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Asset Tag: fungal genome

July 19, 2019

Mind the gap; seven reasons to close fragmented genome assemblies.

Like other domains of life, research into the biology of filamentous microbes has greatly benefited from the advent of whole-genome sequencing. Next-generation sequencing (NGS) technologies have revolutionized sequencing, making genomic sciences accessible to many academic laboratories including those that study non-model organisms. Thus, hundreds of fungal genomes have been sequenced and are publically available today, although these initiatives have typically yielded considerably fragmented genome assemblies that often lack large contiguous genomic regions. Many important genomic features are contained in intergenic DNA that is often missing in current genome assemblies, and recent studies underscore the significance of non-coding regions and repetitive elements for the life style, adaptability and evolution of many organisms. The study of particular types of genetic elements, such as telomeres, centromeres, repetitive elements, effectors, and clusters of co-regulated genes, but also of phenomena such as structural rearrangements, genome compartmentalization and epigenetics, greatly benefits from having a contiguous and high-quality, preferably even complete and gapless, genome assembly. Here we discuss a number of important reasons to produce gapless, finished, genome assemblies to help answer important biological questions. Copyright © 2015 Elsevier Inc. All rights reserved.

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July 19, 2019

Living apart together: crosstalk between the core and supernumerary genomes in a fungal plant pathogen.

Eukaryotes display remarkable genome plasticity, which can include supernumerary chromosomes that differ markedly from the core chromosomes. Despite the widespread occurrence of supernumerary chromosomes in fungi, their origin, relation to the core genome and the reason for their divergent characteristics are still largely unknown. The complexity of genome assembly due to the presence of repetitive DNA partially accounts for this.Here we use single-molecule real-time (SMRT) sequencing to assemble the genome of a prominent fungal wheat pathogen, Fusarium poae, including at least one supernumerary chromosome. The core genome contains limited transposable elements (TEs) and no gene duplications, while the supernumerary genome holds up to 25 % TEs and multiple gene duplications. The core genome shows all hallmarks of repeat-induced point mutation (RIP), a defense mechanism against TEs, specific for fungi. The absence of RIP on the supernumerary genome accounts for the differences between the two (sub)genomes, and results in a functional crosstalk between them. The supernumerary genome is a reservoir for TEs that migrate to the core genome, and even large blocks of supernumerary sequence (>200 kb) have recently translocated to the core. Vice versa, the supernumerary genome acts as a refuge for genes that are duplicated from the core genome.For the first time, a mechanism was determined that explains the differences that exist between the core and supernumerary genome in fungi. Different biology rather than origin was shown to be responsible. A “living apart together” crosstalk exists between the core and supernumerary genome, accelerating chromosomal and organismal evolution.

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July 19, 2019

Condition-dependent co-regulation of genomic clusters of virulence factors in the grapevine trunk pathogen Neofusicoccum parvum.

The ascomycete Neofusicoccum parvum, one of the causal agents of Botryosphaeria dieback, is a destructive wood-infecting fungus and a serious threat to grape production worldwide. The capability to colonize woody tissue, combined with the secretion of phytotoxic compounds, is thought to underlie its pathogenicity and virulence. Here, we describe the repertoire of virulence factors and their transcriptional dynamics as the fungus feeds on different substrates and colonizes the woody stem. We assembled and annotated a highly contiguous genome using single-molecule real-time DNA sequencing. Transcriptome profiling by RNA sequencing determined the genome-wide patterns of expression of virulence factors both in vitro (potato dextrose agar or medium amended with grape wood as substrate) and in planta. Pairwise statistical testing of differential expression, followed by co-expression network analysis, revealed that physically clustered genes coding for putative virulence functions were induced depending on the substrate or stage of plant infection. Co-expressed gene clusters were significantly enriched not only in genes associated with secondary metabolism, but also in those associated with cell wall degradation, suggesting that dynamic co-regulation of transcriptional networks contributes to multiple aspects of N. parvum virulence. In most of the co-expressed clusters, all genes shared at least a common motif in their promoter region, indicative of co-regulation by the same transcription factor. Co-expression analysis also identified chromatin regulators with correlated expression with inducible clusters of virulence factors, suggesting a complex, multi-layered regulation of the virulence repertoire of N. parvum.© 2016 BSPP AND JOHN WILEY & SONS LTD.

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July 19, 2019

Multiple origins of the pathogenic yeast Candida orthopsilosis by separate hybridizations between two parental species.

Mating between different species produces hybrids that are usually asexual and stuck as diploids, but can also lead to the formation of new species. Here, we report the genome sequences of 27 isolates of the pathogenic yeast Candida orthopsilosis. We find that most isolates are diploid hybrids, products of mating between two unknown parental species (A and B) that are 5% divergent in sequence. Isolates vary greatly in the extent of homogenization between A and B, making their genomes a mosaic of highly heterozygous regions interspersed with homozygous regions. Separate phylogenetic analyses of SNPs in the A- and B-derived portions of the genome produces almost identical trees of the isolates with four major clades. However, the presence of two mutually exclusive genotype combinations at the mating type locus, and recombinant mitochondrial genomes diagnostic of inter-clade mating, shows that the species C. orthopsilosis does not have a single evolutionary origin but was created at least four times by separate interspecies hybridizations between parents A and B. Older hybrids have lost more heterozygosity. We also identify two isolates with homozygous genomes derived exclusively from parent A, which are pure non-hybrid strains. The parallel emergence of the same hybrid species from multiple independent hybridization events is common in plant evolution, but is much less documented in pathogenic fungi.

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July 19, 2019

Widespread adenine N6-methylation of active genes in fungi.

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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July 19, 2019

Comparative genomics of two sequential Candida glabrata clinical isolates.

Candida glabrata is an important fungal pathogen which develops rapid antifungal resistance in treated patients. It is known that azole treatments lead to antifungal resistance in this fungal species and that multidrug efflux transporters are involved in this process. Specific mutations in the transcriptional regulator PDR1 result in upregulation of the transporters. In addition, we showed that the PDR1 mutations can contribute to enhance virulence in animal models. In this study, we were interested to compare genomes of two specific C. glabrata-related isolates, one of which was azole susceptible (DSY562) while the other was azole resistant (DSY565). DSY565 contained a PDR1 mutation (L280F) and was isolated after a time-lapse of 50 d of azole therapy. We expected that genome comparisons between both isolates could reveal additional mutations reflecting host adaptation or even additional resistance mechanisms. The PacBio technology used here yielded 14 major contigs (sizes 0.18-1.6 Mb) and mitochondrial genomes from both DSY562 and DSY565 isolates that were highly similar to each other. Comparisons of the clinical genomes with the published CBS138 genome indicated important genome rearrangements, but not between the clinical strains. Among the unique features, several retrotransposons were identified in the genomes of the investigated clinical isolates. DSY562 and DSY565 each contained a large set of adhesin-like genes (101 and 107, respectively), which exceed by far the number of reported adhesins (63) in the CBS138 genome. Comparison between DSY562 and DSY565 yielded 17 nonsynonymous SNPs (among which the was the expected PDR1 mutation) as well as small size indels in coding regions (11) but mainly in adhesin-like genes. The genomes contained a DNA mismatch repair allele of MSH2 known to be involved in the so-called hyper-mutator phenotype of this yeast species and the number of accumulated mutations between both clinical isolates is consistent with the presence of a MSH2 defect. In conclusion, this study is the first to compare genomes of C. glabrata sequential clinical isolates using the PacBio technology as an approach. The genomes of these isolates taken in the same patient at two different time points exhibited limited variations, even if submitted to the host pressure. Copyright © 2017 Vale-Silva et al.

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July 19, 2019

Iterative optimization of xylose catabolism in Saccharomyces cerevisiae using combinatorial expression tuning.

A common challenge in metabolic engineering is rapidly identifying rate-controlling enzymes in heterologous pathways for subsequent production improvement. We demonstrate a workflow to address this challenge and apply it to improving xylose utilization in Saccharomyces cerevisiae. For eight reactions required for conversion of xylose to ethanol, we screened enzymes for functional expression in S. cerevisiae, followed by a combinatorial expression analysis to achieve pathway flux balancing and identification of limiting enzymatic activities. In the next round of strain engineering, we increased the copy number of these limiting enzymes and again tested the eight-enzyme combinatorial expression library in this new background. This workflow yielded a strain that has a ~70% increase in biomass yield and ~240% increase in xylose utilization. Finally, we chromosomally integrated the expression library. This library enriched for strains with multiple integrations of the pathway, which likely were the result of tandem integrations mediated by promoter homology. Biotechnol. Bioeng. 2017;114: 1301-1309. © 2017 Wiley Periodicals, Inc.© 2017 Wiley Periodicals, Inc.

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July 19, 2019

The complete genome sequence of the phytopathogenic fungus Sclerotinia sclerotiorum reveals insights into the genome architecture of broad host range pathogens.

Sclerotinia sclerotiorum is a phytopathogenic fungus with over 400 hosts including numerous economically important cultivated species. This contrasts many economically destructive pathogens that only exhibit a single or very few hosts. Many plant pathogens exhibit a “two-speed” genome. So described because their genomes contain alternating gene rich, repeat sparse and gene poor, repeat-rich regions. In fungi, the repeat-rich regions may be subjected to a process termed repeat-induced point mutation (RIP). Both repeat activity and RIP are thought to play a significant role in evolution of secreted virulence proteins, termed effectors. We present a complete genome sequence of S. sclerotiorum generated using Single Molecule Real-Time Sequencing technology with highly accurate annotations produced using an extensive RNA sequencing data set. We identified 70 effector candidates and have highlighted their in planta expression profiles. Furthermore, we characterized the genome architecture of S. sclerotiorum in comparison to plant pathogens that exhibit “two-speed” genomes. We show that there is a significant association between positions of secreted proteins and regions with a high RIP index in S. sclerotiorum but we did not detect a correlation between secreted protein proportion and GC content. Neither did we detect a negative correlation between CDS content and secreted protein proportion across the S. sclerotiorum genome. We conclude that S. sclerotiorum exhibits subtle signatures of enhanced mutation of secreted proteins in specific genomic compartments as a result of transposition and RIP activity. However, these signatures are not observable at the whole-genome scale.

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July 19, 2019

Evolutionary restoration of fertility in an interspecies hybrid yeast, by whole-genome duplication after a failed mating-type switch.

Many interspecies hybrids have been discovered in yeasts, but most of these hybrids are asexual and can replicate only mitotically. Whole-genome duplication has been proposed as a mechanism by which interspecies hybrids can regain fertility, restoring their ability to perform meiosis and sporulate. Here, we show that this process occurred naturally during the evolution of Zygosaccharomyces parabailii, an interspecies hybrid that was formed by mating between 2 parents that differed by 7% in genome sequence and by many interchromosomal rearrangements. Surprisingly, Z. parabailii has a full sexual cycle and is genetically haploid. It goes through mating-type switching and autodiploidization, followed by immediate sporulation. We identified the key evolutionary event that enabled Z. parabailii to regain fertility, which was breakage of 1 of the 2 homeologous copies of the mating-type (MAT) locus in the hybrid, resulting in a chromosomal rearrangement and irreparable damage to 1 MAT locus. This rearrangement was caused by HO endonuclease, which normally functions in mating-type switching. With 1 copy of MAT inactivated, the interspecies hybrid now behaves as a haploid. Our results provide the first demonstration that MAT locus damage is a naturally occurring evolutionary mechanism for whole-genome duplication and restoration of fertility to interspecies hybrids. The events that occurred in Z. parabailii strongly resemble those postulated to have caused ancient whole-genome duplication in an ancestor of Saccharomyces cerevisiae.

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July 19, 2019

A mobile pathogenicity chromosome in Fusarium oxysporum for infection of multiple cucurbit species.

The genome of Fusarium oxysporum (Fo) consists of a set of eleven ‘core’ chromosomes, shared by most strains and responsible for housekeeping, and one or several accessory chromosomes. We sequenced a strain of Fo f.sp. radicis-cucumerinum (Forc) using PacBio SMRT sequencing. All but one of the core chromosomes were assembled into single contigs, and a chromosome that shows all the hallmarks of a pathogenicity chromosome comprised two contigs. A central part of this chromosome contains all identified candidate effector genes, including homologs of SIX6, SIX9, SIX11 and SIX 13. We show that SIX6 contributes to virulence of Forc. Through horizontal chromosome transfer (HCT) to a non-pathogenic strain, we also show that the accessory chromosome containing the SIX gene homologs is indeed a pathogenicity chromosome for cucurbit infection. Conversely, complete loss of virulence was observed in Forc016 strains that lost this chromosome. We conclude that also a non-wilt-inducing Fo pathogen relies on effector proteins for successful infection and that the Forc pathogenicity chromosome contains all the information necessary for causing root rot of cucurbits. Three out of nine HCT strains investigated have undergone large-scale chromosome alterations, reflecting the remarkable plasticity of Fo genomes.

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July 19, 2019

PacBio sequencing reveals transposable element as a key contributor to genomic plasticity and virulence variation in Magnaporthe oryzae.

The sustainable cultivation of rice, which serves as staple food crop for more than half of the world’s population, is under serious threat due to the huge yield losses inflicted by rice blast disease caused by the globally destructive fungus Magnaporthe oryzae (Pyricularia oryzae) (Dean et al., 2012, Nalley et al., 2016, Deng et al., 2017). This filamentous ascomycete fungus is also capable of causing blast infection on other economically important cereal crops, including wheat, millet, and barley, making it the world’s most important plant pathogenic fungus (Zhong et al., 2016). The advent of whole-genome sequencing technology and the subsequent deployment of next-generation sequencing (NGS) strategies have successfully generated genome assemblies for over 50 isolates of M. oryzae, which have played an instrumental role in enhancing our understanding of how rice blast fungus undertakes host adaptation, host specificity, and host range expansion to overcome host resistance (Dean et al., 2005, Xue et al., 2012, Wu et al., 2015, Zhang et al., 2016). However, research findings obtained from comparative genomic studies conducted using the NGS-assembled genome do not present an in-depth account of the genomic features that contribute to the prevailing genomic variations among M. oryzae species, because NGS assemblies are highly fragmented and lack most of the lineage-specific (LS) regions, which are more plastic than the core genome and enriched with repeats and effector proteins (Raffaele and Kamoun, 2012, Faino et al., 2016).

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July 19, 2019

Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters.

The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture. Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7. The more accurate gene annotation made possible by this new assembly revealed a large repertoire of secondary metabolism (SM) key genes (89) and putative biosynthetic pathways (77 SM gene clusters). The two mini-chromosomes differed from the ten core chromosomes in being repeat- and AT-rich and gene-poor but were significantly enriched with genes encoding putative secreted effector proteins. Transposable elements (TEs) were found to occupy 7% of the genome by length. Certain TE families showed a statistically significant association with effector genes and SM cluster genes and were transcriptionally active at particular stages of fungal development. All 24 subtelomeres were found to contain one of three highly-conserved repeat elements which, by providing sites for homologous recombination, were probably instrumental in four segmental duplications.The gapless genome of C. higginsianum provides access to repeat-rich regions that were previously poorly assembled, notably the mini-chromosomes and subtelomeres, and allowed prediction of the complete SM gene repertoire. It also provides insights into the potential role of TEs in gene and genome evolution and host adaptation in this asexual pathogen.

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July 19, 2019

The distribution of miniature impala elements and SIX genes in the Fusarium genus is suggestive of horizontal gene transfer.

The mimp family of miniature inverted-repeat transposable elements was previously found only in genomes of Fusarium oxysporum and is contextually associated with virulence genes in this species. Through extensive comparative analysis of 83 F. oxysporum and 52 other Fusarium genomes, we uncovered the distribution of different mimp families throughout the genus. We show that (i) mimps are not exclusive to F. oxysporum; (ii) pathogenic isolates generally possess more mimps than non-pathogenic strains and (iii) two isolates of F. hostae and one F. proliferatum isolate display evidence for horizontal transfer of genetic material to or from F. oxysporum. Multiple instances of mimp elements identical to F. oxysporum mimps were encountered in the genomes of these isolates. Moreover, homologs of effector genes (SIX1, 2, 6, 7, 11 and FomAVR2) were discovered here, several with very high (97-100%) pairwise nucleotide sequence identity scores. These three strains were isolated from infected flower bulbs (Hyacinthus and Lilium spp.). Their ancestors may thus have lived in close proximity to pathogenic strains of F. oxysporum f. sp. hyacinthi and f. sp. lilii. The Fo f. sp. lycopersici SIX2 effector gene was found to be widely distributed (15/18 isolates) throughout the F. fujikuroi species complex, exhibiting a predominantly vertical inheritance pattern. These findings shed light on the potential evolutionary mechanism underlying plant-pathogenicity in Fusarium and show that interspecies horizontal gene transfer may have occurred.

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July 19, 2019

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

The fungal genus ofAspergillusis 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 diverseAspergillusspecies (A. campestris,A. novofumigatus,A. ochraceoroseus, andA. steynii) have been whole-genome PacBio sequenced to provide genetic references in threeAspergillussections.A. taichungensisandA. candidusalso were sequenced for SM elucidation. ThirteenAspergillusgenomes 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,A. novofumigatuswas compared with the pathogenic speciesA. fumigatusThis suggests thatA. novofumigatuscan produce most of the same allergens, virulence, and pathogenicity factors asA. fumigatus, suggesting thatA. novofumigatuscould be as pathogenic asA. fumigatusFurthermore, 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 inA. ochraceoroseus,A. campestris, andA. steynii, respectively, and novofumigatonin,ent-cycloechinulin, andepi-aszonalenins inA. novofumigatusOur study delivers six fungal genomes, showing the large diversity found in theAspergillusgenus; highlights the potential for discovery of beneficial or harmful SMs; and supports reports ofA. novofumigatuspathogenicity. 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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July 19, 2019

Extreme sensitivity to ultraviolet light in the fungal pathogen causing white-nose syndrome of bats.

Bat white-nose syndrome (WNS), caused by the fungal pathogen Pseudogymnoascus destructans, has decimated North American hibernating bats since its emergence in 2006. Here, we utilize comparative genomics to examine the evolutionary history of this pathogen in comparison to six closely related nonpathogenic species. P. destructans displays a large reduction in carbohydrate-utilizing enzymes (CAZymes) and in the predicted secretome (~50%), and an increase in lineage-specific genes. The pathogen has lost a key enzyme, UVE1, in the alternate excision repair (AER) pathway, which is known to contribute to repair of DNA lesions induced by ultraviolet (UV) light. Consistent with a nonfunctional AER pathway, P. destructans is extremely sensitive to UV light, as well as the DNA alkylating agent methyl methanesulfonate (MMS). The differential susceptibility of P. destructans to UV light in comparison to other hibernacula-inhabiting fungi represents a potential “Achilles’ heel” of P. destructans that might be exploited for treatment of bats with WNS.

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