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Asset Tag: Pathogen

September 22, 2019  |  

Analysis of the hybrid genomes of two field isolates of the soil-borne fungal species Verticillium longisporum.

Brassica plant species are attacked by a number of pathogens; among them, the ones with a soil-borne lifestyle have become increasingly important. Verticillium stem stripe caused by Verticillium longisporum is one example. This fungal species is thought to be of a hybrid origin, having a genome composed of combinations of lineages denominated A and D. In this study we report the draft genomes of 2 V. longisporum field isolates sequenced using the Illumina technology. Genomic characterization and lineage composition, followed by selected gene analysis to facilitate the comprehension of its genomic features and potential effector categories were performed.The draft genomes of 2 Verticillium longisporum single spore isolates (VL1 and VL2) have an estimated ungapped size of about 70 Mb. The total number of protein encoding genes identified in VL1 was 20,793, whereas 21,072 gene models were predicted in VL2. The predicted genome size, gene contents, including the gene families coding for carbohydrate active enzymes were almost double the numbers found in V. dahliae and V. albo-atrum. Single nucleotide polymorphisms (SNPs) were frequently distributed in the two genomes but the distribution of heterozygosity and depth was not independent. Further analysis of potential parental lineages suggests that the V. longisporum genome is composed of two parts, A1 and D1, where A1 is more ancient than the parental lineage genome D1, the latter being more closer related to V. dahliae. Presence of the mating-type genes MAT1-1-1 and MAT1-2-1 in the V. longisporum genomes were confirmed. However, the MAT genes in V. dahliae, V. albo-atrum and V. longisporum have experienced extensive nucleotide changes at least partly explaining the present asexual nature of these fungal species.The established draft genome of V. longisporum is comparatively large compared to other studied ascomycete fungi. Consequently, high numbers of genes were predicted in the two V. longisporum genomes, among them many secreted proteins and carbohydrate active enzyme (CAZy) encoding genes. The genome is composed of two parts, where one lineage is more ancient than the part being more closely related to V. dahliae. Dissimilar mating-type sequences were identified indicating possible ancient hybridization events.

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September 22, 2019  |  

The non-specific adenine DNA methyltransferase M.EcoGII.

We describe the cloning, expression and characterization of the first truly non-specific adenine DNA methyltransferase, M.EcoGII. It is encoded in the genome of the pathogenic strain Escherichia coli O104:H4 C227-11, where it appears to reside on a cryptic prophage, but is not expressed. However, when the gene encoding M.EcoGII is expressed in vivo – using a high copy pRRS plasmid vector and a methylation-deficient E. coli host-extensive in vivo adenine methylation activity is revealed. M.EcoGII methylates adenine residues in any DNA sequence context and this activity extends to dA and rA bases in either strand of a DNA:RNA-hybrid oligonucleotide duplex and to rA bases in RNAs prepared by in vitro transcription. Using oligonucleotide and bacteriophage M13mp18 virion DNA substrates, we find that M.EcoGII also methylates single-stranded DNA in vitro and that this activity is only slightly less robust than that observed using equivalent double-stranded DNAs. In vitro assays, using purified recombinant M.EcoGII enzyme, demonstrate that up to 99% of dA bases in duplex DNA substrates can be methylated thereby rendering them insensitive to cleavage by multiple restriction endonucleases. These properties suggest that the enzyme could also be used for high resolution mapping of protein binding sites in DNA and RNA substrates.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019  |  

Complete genome sequencing of the luminescent bacterium, Vibrio qinghaiensis sp. Q67 using PacBio technology.

Vibrio qinghaiensis sp.-Q67 (Vqin-Q67) is a freshwater luminescent bacterium that continuously emits blue-green light (485?nm). The bacterium has been widely used for detecting toxic contaminants. Here, we report the complete genome sequence of Vqin-Q67, obtained using third-generation PacBio sequencing technology. Continuous long reads were attained from three PacBio sequencing runs and reads >500?bp with a quality value of >0.75 were merged together into a single dataset. This resultant highly-contiguous de novo assembly has no genome gaps, and comprises two chromosomes with substantial genetic information, including protein-coding genes, non-coding RNA, transposon and gene islands. Our dataset can be useful as a comparative genome for evolution and speciation studies, as well as for the analysis of protein-coding gene families, the pathogenicity of different Vibrio species in fish, the evolution of non-coding RNA and transposon, and the regulation of gene expression in relation to the bioluminescence of Vqin-Q67.

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September 22, 2019  |  

Extreme haplotype variation in the desiccation-tolerant clubmoss Selaginella lepidophylla.

Plant genome size varies by four orders of magnitude, and most of this variation stems from dynamic changes in repetitive DNA content. Here we report the small 109?Mb genome of Selaginella lepidophylla, a clubmoss with extreme desiccation tolerance. Single-molecule sequencing enables accurate haplotype assembly of a single heterozygous S. lepidophylla plant, revealing extensive structural variation. We observe numerous haplotype-specific deletions consisting of largely repetitive and heavily methylated sequences, with enrichment in young Gypsy LTR retrotransposons. Such elements are active but rapidly deleted, suggesting “bloat and purge” to maintain a small genome size. Unlike all other land plant lineages, Selaginella has no evidence of a whole-genome duplication event in its evolutionary history, but instead shows unique tandem gene duplication patterns reflecting adaptation to extreme drying. Gene expression changes during desiccation in S. lepidophylla mirror patterns observed across angiosperm resurrection plants.

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September 22, 2019  |  

Reference assembly and annotation of the Pyrenophora teres f. teres isolate 0-1.

Pyrenophora teres f.teres, the causal agent of net form net blotch (NFNB) of barley, is a destructive pathogen in barley-growing regions throughout the world. Typical yield losses due to NFNB range from 10 to 40%; however, complete loss has been observed on highly susceptible barley lines where environmental conditions favor the pathogen. Currently, genomic resources for this economically important pathogen are limited to a fragmented draft genome assembly and annotation, with limited RNA support of theP. teresf.teresisolate 0-1. This research presents an updated 0-1 reference assembly facilitated by long-read sequencing and scaffolding with the assistance of genetic linkage maps. Additionally, genome annotation was mediated by RNAseq analysis using three infection time points and a pure culture sample, resulting in 11,541 high-confidence gene models. The 0-1 genome assembly and annotation presented here now contains the majority of the repetitive content of the genome. Analysis of the 0-1 genome revealed classic characteristics of a “two-speed” genome, being compartmentalized into GC-equilibrated and AT-rich compartments. The assembly of repetitive AT-rich regions will be important for future investigation of genes known as effectors, which often reside in close proximity to repetitive regions. These effectors are responsible for manipulation of the host defense during infection. This updatedP. teresf.teresisolate 0-1 reference genome assembly and annotation provides a robust resource for the examination of the barley-P. teresf.tereshost-pathogen coevolution. Copyright © 2018 Wyatt et al.

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September 22, 2019  |  

Functional characterization of the mucus barrier on the Xenopus tropicalis skin surface.

Mucosal surfaces represent critical routes for entry and exit of pathogens. As such, animals have evolved strategies to combat infection at these sites, in particular the production of mucus to prevent attachment and to promote subsequent movement of the mucus/microbe away from the underlying epithelial surface. Using biochemical, biophysical, and infection studies, we have investigated the host protective properties of the skin mucus barrier of the Xenopus tropicalis tadpole. Specifically, we have characterized the major structural component of the barrier and shown that it is a mucin glycoprotein (Otogelin-like or Otogl) with similar sequence, domain organization, and structural properties to human gel-forming mucins. This mucin forms the structural basis of a surface barrier (~6 µm thick), which is depleted through knockdown of Otogl. Crucially, Otogl knockdown leads to susceptibility to infection by the opportunistic pathogen Aeromonas hydrophila To more accurately reflect its structure, tissue localization, and function, we have renamed Otogl as Xenopus Skin Mucin, or MucXS. Our findings characterize an accessible and tractable model system to define mucus barrier function and host-microbe interactions. Copyright © 2018 the Author(s). Published by PNAS.

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September 22, 2019  |  

Comparative genomics reveals cotton-specific virulence factors in flexible genomic regions in Verticillium dahliae and evidence of horizontal gene transfer from Fusarium.

Verticillium dahliae isolates are most virulent on the host from which they were originally isolated. Mechanisms underlying these dominant host adaptations are currently unknown. We sequenced the genome of V. dahliae Vd991, which is highly virulent on its original host, cotton, and performed comparisons with the reference genomes of JR2 (from tomato) and VdLs.17 (from lettuce). Pathogenicity-related factor prediction, orthology and multigene family classification, transcriptome analyses, phylogenetic analyses, and pathogenicity experiments were performed. The Vd991 genome harbored several exclusive, lineage-specific (LS) genes within LS regions (LSRs). Deletion mutants of the seven genes within one LSR (G-LSR2) in Vd991 were less virulent only on cotton. Integration of G-LSR2 genes individually into JR2 and VdLs.17 resulted in significantly enhanced virulence on cotton but did not affect virulence on tomato or lettuce. Transcription levels of the seven LS genes in Vd991 were higher during the early stages of cotton infection, as compared with other hosts. Phylogenetic analyses suggested that G-LSR2 was acquired from Fusarium oxysporum f. sp. vasinfectum through horizontal gene transfer. Our results provide evidence that horizontal gene transfer from Fusarium to Vd991 contributed significantly to its adaptation to cotton and may represent a significant mechanism in the evolution of an asexual plant pathogen.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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September 22, 2019  |  

Complete genome sequence of Lactobacillus pentosus SLC13, isolated from mustard pickles, a potential probiotic strain with antimicrobial activity against foodborne pathogenic microorganisms.

Lactobacillus pentosus SLC13 is a high exopolysaccharide (EPS)-producing strain with broad-spectrum antimicrobial activity and the ability to grow in simulated gastrointestinal conditions. SLC13 was isolated from mustard pickles in Taiwan for potential probiotic applications. To better understand the molecular base for its antimicrobial activity and high EPS production, entire genome of SLC13 was determined by PacBio SMRT sequencing.L. pentosus SLC13 contains a genome with a 3,520,510-bp chromosome and a 62,498-bp plasmid. GC content of the complete genome was 46.5% and that of plasmid pSLC13 was 41.3%. Sequences were annotated at the RAST prokaryotic genome annotation server, and the results showed that the genome contained 3172 coding sequences and 82 RNA genes. Seventy-six protein-coding sequences were identified on the plasmid pSLC13. A plantaricin gene cluster, which is responsible for bacteriosins biosynthesis and could be associated with its broad-spectrum antimicrobial activity, was identified based on comparative genomic analysis. Two gene clusters involved in EPS production were also identified.This genomic sequence might contribute to a future application of this strain as probiotic in productive livestock potentially inhibiting competing and pathogenic organisms.

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September 22, 2019  |  

The genomes of Crithidia bombi and C. expoeki, common parasites of bumblebees.

Trypanosomatids (Trypanosomatidae, Kinetoplastida) are flagellated protozoa containing many parasites of medical or agricultural importance. Among those, Crithidia bombi and C. expoeki, are common parasites in bumble bees around the world, and phylogenetically close to Leishmania and Leptomonas. They have a simple and direct life cycle with one host, and partially castrate the founding queens greatly reducing their fitness. Here, we report the nuclear genome sequences of one clone of each species, extracted from a field-collected infection. Using a combination of Roche 454 FLX Titanium, Pacific Biosciences PacBio RS, and Illumina GA2 instruments for C. bombi, and PacBio for C. expoeki, we could produce high-quality and well resolved sequences. We find that these genomes are around 32 and 34 MB, with 7,808 and 7,851 annotated genes for C. bombi and C. expoeki, respectively-which is somewhat less than reported from other trypanosomatids, with few introns, and organized in polycistronic units. A large fraction of genes received plausible functional support in comparison primarily with Leishmania and Trypanosoma. Comparing the annotated genes of the two species with those of six other trypanosomatids (C. fasciculata, L. pyrrhocoris, L. seymouri, B. ayalai, L. major, and T. brucei) shows similar gene repertoires and many orthologs. Similar to other trypanosomatids, we also find signs of concerted evolution in genes putatively involved in the interaction with the host, a high degree of synteny between C. bombi and C. expoeki, and considerable overlap with several other species in the set. A total of 86 orthologous gene groups show signatures of positive selection in the branch leading to the two Crithidia under study, mostly of unknown function. As an example, we examined the initiating glycosylation pathway of surface components in C. bombi, finding it deviates from most other eukaryotes and also from other kinetoplastids, which may indicate rapid evolution in the extracellular matrix that is involved in interactions with the host. Bumble bees are important pollinators and Crithidia-infections are suspected to cause substantial selection pressure on their host populations. These newly sequenced genomes provide tools that should help better understand host-parasite interactions in these pollinator pathogens.

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September 22, 2019  |  

An ancient integration in a plant NLR is maintained as a trans-species polymorphism

Plant immune receptors are under constant selective pressure to maintain resistance to plant pathogens. Nucleotide-binding leucine-rich repeat (NLR) proteins are one class of cytoplasmic immune receptors whose genes commonly show signatures of adaptive evolution. While it is known that balancing selection contributes to maintaining high intraspecific allelic diversity, the evolutionary mechanism that influences the transmission of alleles during speciation remains unclear. The barley Mla locus has over 30 described alleles conferring isolate-specific resistance to barley powdery mildew and contains three NLR families (RGH1, RGH2, and RGH3). We discovered (using sequence capture and RNAseq) the presence of a novel integrated Exo70 domain in RGH2 in the Mla3 haplotype. Allelic variation across barley accessions includes presence/absence of the integrated domain in RGH2. Expanding our search to several Poaceae species, we found shared interspecific conservation in the RGH2-Exo70 integration. We hypothesise that balancing selection has maintained allelic variation at Mla as a trans-species polymorphism over 24 My, thus contributing to and preserving interspecific allelic diversity during speciation.

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September 22, 2019  |  

Pangenome analyses of the wheat pathogen Zymoseptoria tritici reveal the structural basis of a highly plastic eukaryotic genome.

Structural variation contributes substantially to polymorphism within species. Chromosomal rearrangements that impact genes can lead to functional variation among individuals and influence the expression of phenotypic traits. Genomes of fungal pathogens show substantial chromosomal polymorphism that can drive virulence evolution on host plants. Assessing the adaptive significance of structural variation is challenging, because most studies rely on inferences based on a single reference genome sequence.We constructed and analyzed the pangenome of Zymoseptoria tritici, a major pathogen of wheat that evolved host specialization by chromosomal rearrangements and gene deletions. We used single-molecule real-time sequencing and high-density genetic maps to assemble multiple genomes. We annotated the gene space based on transcriptomics data that covered the infection life cycle of each strain. Based on a total of five telomere-to-telomere genomes, we constructed a pangenome for the species and identified a core set of 9149 genes. However, an additional 6600 genes were exclusive to a subset of the isolates. The substantial accessory genome encoded on average fewer expressed genes but a larger fraction of the candidate effector genes that may interact with the host during infection. We expanded our analyses of the pangenome to a worldwide collection of 123 isolates of the same species. We confirmed that accessory genes were indeed more likely to show deletion polymorphisms and loss-of-function mutations compared to core genes.The pangenome construction of a highly polymorphic eukaryotic pathogen showed that a single reference genome significantly underestimates the gene space of a species. The substantial accessory genome provides a cradle for adaptive evolution.

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September 22, 2019  |  

Nuclear and mitochondrial genomes of the hybrid fungal plant pathogen Verticillium longisporum display a mosaic structure

Allopolyploidization, genome duplication through interspecific hybridization, is an important evolutionary mechanism that can enable organisms to adapt to environmental changes or stresses. This increased adaptive potential of allopolyploids can be particularly relevant for plant pathogens in their quest for host immune response evasion. Allodiploidization likely caused the shift in host range of the fungal pathogen plant Verticillium longisporum, as V. longisporum mainly infects Brassicaceae plants in contrast to haploid Verticillium spp. In this study, we investigated the allodiploid genome structure of V. longisporum and its evolution in the hybridization aftermath. The nuclear genome of V. longisporum displays a mosaic structure, as numerous contigs consists of sections of both parental origins. V. longisporum encountered extensive genome rearrangements, whereas the contribution of gene conversion is negligible. Thus, the mosaic genome structure mainly resulted from genomic rearrangements between parental chromosome sets. Furthermore, a mosaic structure was also found in the mitochondrial genome, demonstrating its bi-parental inheritance. In conclusion, the nuclear and mitochondrial genomes of V. longisporum parents interacted dynamically in the hybridization aftermath. Conceivably, novel combinations of DNA sequence of different parental origin facilitated genome stability after hybridization and consecutive niche adaptation of V. longisporum.

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September 22, 2019  |  

Genetic basis of emerging vancomycin, linezolid, and daptomycin heteroresistance in a case of persistent Enterococcus faecium bacteremia.

Whole-genome sequencing was used to examine a persistent Enterococcus faecium bacteremia that acquired heteroresistance to three antibiotics in response to prolonged multidrug therapy. A comparison of the complete genomes before and after each change revealed the emergence of known resistance determinants for vancomycin and linezolid and suggested that a novel mutation in fabF, encoding a fatty acid synthase, was responsible for daptomycin nonsusceptibility. Plasmid recombination contributed to the progressive loss of vancomycin resistance after withdrawal of the drug. Copyright © 2018 Chacko et al.

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September 22, 2019  |  

Identification of candidate genes at the Dp-fl locus conferring resistance against the rosy apple aphid Dysaphis plantaginea

The cultivated apple is susceptible to several pests including the rosy apple aphid (RAA; Dysaphis plantaginea Passerini), control of which is mainly based on chemical treatments. A few cases of resistance to aphids have been described in apple germplasm resources, laying the basis for the development of new resistant cultivars by breeding. The cultivar ‘Florina’ is resistant to RAA, and recently, the Dp-fl locus responsible for its resistance was mapped on linkage group 8 of the apple genome. In this paper, a chromosome walking approach was performed by using a ‘Florina’ bacterial artificial chromosome (BAC) library. The walking started from the available tightly linked molecular markers flanking the resistance region. Various walking steps were performed in order to identify the minimum tiling path of BAC clones covering the Dp-fl region from both the “resistant” and “susceptible” chromosomes of ‘Florina’. A genomic region of about 279 Kb encompassing the Dp-fl resistance locus was fully sequenced by the PacBio technology. Through the development of new polymorphic markers, the mapping interval around the resistance locus was narrowed down to a physical region of 95 Kb. The annotation of this sequence resulted in the identification of four candidate genes putatively involved in the RAA resistance response.

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September 22, 2019  |  

The novel phages phiCD5763 and phiCD2955 represent two groups of big plasmidial Siphoviridae phages of Clostridium difficile.

Until recently, Clostridium difficile phages were limited to Myoviruses and Siphoviruses of medium genome length (32–57 kb). Here we report the finding of phiCD5763, a Siphovirus with a large extrachromosomal circular genome (132.5 kb, 172 ORFs) and a large capsid (205.6 ± 25.6 nm in diameter) infecting MLST Clade 1 strains of C. difficile. Two subgroups of big phage genomes similar to phiCD5763 were identified in 32 NAPCR1/RT012/ST-54 C. difficile isolates from Costa Rica and in whole genome sequences (WGS) of 41 C. difficile isolates of Clades 1, 2, 3, and 4 from Canada, USA, UK, Belgium, Iraq, and China. Through comparative genomics we discovered another putative big phage genome in a non-NAPCR1 isolate from Costa Rica, phiCD2955, which represents other big phage genomes found in 130 WGS of MLST Clade 1 and 2 isolates from Canada, USA, Hungary, France, Austria, and UK. phiCD2955 (131.6 kb, 172 ORFs) is related to a previously reported C. difficile phage genome, phiCD211/phiCDIF1296T. Detailed genome analyses of phiCD5763, phiCD2955, phiCD211/phiCDIF1296T, and seven other putative C. difficile big phage genome sequences of 131–136 kb reconstructed from publicly available WGS revealed a modular gene organization and high levels of sequence heterogeneity at several hotspots, suggesting that these genomes correspond to biological entities undergoing recombination. Compared to other C. difficile phages, these big phages have unique predicted terminase, capsid, portal, neck and tail proteins, receptor binding proteins (RBPs), recombinases, resolvases, primases, helicases, ligases, and hypothetical proteins. Moreover, their predicted gene load suggests a complex regulation of both phage and host functions. Overall, our results indicate that the prevalence of C. difficile big bacteriophages is more widespread than realized and open new avenues of research aiming to decipher how these viral elements influence the biology of this emerging pathogen.

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