this gene may have a role in nutrient uptake from the host. The low abundance of selective sweeps in the S. sclerotiorum genome contrasts the numerous examples in the genomes of other fungal pathogens. This may be a result of its slow rate of evolution and low effective recombination rate due to self-fertilisation and vegetative reproduction.

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April 21, 2020  |  

Genome Sequence of the Black Yeast Exophiala lecanii-corni.

The genome sequence of Exophiala lecanii-corni, a melanized dimorphic fungus with the capability of degrading several volatile organic compounds, was sequenced using PacBio single-molecule real-time (SMRT) sequencing to assist with understanding the molecular basis of its uncommon morphological and metabolic characteristics. The assembled draft genome is presented here.

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April 21, 2020  |  

Complete Genome Sequence of Malassezia restricta CBS 7877, an Opportunist Pathogen Involved in Dandruff and Seborrheic Dermatitis.

Malassezia restricta, one of the predominant basidiomycetous yeasts present on human skin, is involved in scalp disorders. Here, we report the complete genome sequence of the lipophilic Malassezia restricta CBS 7877 strain, which will facilitate the study of the mechanisms underlying its commensal and pathogenic roles within the skin microbiome.

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April 21, 2020  |  

The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications.

UNITE (https://unite.ut.ee/) is a web-based database and sequence management environment for the molecular identification of fungi. It targets the formal fungal barcode-the nuclear ribosomal internal transcribed spacer (ITS) region-and offers all ~1 000 000 public fungal ITS sequences for reference. These are clustered into ~459 000 species hypotheses and assigned digital object identifiers (DOIs) to promote unambiguous reference across studies. In-house and web-based third-party sequence curation and annotation have resulted in more than 275 000 improvements to the data over the past 15 years. UNITE serves as a data provider for a range of metabarcoding software pipelines and regularly exchanges data with all major fungal sequence databases and other community resources. Recent improvements include redesigned handling of unclassifiable species hypotheses, integration with the taxonomic backbone of the Global Biodiversity Information Facility, and support for an unlimited number of parallel taxonomic classification systems.

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April 21, 2020  |  

Estimating the Fitness Effect of Deleterious Mutations During the Two Phases of the Life Cycle: A New Method Applied to the Root-Rot Fungus Heterobasidion parviporum.

Many eukaryote species, including taxa such as fungi or algae, have a lifecycle with substantial haploid and diploid phases. A recent theoretical model predicts that such haploid-diploid lifecycles are stable over long evolutionary time scales when segregating deleterious mutations have stronger effects in homozygous diploids than in haploids and when they are partially recessive in heterozygous diploids. The model predicts that effective dominance-a measure that accounts for these two effects-should be close to 0.5 in these species. It also predicts that diploids should have higher fitness than haploids on average. However, an appropriate statistical framework to conjointly investigate these predictions is currently lacking. In this study, we derive a new quantitative genetic model to test these predictions using fitness data of two haploid parents and their diploid offspring, and genome-wide genetic distance between haploid parents. We apply this model to the root-rot basidiomycete fungus Heterobasidion parviporum-a species where the heterokaryotic (equivalent to the diploid) phase is longer than the homokaryotic (haploid) phase. We measured two fitness-related traits (mycelium growth rate and the ability to degrade wood) in both homokaryons and heterokaryons, and we used whole-genome sequencing to estimate nuclear genetic distance between parents. Possibly due to a lack of power, we did not find that deleterious mutations were recessive or more deleterious when expressed during the heterokaryotic phase. Using this model to compare effective dominance among haploid-diploid species where the relative importance of the two phases varies should help better understand the evolution of haploid-diploid life cycles. Copyright © 2019 by the Genetics Society of America.

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April 21, 2020  |  

A High-Quality Grapevine Downy Mildew Genome Assembly Reveals Rapidly Evolving and Lineage-Specific Putative Host Adaptation Genes.

Downy mildews are obligate biotrophic oomycete pathogens that cause devastating plant diseases on economically important crops. Plasmopara viticola is the causal agent of grapevine downy mildew, a major disease in vineyards worldwide. We sequenced the genome of Pl. viticola with PacBio long reads and obtained a new 92.94?Mb assembly with high contiguity (359 scaffolds for a N50 of 706.5?kb) due to a better resolution of repeat regions. This assembly presented a high level of gene completeness, recovering 1,592 genes encoding secreted proteins involved in plant-pathogen interactions. Plasmopara viticola had a two-speed genome architecture, with secreted protein-encoding genes preferentially located in gene-sparse, repeat-rich regions and evolving rapidly, as indicated by pairwise dN/dS values. We also used short reads to assemble the genome of Plasmopara muralis, a closely related species infecting grape ivy (Parthenocissus tricuspidata). The lineage-specific proteins identified by comparative genomics analysis included a large proportion of RxLR cytoplasmic effectors and, more generally, genes with high dN/dS values. We identified 270 candidate genes under positive selection, including several genes encoding transporters and components of the RNA machinery potentially involved in host specialization. Finally, the Pl. viticola genome assembly generated here will allow the development of robust population genomics approaches for investigating the mechanisms involved in adaptation to biotic and abiotic selective pressures in this species. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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