Publications by authors named "Sajeet Haridas"

31 Publications

Unique genomic traits for cold adaptation in Naganishia vishniacii, a polyextremophile yeast isolated from Antarctica.

FEMS Yeast Res 2021 Jan;21(1)

Centro de Referencia en Levaduras y Tecnologı́a Cervecera (CRELTEC), Instituto Andino Patagónico de Tecnologı́as Biológicas y Geoambientales (IPATEC) - CONICET / Universidad Nacional del Comahue, San Carlos de Bariloche, Rı́o Negro 8400, Argentina.

Cold environments impose challenges to organisms. Polyextremophile microorganisms can survive in these conditions thanks to an array of counteracting mechanisms. Naganishia vishniacii, a yeast species hitherto only isolated from McMurdo Dry Valleys, Antarctica, is an example of a polyextremophile. Here we present the first draft genomic sequence of N. vishniacii. Using comparative genomics, we unraveled unique characteristics of cold associated adaptations. 336 putative genes (total: 6183) encoding solute transfers and chaperones, among others, were absent in sister species. Among genes shared by N. vishniacii and its closest related species we found orthologs encompassing possible evidence of positive selection (dN/dS > 1). Genes associated with photoprotection were found in agreement with high solar irradiation exposure. Also genes coding for desaturases and genomic features associated with cold tolerance (i.e. trehalose synthesis and lipid metabolism) were explored. Finally, biases in amino acid usage (namely an enrichment of glutamine and a trend in proline reduction) were observed, possibly conferring increased protein flexibility. To the best of our knowledge, such a combination of mechanisms for cold tolerance has not been previously reported in fungi, making N. vishniacii a unique model for the study of the genetic basis and evolution of cold adaptation strategies.
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http://dx.doi.org/10.1093/femsyr/foaa056DOI Listing
January 2021

The Architecture of Metabolism Maximizes Biosynthetic Diversity in the Largest Class of Fungi.

Mol Biol Evol 2020 10;37(10):2838-2856

Department of Plant Pathology, The Ohio State University, Columbus, OH.

Ecological diversity in fungi is largely defined by metabolic traits, including the ability to produce secondary or "specialized" metabolites (SMs) that mediate interactions with other organisms. Fungal SM pathways are frequently encoded in biosynthetic gene clusters (BGCs), which facilitate the identification and characterization of metabolic pathways. Variation in BGC composition reflects the diversity of their SM products. Recent studies have documented surprising diversity of BGC repertoires among isolates of the same fungal species, yet little is known about how this population-level variation is inherited across macroevolutionary timescales. Here, we applied a novel linkage-based algorithm to reveal previously unexplored dimensions of diversity in BGC composition, distribution, and repertoire across 101 species of Dothideomycetes, which are considered the most phylogenetically diverse class of fungi and known to produce many SMs. We predicted both complementary and overlapping sets of clustered genes compared with existing methods and identified novel gene pairs that associate with known secondary metabolite genes. We found that variation among sets of BGCs in individual genomes is due to nonoverlapping BGC combinations and that several BGCs have biased ecological distributions, consistent with niche-specific selection. We observed that total BGC diversity scales linearly with increasing repertoire size, suggesting that secondary metabolites have little structural redundancy in individual fungi. We project that there is substantial unsampled BGC diversity across specific families of Dothideomycetes, which will provide a roadmap for future sampling efforts. Our approach and findings lend new insight into how BGC diversity is generated and maintained across an entire fungal taxonomic class.
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http://dx.doi.org/10.1093/molbev/msaa122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530617PMC
October 2020

A comparative genomics study of 23 Aspergillus species from section Flavi.

Nat Commun 2020 02 27;11(1):1106. Epub 2020 Feb 27.

Department of Biotechnology and Bioengineering, Technical University of Denmark, Søltoft Plads 223, 2800, Kongens Lyngby, Denmark.

Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.
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http://dx.doi.org/10.1038/s41467-019-14051-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046712PMC
February 2020

Draft Genome Assemblies of Ionic Liquid-Resistant Yarrowia lipolytica PO1f and Its Superior Evolved Strain, YlCW001.

Microbiol Resour Announc 2020 Feb 27;9(9). Epub 2020 Feb 27.

Department of Chemical and Biomolecular Engineering, The University of Tennessee, Knoxville, Tennessee, USA

Adaptive laboratory evolution of PO1f in the benchmark ionic liquid (IL; 1-ethyl-3-methylimidazolium acetate) produced a superior IL-tolerant microorganism, strain YlCW001. Here, we report the genome sequences of PO1f and YlCW001 to study the robustness of and its potential use as a microbial platform for producing fuels and chemicals.
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http://dx.doi.org/10.1128/MRA.01356-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046820PMC
February 2020

Draft Genome Sequence of the Ectomycorrhizal Ascomycete .

Microbiol Resour Announc 2019 Dec 12;8(50). Epub 2019 Dec 12.

Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, Michigan, USA.

is a pioneer ectomycorrhizal fungus with facultative saprophytic capacities. Here, we sequenced the genome of strain Sb_GMNB300, which is estimated at 51.6 Mb in size with 872 assembled contigs accounting for 12,597 predicted coding genes. This genome will be useful for comparative studies of Pezizales ectomycorrhizal symbioses.
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http://dx.doi.org/10.1128/MRA.00857-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908789PMC
December 2019

Phyllosticta citricarpa and sister species of global importance to Citrus.

Mol Plant Pathol 2019 12 11;20(12):1619-1635. Epub 2019 Sep 11.

Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands.

Several Phyllosticta species are known as pathogens of Citrus spp., and are responsible for various disease symptoms including leaf and fruit spots. One of the most important species is P. citricarpa, which causes a foliar and fruit disease called citrus black spot. The Phyllosticta species occurring on citrus can most effectively be distinguished from P. citricarpa by means of multilocus DNA sequence data. Recent studies also demonstrated P. citricarpa to be heterothallic, and reported successful mating in the laboratory. Since the domestication of citrus, different clones of P. citricarpa have escaped Asia to other continents via trade routes, with obvious disease management consequences. This pathogen profile represents a comprehensive literature review of this pathogen and allied taxa associated with citrus, focusing on identification, distribution, genomics, epidemiology and disease management. This review also considers the knowledge emerging from seven genomes of Phyllosticta spp., demonstrating unknown aspects of these species, including their mating behaviour.

Taxonomy: Phyllosticta citricarpa (McAlpine) Aa, 1973. Kingdom Fungi, Phylum Ascomycota, Class Dothideomycetes, Order Botryosphaeriales, Family Phyllostictaceae, Genus Phyllosticta, Species citricarpa.

Host Range: Confirmed on more than 12 Citrus species, Phyllosticta citricarpa has only been found on plant species in the Rutaceae.

Disease Symptoms: P. citricarpa causes diverse symptoms such as hard spot, virulent spot, false melanose and freckle spot on fruit, and necrotic lesions on leaves and twigs.

Useful Websites: DOE Joint Genome Institute MycoCosm portals for the Phyllosticta capitalensis (https://genome.jgi.doe.gov/Phycap1), P. citriasiana (https://genome.jgi.doe.gov/Phycit1), P. citribraziliensis (https://genome.jgi.doe.gov/Phcit1), P. citrichinaensis (https://genome.jgi.doe.gov/Phcitr1), P. citricarpa (https://genome.jgi.doe.gov/Phycitr1, https://genome.jgi.doe.gov/Phycpc1), P. paracitricarpa (https://genome.jgi.doe.gov/Phy27169) genomes. All available Phyllosticta genomes on MycoCosm can be viewed at https://genome.jgi.doe.gov/Phyllosticta.
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http://dx.doi.org/10.1111/mpp.12861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859488PMC
December 2019

Megaphylogeny resolves global patterns of mushroom evolution.

Nat Ecol Evol 2019 04 18;3(4):668-678. Epub 2019 Mar 18.

Synthetic and Systems Biology Unit, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.

Mushroom-forming fungi (Agaricomycetes) have the greatest morphological diversity and complexity of any group of fungi. They have radiated into most niches and fulfil diverse roles in the ecosystem, including wood decomposers, pathogens or mycorrhizal mutualists. Despite the importance of mushroom-forming fungi, large-scale patterns of their evolutionary history are poorly known, in part due to the lack of a comprehensive and dated molecular phylogeny. Here, using multigene and genome-based data, we assemble a 5,284-species phylogenetic tree and infer ages and broad patterns of speciation/extinction and morphological innovation in mushroom-forming fungi. Agaricomycetes started a rapid class-wide radiation in the Jurassic, coinciding with the spread of (sub)tropical coniferous forests and a warming climate. A possible mass extinction, several clade-specific adaptive radiations and morphological diversification of fruiting bodies followed during the Cretaceous and the Paleogene, convergently giving rise to the classic toadstool morphology, with a cap, stalk and gills (pileate-stipitate morphology). This morphology is associated with increased rates of lineage diversification, suggesting it represents a key innovation in the evolution of mushroom-forming fungi. The increase in mushroom diversity started during the Mesozoic-Cenozoic radiation event, an era of humid climate when terrestrial communities dominated by gymnosperms and reptiles were also expanding.
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http://dx.doi.org/10.1038/s41559-019-0834-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443077PMC
April 2019

Draft Genome Assemblies of Five Robust Yarrowia lipolytica Strains Exhibiting High Lipid Production, Pentose Sugar Utilization, and Sugar Alcohol Secretion from Undetoxified Lignocellulosic Biomass Hydrolysates.

Microbiol Resour Announc 2018 Sep 27;7(12). Epub 2018 Sep 27.

Department of Chemical and Biomolecular Engineering, The University of Tennessee, Knoxville, Tennessee, USA.

Screening the genetic diversity of 45 Yarrowia lipolytica strains identified five candidates with unique metabolic capability and robustness in undetoxified switchgrass hydrolysates, including superior lipid production and efficient pentose sugar utilization. Here, we report the genome sequences of these strains to study their robustness and potential to produce fuels and chemicals.
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http://dx.doi.org/10.1128/MRA.01040-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256683PMC
September 2018

Regulation of Yeast-to-Hyphae Transition in Yarrowia lipolytica.

mSphere 2018 12 5;3(6). Epub 2018 Dec 5.

Environmental Molecular Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA

The yeast undergoes a morphological transition from yeast-to-hyphal growth in response to environmental conditions. A forward genetic screen was used to identify mutants that reliably remain in the yeast phase, which were then assessed by whole-genome sequencing. All the mutants identified, so named because of their colony morphology, exhibit independent loss of DNA at a repetitive locus made up of interspersed ribosomal DNA and short 10- to 40-mer telomere-like repeats. The loss of repetitive DNA is associated with downregulation of genes with stress response elements (5'-CCCCT-3') and upregulation of genes with cell cycle box (5'-ACGCG-3') motifs in their promoter region. The stress response element is bound by the transcription factor Msn2p in We confirmed that the (Yl) ortholog is required for hyphal growth and found that overexpression of Yl enables hyphal growth in strains. The cell cycle box is bound by the Mbp1p/Swi6p complex in to regulate G-to-S phase progression. We found that overexpression of either the Yl or Yl homologs decreased hyphal growth and that deletion of either Yl or Yl promotes hyphal growth in strains. A second forward genetic screen for reversion to hyphal growth was performed with the mutant to identify additional genetic factors regulating hyphal growth in Thirteen of the mutants sequenced from this screen had coding mutations in five kinases, including the histidine kinases Yl and Yl and kinases of the high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase cascade Yl, Yl, and Yl Together, these results demonstrate that transitions to hyphal growth in response to stress through multiple signaling pathways. Many yeasts undergo a morphological transition from yeast-to-hyphal growth in response to environmental conditions. We used forward and reverse genetic techniques to identify genes regulating this transition in We confirmed that the transcription factor Yl is required for the transition to hyphal growth and found that signaling by the histidine kinases Yl and Yl as well as the MAP kinases of the HOG pathway (Yl, Yl, and Yl) regulates the transition to hyphal growth. These results suggest that transitions to hyphal growth in response to stress through multiple kinase pathways. Intriguingly, we found that a repetitive portion of the genome containing telomere-like and rDNA repeats may be involved in the transition to hyphal growth, suggesting a link between this region and the general stress response.
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http://dx.doi.org/10.1128/mSphere.00541-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282006PMC
December 2018

Investigation of inter- and intraspecies variation through genome sequencing of Aspergillus section Nigri.

Nat Genet 2018 12 22;50(12):1688-1695. Epub 2018 Oct 22.

Department of Biotechnology and Bioengineering, Technical University of Denmark, Kongens Lyngby, Denmark.

Aspergillus section Nigri comprises filamentous fungi relevant to biomedicine, bioenergy, health, and biotechnology. To learn more about what genetically sets these species apart, as well as about potential applications in biotechnology and biomedicine, we sequenced 23 genomes de novo, forming a full genome compendium for the section (26 species), as well as 6 Aspergillus niger isolates. This allowed us to quantify both inter- and intraspecies genomic variation. We further predicted 17,903 carbohydrate-active enzymes and 2,717 secondary metabolite gene clusters, which we condensed into 455 distinct families corresponding to compound classes, 49% of which are only found in single species. We performed metabolomics and genetic engineering to correlate genotypes to phenotypes, as demonstrated for the metabolite aurasperone, and by heterologous transfer of citrate production to Aspergillus nidulans. Experimental and computational analyses showed that both secondary metabolism and regulation are key factors that are significant in the delineation of Aspergillus species.
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http://dx.doi.org/10.1038/s41588-018-0246-1DOI Listing
December 2018

An analysis of codon bias in six red yeast species.

Yeast 2019 01 16;36(1):53-64. Epub 2018 Oct 16.

Dipartimento di Agraria, Università degli Studi di Sassari, Sassari, Italy.

Red yeasts, primarily species of Rhodotorula, Sporobolomyces, and other genera of Pucciniomycotina, are traditionally considered proficient systems for lipid and terpene production, and only recently have also gained consideration for the production of a wider range of molecules of biotechnological potential. Improvements of transgene delivery protocols and regulated gene expression systems have been proposed, but a dearth of information on compositional and/or structural features of genes has prevented transgene sequence optimization efforts for high expression levels. Here, the codon compositional features of genes in six red yeast species were characterized, and the impact that evolutionary forces may have played in shaping this compositional bias was dissected by using several computational approaches. Results obtained are compatible with the hypothesis that mutational bias, although playing a significant role, cannot alone explain synonymous codon usage bias of genes. Nevertheless, several lines of evidences indicated a role for translational selection in driving the synonymous codons that allow high expression efficiency. These optimal synonymous codons are identified for each of the six species analyzed. Moreover, the presence of intragenic patterns of codon usage, which are thought to facilitate polyribosome formation, was highlighted. The information presented should be taken into consideration for transgene design for optimal expression in red yeast species.
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http://dx.doi.org/10.1002/yea.3359DOI Listing
January 2019

Fungal Genome Annotation.

Methods Mol Biol 2018 ;1775:171-184

United States Department of Energy Joint Genome Institute, Walnut Creek, CA, USA.

The term "genome annotation" includes identification of protein-coding and noncoding sequences (e.g., repeats, rDNA, and ncRNA) in genome assemblies and attaching functional information (metadata) to these annotated features. Here, we describe the basic outline of fungal nuclear and mitochondrial genome annotation as performed at the US Department of Energy Joint Genome Institute (JGI).
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http://dx.doi.org/10.1007/978-1-4939-7804-5_15DOI Listing
February 2019

Linking secondary metabolites to gene clusters through genome sequencing of six diverse species.

Proc Natl Acad Sci U S A 2018 01 9;115(4):E753-E761. Epub 2018 Jan 9.

Department of Biotechnology and Biomedicine, Technical University of Denmark, 2800 Lyngby, Denmark;

The fungal genus of is highly interesting, containing everything from industrial cell factories, model organisms, and human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverse species (, , , and ) have been whole-genome PacBio sequenced to provide genetic references in three sections. and also were sequenced for SM elucidation. Thirteen genomes were analyzed with comparative genomics to determine phylogeny and genetic diversity, showing that each presented genome contains 15-27% genes not found in other sequenced Aspergilli. In particular, was compared with the pathogenic species This suggests that can produce most of the same allergens, virulence, and pathogenicity factors as , suggesting that could be as pathogenic as Furthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences, and predictive algorithms. We thus identify putative SM clusters for aflatoxin, chlorflavonin, and ochrindol in , , and , respectively, and novofumigatonin, -cycloechinulin, and -aszonalenins in Our study delivers six fungal genomes, showing the large diversity found in the genus; highlights the potential for discovery of beneficial or harmful SMs; and supports reports of pathogenicity. It also shows how biological, biochemical, and genomic information can be combined to identify genes involved in the biosynthesis of specific SMs.
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http://dx.doi.org/10.1073/pnas.1715954115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789934PMC
January 2018

Comparative genomics of Coniophora olivacea reveals different patterns of genome expansion in Boletales.

BMC Genomics 2017 Nov 16;18(1):883. Epub 2017 Nov 16.

Genetics and Microbiology Research Group, Department of Agrarian Production, Public University of Navarre, 31006, Pamplona, Navarre, Spain.

Background: Coniophora olivacea is a basidiomycete fungus belonging to the order Boletales that produces brown-rot decay on dead wood of conifers. The Boletales order comprises a diverse group of species including saprotrophs and ectomycorrhizal fungi that show important differences in genome size.

Results: In this study we report the 39.07-megabase (Mb) draft genome assembly and annotation of C. olivacea. A total of 14,928 genes were annotated, including 470 putatively secreted proteins enriched in functions involved in lignocellulose degradation. Using similarity clustering and protein structure prediction we identified a new family of 10 putative lytic polysaccharide monooxygenase genes. This family is conserved in basidiomycota and lacks of previous functional annotation. Further analyses showed that C. olivacea has a low repetitive genome, with 2.91% of repeats and a restrained content of transposable elements (TEs). The annotation of TEs in four related Boletales yielded important differences in repeat content, ranging from 3.94 to 41.17% of the genome size. The distribution of insertion ages of LTR-retrotransposons showed that differential expansions of these repetitive elements have shaped the genome architecture of Boletales over the last 60 million years.

Conclusions: Coniophora olivacea has a small, compact genome that shows macrosynteny with Coniophora puteana. The functional annotation revealed the enzymatic signature of a canonical brown-rot. The annotation and comparative genomics of transposable elements uncovered their particular contraction in the Coniophora genera, highlighting their role in the differential genome expansions found in Boletales species.
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http://dx.doi.org/10.1186/s12864-017-4243-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5689174PMC
November 2017

Widespread adenine N6-methylation of active genes in fungi.

Nat Genet 2017 Jun 8;49(6):964-968. Epub 2017 May 8.

US Department of Energy Joint Genome Institute, Walnut Creek, California, USA.

N6-methyldeoxyadenine (6mA) is a noncanonical DNA base modification present at low levels in plant and animal genomes, but its prevalence and association with genome function in other eukaryotic lineages remains poorly understood. Here we report that abundant 6mA is associated with transcriptionally active genes in early-diverging fungal lineages. Using single-molecule long-read sequencing of 16 diverse fungal genomes, we observed that up to 2.8% of all adenines were methylated in early-diverging fungi, far exceeding levels observed in other eukaryotes and more derived fungi. 6mA occurred symmetrically at ApT dinucleotides and was concentrated in dense methylated adenine clusters surrounding the transcriptional start sites of expressed genes; its distribution was inversely correlated with that of 5-methylcytosine. Our results show a striking contrast in the genomic distributions of 6mA and 5-methylcytosine and reinforce a distinct role for 6mA as a gene-expression-associated epigenomic mark in eukaryotes.
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http://dx.doi.org/10.1038/ng.3859DOI Listing
June 2017

Ectomycorrhizal ecology is imprinted in the genome of the dominant symbiotic fungus Cenococcum geophilum.

Nat Commun 2016 09 7;7:12662. Epub 2016 Sep 7.

INRA, UMR INRA-Université de Lorraine 'Interactions Arbres/Microorganismes', Laboratoire d'Excellence ARBRE, INRA-Nancy, 54280 Champenoux, France.

The most frequently encountered symbiont on tree roots is the ascomycete Cenococcum geophilum, the only mycorrhizal species within the largest fungal class Dothideomycetes, a class known for devastating plant pathogens. Here we show that the symbiotic genomic idiosyncrasies of ectomycorrhizal basidiomycetes are also present in C. geophilum with symbiosis-induced, taxon-specific genes of unknown function and reduced numbers of plant cell wall-degrading enzymes. C. geophilum still holds a significant set of genes in categories known to be involved in pathogenesis and shows an increased genome size due to transposable elements proliferation. Transcript profiling revealed a striking upregulation of membrane transporters, including aquaporin water channels and sugar transporters, and mycorrhiza-induced small secreted proteins (MiSSPs) in ectomycorrhiza compared with free-living mycelium. The frequency with which this symbiont is found on tree roots and its possible role in water and nutrient transport in symbiosis calls for further studies on mechanisms of host and environmental adaptation.
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http://dx.doi.org/10.1038/ncomms12662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023957PMC
September 2016

Comparative genomics of biotechnologically important yeasts.

Proc Natl Acad Sci U S A 2016 08 17;113(35):9882-7. Epub 2016 Aug 17.

Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706;

Ascomycete yeasts are metabolically diverse, with great potential for biotechnology. Here, we report the comparative genome analysis of 29 taxonomically and biotechnologically important yeasts, including 16 newly sequenced. We identify a genetic code change, CUG-Ala, in Pachysolen tannophilus in the clade sister to the known CUG-Ser clade. Our well-resolved yeast phylogeny shows that some traits, such as methylotrophy, are restricted to single clades, whereas others, such as l-rhamnose utilization, have patchy phylogenetic distributions. Gene clusters, with variable organization and distribution, encode many pathways of interest. Genomics can predict some biochemical traits precisely, but the genomic basis of others, such as xylose utilization, remains unresolved. Our data also provide insight into early evolution of ascomycetes. We document the loss of H3K9me2/3 heterochromatin, the origin of ascomycete mating-type switching, and panascomycete synteny at the MAT locus. These data and analyses will facilitate the engineering of efficient biosynthetic and degradative pathways and gateways for genomic manipulation.
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http://dx.doi.org/10.1073/pnas.1603941113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024638PMC
August 2016

Comparative Analysis of Secretome Profiles of Manganese(II)-Oxidizing Ascomycete Fungi.

PLoS One 2016 19;11(7):e0157844. Epub 2016 Jul 19.

Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, United States of America.

Fungal secretomes contain a wide range of hydrolytic and oxidative enzymes, including cellulases, hemicellulases, pectinases, and lignin-degrading accessory enzymes, that synergistically drive litter decomposition in the environment. While secretome studies of model organisms such as Phanerochaete chrysosporium and Aspergillus species have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or conducted side-by-side comparisons of diverse species. Thus, the mechanisms of carbon degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of LC-MS/MS, genomic, and bioinformatic analyses to characterize and compare the protein composition of the secretomes of four recently isolated, cosmopolitan, Mn(II)-oxidizing Ascomycetes (Alternaria alternata SRC1lrK2f, Stagonospora sp. SRC1lsM3a, Pyrenochaeta sp. DS3sAY3a, and Paraconiothyrium sporulosum AP3s5-JAC2a). We demonstrate that the organisms produce a rich yet functionally similar suite of extracellular enzymes, with species-specific differences in secretome composition arising from unique amino acid sequences rather than overall protein function. Furthermore, we identify not only a wide range of carbohydrate-active enzymes that can directly oxidize recalcitrant carbon, but also an impressive suite of redox-active accessory enzymes that suggests a role for Fenton-based hydroxyl radical formation in indirect, non-specific lignocellulose attack. Our findings highlight the diverse oxidative capacity of these environmental isolates and enhance our understanding of the role of filamentous Ascomycetes in carbon turnover in the environment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0157844PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4951024PMC
July 2017

Draft Genome Sequence of Microdochium bolleyi, a Dark Septate Fungal Endophyte of Beach Grass.

Genome Announc 2016 Apr 28;4(2). Epub 2016 Apr 28.

Department of Ecology, Evolution and Behavior, University of Minnesota, Saint Paul, Minnesota, USA Department of Plant Biology, University of Minnesota, Saint Paul, Minnesota, USA

Here, we present the genome sequence of the dark septate fungal endophyte Microdochium bolleyi (Ascomycota, Sordariomycetes, Xylariales). The assembled genome size was 38.84 Mbp and consisted of 173 scaffolds and 13,177 predicted genes.
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http://dx.doi.org/10.1128/genomeA.00270-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850852PMC
April 2016

Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis cinerea.

PLoS One 2015 28;10(10):e0141586. Epub 2015 Oct 28.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599-3280, United States of America.

The basidiomycete fungus Coprinopsis cinerea is an important model system for multicellular development. Fruiting bodies of C. cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC). To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141586PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624876PMC
June 2016

Draft Genome Sequence of Neurospora crassa Strain FGSC 73.

Genome Announc 2015 Apr 2;3(2). Epub 2015 Apr 2.

Fungal Genetics Stock Center, Kansas State University, Manhattan, Kansas, USA

We report the elucidation of the complete genome of the Neurospora crassa (Shear and Dodge) strain FGSC 73, a mat-a, trp-3 mutant strain. The genome sequence around the idiotypic mating type locus represents the only publicly available sequence for a mat-a strain. 40.42 Megabases are assembled into 358 scaffolds carrying 11,978 gene models.
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http://dx.doi.org/10.1128/genomeA.00074-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384475PMC
April 2015

Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii.

Fungal Genet Biol 2015 Mar 12;76:78-92. Epub 2015 Feb 12.

Department of Biology, Clark University, 950 Main St, Worcester 01610, MA, USA. Electronic address:

Wood decay mechanisms in Agaricomycotina have been traditionally separated in two categories termed white and brown rot. Recently the accuracy of such a dichotomy has been questioned. Here, we present the genome sequences of the white-rot fungus Cylindrobasidium torrendii and the brown-rot fungus Fistulina hepatica both members of Agaricales, combining comparative genomics and wood decay experiments. C. torrendii is closely related to the white-rot root pathogen Armillaria mellea, while F. hepatica is related to Schizophyllum commune, which has been reported to cause white rot. Our results suggest that C. torrendii and S. commune are intermediate between white-rot and brown-rot fungi, but at the same time they show characteristics of decay that resembles soft rot. Both species cause weak wood decay and degrade all wood components but leave the middle lamella intact. Their gene content related to lignin degradation is reduced, similar to brown-rot fungi, but both have maintained a rich array of genes related to carbohydrate degradation, similar to white-rot fungi. These characteristics appear to have evolved from white-rot ancestors with stronger ligninolytic ability. F. hepatica shows characteristics of brown rot both in terms of wood decay genes found in its genome and the decay that it causes. However, genes related to cellulose degradation are still present, which is a plesiomorphic characteristic shared with its white-rot ancestors. Four wood degradation-related genes, homologs of which are frequently lost in brown-rot fungi, show signs of pseudogenization in the genome of F. hepatica. These results suggest that transition toward a brown-rot lifestyle could be an ongoing process in F. hepatica. Our results reinforce the idea that wood decay mechanisms are more diverse than initially thought and that the dichotomous separation of wood decay mechanisms in Agaricomycotina into white rot and brown rot should be revisited.
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http://dx.doi.org/10.1016/j.fgb.2015.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399860PMC
March 2015

Comparative genomics of the pine pathogens and beetle symbionts in the genus Grosmannia.

Mol Biol Evol 2014 Jun 12;31(6):1454-74. Epub 2014 Mar 12.

Department of Wood Science, University of British Columbia, Vancouver, British Columbia, Canada

Studies on beetle/tree fungal symbionts typically characterize the ecological and geographic distributions of the fungal populations. There is limited understanding of the genome-wide evolutionary processes that act within and between species as such fungi adapt to different environments, leading to physiological differences and reproductive isolation. Here, we assess genomic evidence for such evolutionary processes by extending our recent work on Grosmannia clavigera, which is vectored by the mountain pine beetle and jeffrey pine beetle. We report the genome sequences of an additional 11 G. clavigera (Gc) sensu lato strains from the two known sibling species, Grosmannia sp. (Gs) and Gc. The 12 fungal genomes are structurally similar, showing large-scale synteny within and between species. We identified 103,430 single-nucleotide variations that separated the Grosmannia strains into divergent Gs and Gc clades, and further divided each of these clades into two subclades, one of which may represent an additional species. Comparing variable genes between these lineages, we identified truncated genes and potential pseudogenes, as well as seven genes that show evidence of positive selection. As these variable genes are involved in secondary metabolism and in detoxifying or utilizing host-tree defense chemicals (e.g., polyketide synthases, oxidoreductases, and mono-oxygenases), their variants may reflect adaptation to the specific chemistries of the host trees Pinus contorta, P. ponderosa, and P. jeffreyi. This work provides a comprehensive resource for developing informative markers for landscape population genomics of these ecologically and economically important fungi, and an approach that could be extended to other beetle-tree-associated fungi.
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http://dx.doi.org/10.1093/molbev/msu102DOI Listing
June 2014

MycoCosm portal: gearing up for 1000 fungal genomes.

Nucleic Acids Res 2014 Jan 1;42(Database issue):D699-704. Epub 2013 Dec 1.

US Department of Energy Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA 94598, USA.

MycoCosm is a fungal genomics portal (http://jgi.doe.gov/fungi), developed by the US Department of Energy Joint Genome Institute to support integration, analysis and dissemination of fungal genome sequences and other 'omics' data by providing interactive web-based tools. MycoCosm also promotes and facilitates user community participation through the nomination of new species of fungi for sequencing, and the annotation and analysis of resulting data. By efficiently filling gaps in the Fungal Tree of Life, MycoCosm will help address important problems associated with energy and the environment, taking advantage of growing fungal genomics resources.
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http://dx.doi.org/10.1093/nar/gkt1183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965089PMC
January 2014

The genome and transcriptome of the pine saprophyte Ophiostoma piceae, and a comparison with the bark beetle-associated pine pathogen Grosmannia clavigera.

BMC Genomics 2013 Jun 2;14:373. Epub 2013 Jun 2.

Department of Wood Science, University of British Columbia, Vancouver, BC, Canada.

Background: Ophiostoma piceae is a wood-staining fungus that grows in the sapwood of conifer logs and lumber. We sequenced its genome and analyzed its transcriptomes under a range of growth conditions. A comparison with the genome and transcriptomes of the mountain pine beetle-associated pathogen Grosmannia clavigera highlights differences between a pathogen that colonizes and kills living pine trees and a saprophyte that colonizes wood and the inner bark of dead trees.

Results: We assembled a 33 Mbp genome in 45 scaffolds, and predicted approximately 8,884 genes. The genome size and gene content were similar to those of other ascomycetes. Despite having similar ecological niches, O. piceae and G. clavigera showed no large-scale synteny. We identified O. piceae genes involved in the biosynthesis of melanin, which causes wood discoloration and reduces the commercial value of wood products. We also identified genes and pathways involved in growth on simple carbon sources and in sapwood, O. piceae's natural substrate. Like the pathogen, the saprophyte is able to tolerate terpenes, which are a major class of pine tree defense compounds; unlike the pathogen, it cannot utilize monoterpenes as a carbon source.

Conclusions: This work makes available the second annotated genome of a softwood ophiostomatoid fungus, and suggests that O. piceae's tolerance to terpenes may be due in part to these chemicals being removed from the cells by an ABC transporter that is highly induced by terpenes. The data generated will provide the research community with resources for work on host-vector-fungus interactions for wood-inhabiting, beetle-associated saprophytes and pathogens.
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http://dx.doi.org/10.1186/1471-2164-14-373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3680317PMC
June 2013

The cytochromes P450 of Grosmannia clavigera: Genome organization, phylogeny, and expression in response to pine host chemicals.

Fungal Genet Biol 2013 Jan 27;50:72-81. Epub 2012 Oct 27.

Department of Wood Sciences, University of British Columbia, Vancouver, BC, Canada.

Grosmannia clavigera is a fungal associate of the mountain pine beetle (Dendroctonus ponderosae) and a pathogen of lodgepole pine (Pinus contorta) that must overcome terpenoid oleoresin and phenolic defenses of host trees. G. clavigera responds to monoterpene influx with complementary mechanisms that include export and the use of these compounds as a carbon source. Cytochromes P450 (CYPs) may also be involved in the metabolism of host defense compounds. We have identified and phylogenetically classified G. clavigera CYPs (CYPome). We show that although the G. clavigera CYPome has contracted in evolution, certain CYP families have expanded by duplication. We analyzed RNA-seq data for CYP expression following treatment with terpenes and pine phloem extracts to identify CYPs potentially involved in detoxification of these pine defense compounds. We also used transcriptome analysis of G. clavigera grown on monoterpenes, triglycerides or oleic acid as a carbon source to identify up-regulated CYPs that may be involved in the utilization of these compounds to support fungal growth. Finally, we identify secondary metabolite biosynthetic gene clusters that contain CYPs, and CYPs in clusters that may be involved in conversion of host chemicals.
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http://dx.doi.org/10.1016/j.fgb.2012.10.002DOI Listing
January 2013

A biologist's guide to de novo genome assembly using next-generation sequence data: A test with fungal genomes.

J Microbiol Methods 2011 Sep 3;86(3):368-75. Epub 2011 Jul 3.

Faculty of Forestry, University of British Columbia, Vancouver, B.C., Canada V6T 1Z4.

We offer a guide to de novo genome assembly using sequence data generated by the Illumina platform for biologists working with fungi or other organisms whose genomes are less than 100Mb in size. The guide requires no familiarity with sequencing assembly technology or associated computer programs. It defines commonly used terms in genome sequencing and assembly; provides examples of assembling short-read genome sequence data for four strains of the fungus Grosmannia clavigera using four assembly programs; gives examples of protocols and software; and presents a commented flowchart that extends from DNA preparation for submission to a sequencing center, through to processing and assembly of the raw sequence reads using freely available operating systems and software.
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http://dx.doi.org/10.1016/j.mimet.2011.06.019DOI Listing
September 2011

Genome and transcriptome analyses of the mountain pine beetle-fungal symbiont Grosmannia clavigera, a lodgepole pine pathogen.

Proc Natl Acad Sci U S A 2011 Feb 24;108(6):2504-9. Epub 2011 Jan 24.

Department of Wood Science, University of British Columbia, Vancouver, BC, Canada V6T 1Z4.

In western North America, the current outbreak of the mountain pine beetle (MPB) and its microbial associates has destroyed wide areas of lodgepole pine forest, including more than 16 million hectares in British Columbia. Grosmannia clavigera (Gc), a critical component of the outbreak, is a symbiont of the MPB and a pathogen of pine trees. To better understand the interactions between Gc, MPB, and lodgepole pine hosts, we sequenced the ∼30-Mb Gc genome and assembled it into 18 supercontigs. We predict 8,314 protein-coding genes, and support the gene models with proteome, expressed sequence tag, and RNA-seq data. We establish that Gc is heterothallic, and report evidence for repeat-induced point mutation. We report insights, from genome and transcriptome analyses, into how Gc tolerates conifer-defense chemicals, including oleoresin terpenoids, as they colonize a host tree. RNA-seq data indicate that terpenoids induce a substantial antimicrobial stress in Gc, and suggest that the fungus may detoxify these chemicals by using them as a carbon source. Terpenoid treatment strongly activated a ∼100-kb region of the Gc genome that contains a set of genes that may be important for detoxification of these host-defense chemicals. This work is a major step toward understanding the biological interactions between the tripartite MPB/fungus/forest system.
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http://dx.doi.org/10.1073/pnas.1011289108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3038703PMC
February 2011

The mitochondrial genome of the wood-degrading basidiomycete Trametes cingulata.

FEMS Microbiol Lett 2010 Jul 8;308(1):29-34. Epub 2010 Apr 8.

Department of Plant Biology, University of Minnesota, St Paul, MN, USA.

We present the 91,500 bp mitochondrial genome of the wood-degrading basidiomycete Trametes cingulata and compare it with the mitochondrial genomes of five additional Basidiomycota species. The Trametes mitochondrial genome encodes 15 proteins, 25 tRNAs and the small and large rRNAs. All of the genes, except one tRNA, are found on the same DNA strand. Several additional ORFs have also been identified; however, their sequences have not been conserved across the species we compared and they show no similarity to any known gene, suggesting that they may not correspond to authentic genes. The presence of endonuclease-like sequences in introns suggests a mechanism that explains the diversity of mitochondrial genome sizes that are unrelated to the gene content.
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http://dx.doi.org/10.1111/j.1574-6968.2010.01979.xDOI Listing
July 2010

A transient decrease in reactive oxygen species in roots leads to root hair deformation in the legume-rhizobia symbiosis.

New Phytol 2007 ;173(1):39-49

Department of Plant Biology, University of Minnesota, 250 Biological Sciences Building, 1445 Gortner Avenue, Saint Paul, MN 551088, USA.

* A possible role for reactive oxygen species (ROS) in root hair deformation in response to Nod factor (NF) was investigated using Medicago truncatula nodulation mutants, and an inhibitor and precursors of ROS. * In wild-type roots, ROS efflux transiently decreased approximately 1 h after NF treatment. Transcript accumulation of two NADPH oxidase homologs, respiratory burst oxidase homolog 2 (MtRBOH2) and MtRBOH3, also transiently decreased at 1 h. However, in the nonnodulating mutant Nod factor perception (nfp), transcript accumulation did not change. * Exogenous application of ROS prevented root hair swelling and branching induced by NF. When accumulation of ROS was prevented by diphenylene iodonium (DPI), NF did not induce root hair branching. Root treatment with DPI alone reduced ROS efflux and induced root hair tip swelling. Transient treatment of roots with DPI mimicked NF treatment and resulted in root hair branching in the absence of NF. A transient DPI treatment did not induce root hair branching in the nonlegumes Arabidopsis thaliana and tomato (Lycopersicon esculentum). * The results suggest a role for the transient reduction of ROS accumulation in governing NF-induced root hair deformation in legumes.
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http://dx.doi.org/10.1111/j.1469-8137.2006.01901.xDOI Listing
March 2007