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April 21, 2020  |  

Gut pathobionts underlie intestinal barrier dysfunction and liver T helper 17 cell immune response in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease and its frequent complication with ulcerative colitis highlights the pathogenic role of epithelial barrier dysfunction. Intestinal barrier dysfunction has been implicated in the pathogenesis of PSC, yet its underlying mechanism remains unknown. Here, we identify Klebsiella pneumonia in the microbiota of patients with PSC and demonstrate that K.?pneumoniae disrupts the epithelial barrier to initiate bacterial translocation and liver inflammatory responses. Gnotobiotic mice inoculated with PSC-derived microbiota exhibited T helper 17 (TH17) cell responses in the liver and increased susceptibility to hepatobiliary injuries. Bacterial culture of mesenteric lymph nodes in these mice isolated K.?pneumoniae, Proteus mirabilis and Enterococcus gallinarum, which were prevalently detected in patients with PSC. A bacterial-organoid co-culture system visualized the epithelial-damaging effect of PSC-derived K.?pneumoniae that was associated with bacterial translocation and susceptibility to TH17-mediated hepatobiliary injuries. We also show that antibiotic treatment ameliorated the TH17 immune response induced by PSC-derived microbiota. These results highlight the role of pathobionts in intestinal barrier dysfunction and liver inflammation, providing insights into therapeutic strategies for PSC.

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April 21, 2020  |  

Antarctic blackfin icefish genome reveals adaptations to extreme environments.

Icefishes (suborder Notothenioidei; family Channichthyidae) are the only vertebrates that lack functional haemoglobin genes and red blood cells. Here, we report a high-quality genome assembly and linkage map for the Antarctic blackfin icefish Chaenocephalus aceratus, highlighting evolved genomic features for its unique physiology. Phylogenomic analysis revealed that Antarctic fish of the teleost suborder Notothenioidei, including icefishes, diverged from the stickleback lineage about 77 million years ago and subsequently evolved cold-adapted phenotypes as the Southern Ocean cooled to sub-zero temperatures. Our results show that genes involved in protection from ice damage, including genes encoding antifreeze glycoprotein and zona pellucida proteins, are highly expanded in the icefish genome. Furthermore, genes that encode enzymes that help to control cellular redox state, including members of the sod3 and nqo1 gene families, are expanded, probably as evolutionary adaptations to the relatively high concentration of oxygen dissolved in cold Antarctic waters. In contrast, some crucial regulators of circadian homeostasis (cry and per genes) are absent from the icefish genome, suggesting compromised control of biological rhythms in the polar light environment. The availability of the icefish genome sequence will accelerate our understanding of adaptation to extreme Antarctic environments.

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April 21, 2020  |  

Genome sequence and genetic transformation of a widely distributed and cultivated poplar.

Populus alba is widely distributed and cultivated in Europe and Asia. This species has been used for diverse studies. In this study, we assembled a de novo genome sequence of P. alba var. pyramidalis (= P. bolleana) and confirmed its high transformation efficiency and short transformation time by experiments. Through a process of hybrid genome assembly, a total of 464 M of the genome was assembled. Annotation analyses predicted 37 901 protein-coding genes. This genome is highly collinear to that of P. trichocarpa, with most genes having orthologs in the two species. We found a marked expansion of gene families related to histone and the hormone auxin but loss of disease resistance genes in P. alba if compared with the closely related P. trichocarpa. The genome sequence presented here represents a valuable resource for further molecular functional analyses of this species as a new tree model, poplar breeding practices and comparative genomic analyses across different poplars. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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April 21, 2020  |  

Phylogenetic barriers to horizontal transfer of antimicrobial peptide resistance genes in the human gut microbiota.

The human gut microbiota has adapted to the presence of antimicrobial peptides (AMPs), which are ancient components of immune defence. Despite its medical importance, it has remained unclear whether AMP resistance genes in the gut microbiome are available for genetic exchange between bacterial species. Here, we show that AMP resistance and antibiotic resistance genes differ in their mobilization patterns and functional compatibilities with new bacterial hosts. First, whereas AMP resistance genes are widespread in the gut microbiome, their rate of horizontal transfer is lower than that of antibiotic resistance genes. Second, gut microbiota culturing and functional metagenomics have revealed that AMP resistance genes originating from phylogenetically distant bacteria have only a limited potential to confer resistance in Escherichia coli, an intrinsically susceptible species. Taken together, functional compatibility with the new bacterial host emerges as a key factor limiting the genetic exchange of AMP resistance genes. Finally, our results suggest that AMPs induce highly specific changes in the composition of the human microbiota, with implications for disease risks.

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April 21, 2020  |  

Genetic map-guided genome assembly reveals a virulence-governing minichromosome in the lentil anthracnose pathogen Colletotrichum lentis.

Colletotrichum lentis causes anthracnose, which is a serious disease on lentil and can account for up to 70% crop loss. Two pathogenic races, 0 and 1, have been described in the C. lentis population from lentil. To unravel the genetic control of virulence, an isolate of the virulent race 0 was sequenced at 1481-fold genomic coverage. The 56.10-Mb genome assembly consists of 50 scaffolds with N50 scaffold length of 4.89 Mb. A total of 11 436 protein-coding gene models was predicted in the genome with 237 coding candidate effectors, 43 secondary metabolite biosynthetic enzymes and 229 carbohydrate-active enzymes (CAZymes), suggesting a contraction of the virulence gene repertoire in C. lentis. Scaffolds were assigned to 10 core and two minichromosomes using a population (race 0 × race 1, n = 94 progeny isolates) sequencing-based, high-density (14 312 single nucleotide polymorphisms) genetic map. Composite interval mapping revealed a single quantitative trait locus (QTL), qClVIR-11, located on minichromosome 11, explaining 85% of the variability in virulence of the C. lentis population. The QTL covers a physical distance of 0.84 Mb with 98 genes, including seven candidate effector and two secondary metabolite genes. Taken together, the study provides genetic and physical evidence for the existence of a minichromosome controlling the C. lentis virulence on lentil. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

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April 21, 2020  |  

Genome-Scale Sequence Disruption Following Biolistic Transformation in Rice and Maize.

Biolistic transformation delivers nucleic acids into plant cells by bombarding the cells with microprojectiles, which are micron-scale, typically gold particles. Despite the wide use of this technique, little is known about its effect on the cell’s genome. We biolistically transformed linear 48-kb phage lambda and two different circular plasmids into rice (Oryza sativa) and maize (Zea mays) and analyzed the results by whole genome sequencing and optical mapping. Although some transgenic events showed simple insertions, others showed extreme genome damage in the form of chromosome truncations, large deletions, partial trisomy, and evidence of chromothripsis and breakage-fusion bridge cycling. Several transgenic events contained megabase-scale arrays of introduced DNA mixed with genomic fragments assembled by nonhomologous or microhomology-mediated joining. Damaged regions of the genome, assayed by the presence of small fragments displaced elsewhere, were often repaired without a trace, presumably by homology-dependent repair (HDR). The results suggest a model whereby successful biolistic transformation relies on a combination of end joining to insert foreign DNA and HDR to repair collateral damage caused by the microprojectiles. The differing levels of genome damage observed among transgenic events may reflect the stage of the cell cycle and the availability of templates for HDR. © 2019 American Society of Plant Biologists. All rights reserved.

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April 21, 2020  |  

RADAR-seq: A RAre DAmage and Repair sequencing method for detecting DNA damage on a genome-wide scale.

RAre DAmage and Repair sequencing (RADAR-seq) is a highly adaptable sequencing method that enables the identification and detection of rare DNA damage events for a wide variety of DNA lesions at single-molecule resolution on a genome-wide scale. In RADAR-seq, DNA lesions are replaced with a patch of modified bases that can be directly detected by Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing. RADAR-seq enables dynamic detection over a wide range of DNA damage frequencies, including low physiological levels. Furthermore, without the need for DNA amplification and enrichment steps, RADAR-seq provides sequencing coverage of damaged and undamaged DNA across an entire genome. Here, we use RADAR-seq to measure the frequency and map the location of ribonucleotides in wild-type and RNaseH2-deficient E. coli and Thermococcus kodakarensis strains. Additionally, by tracking ribonucleotides incorporated during in vivo lagging strand DNA synthesis, we determined the replication initiation point in E. coli, and its relation to the origin of replication (oriC). RADAR-seq was also used to map cyclobutane pyrimidine dimers (CPDs) in Escherichia coli (E. coli) genomic DNA exposed to UV-radiation. On a broader scale, RADAR-seq can be applied to understand formation and repair of DNA damage, the correlation between DNA damage and disease initiation and progression, and complex biological pathways, including DNA replication.Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.

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April 21, 2020  |  

Fast and accurate genomic analyses using genome graphs.

The human reference genome serves as the foundation for genomics by providing a scaffold for alignment of sequencing reads, but currently only reflects a single consensus haplotype, thus impairing analysis accuracy. Here we present a graph reference genome implementation that enables read alignment across 2,800 diploid genomes encompassing 12.6 million SNPs and 4.0 million insertions and deletions (indels). The pipeline processes one whole-genome sequencing sample in 6.5?h using a system with 36?CPU cores. We show that using a graph genome reference improves read mapping sensitivity and produces a 0.5% increase in variant calling recall, with unaffected specificity. Structural variations incorporated into a graph genome can be genotyped accurately under a unified framework. Finally, we show that iterative augmentation of graph genomes yields incremental gains in variant calling accuracy. Our implementation is an important advance toward fulfilling the promise of graph genomes to radically enhance the scalability and accuracy of genomic analyses.

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April 21, 2020  |  

Characterization and analysis of the transcriptome in Gymnocypris selincuoensis on the Qinghai-Tibetan Plateau using single-molecule long-read sequencing and RNA-seq.

The lakes on the Qinghai-Tibet Plateau (QTP) are the largest and highest lake group in the world. Gymnocypris selincuoensis is the only cyprinid fish living in lake Selincuo, the largest lake on QTP. However, its genetic resource is still blank, limiting studies on molecular and genetic analysis. In this study, the transcriptome of G. selincuoensis was first generated by using PacBio Iso-Seq and Illumina RNA-seq. A full-length (FL) transcriptome with 75,435 transcripts was obtained by Iso-Seq with N50 length of 3,870 bp. Among all transcripts, 75,016 were annotated to public databases, 64,710 contain complete open reading frames and 2,811 were long non-coding RNAs. Based on all- vs.-all BLAST, 2,069 alternative splicing events were detected, and 80% of them were validated by reverse transcription polymerase chain reaction (RT-PCR). Tissue gene expression atlas showed that the number of detected expressed transcripts ranged from 37,397 in brain to 19,914 in muscle, with 10,488 transcripts detected in all seven tissues. Comparative genomic analysis with other cyprinid fishes identified 77 orthologous genes with potential positive selection (Ka/Ks > 0.3). A total of 56,696 perfect simple sequence repeats were identified from FL transcripts. Our results provide valuable genetic resources for further studies on adaptive evolution, gene expression and population genetics in G. selincuoensis and other congeneric fishes. © The Author(s) 2019. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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April 21, 2020  |  

Dynamic virulence-related regions of the plant pathogenic fungus Verticillium dahliae display enhanced sequence conservation.

Plant pathogens continuously evolve to evade host immune responses. During host colonization, many fungal pathogens secrete effectors to perturb such responses, but these in turn may become recognized by host immune receptors. To facilitate the evolution of effector repertoires, such as the elimination of recognized effectors, effector genes often reside in genomic regions that display increased plasticity, a phenomenon that is captured in the two-speed genome hypothesis. The genome of the vascular wilt fungus Verticillium dahliae displays regions with extensive presence/absence polymorphisms, so-called lineage-specific regions, that are enriched in in planta-induced putative effector genes. As expected, comparative genomics reveals differential degrees of sequence divergence between lineage-specific regions and the core genome. Unanticipated, lineage-specific regions display markedly higher sequence conservation in coding as well as noncoding regions than the core genome. We provide evidence that disqualifies horizontal transfer to explain the observed sequence conservation and conclude that sequence divergence occurs at a slower pace in lineage-specific regions of the V. dahliae genome. We hypothesize that differences in chromatin organisation may explain lower nucleotide substitution rates in the plastic, lineage-specific regions of V. dahliae. © 2019 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

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April 21, 2020  |  

TranscriptClean: variant-aware correction of indels, mismatches and splice junctions in long-read transcripts.

Long-read, single-molecule sequencing platforms hold great potential for isoform discovery and characterization of multi-exon transcripts. However, their high error rates are an obstacle to distinguishing novel transcript isoforms from sequencing artifacts. Therefore, we developed the package TranscriptClean to correct mismatches, microindels and noncanonical splice junctions in mapped transcripts using the reference genome while preserving known variants.Our method corrects nearly all mismatches and indels present in a publically available human PacBio Iso-seq dataset, and rescues 39% of noncanonical splice junctions.All Python and R scripts used in this paper are available at https://github.com/dewyman/TranscriptClean.

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April 21, 2020  |  

The conservation of polyol transporter proteins and their involvement in lichenized Ascomycota.

In lichen symbiosis, polyol transfer from green algae is important for acquiring the fungal carbon source. However, the existence of polyol transporter genes and their correlation with lichenization remain unclear. Here, we report candidate polyol transporter genes selected from the genome of the lichen-forming fungus (LFF) Ramalina conduplicans. A phylogenetic analysis using characterized polyol and monosaccharide transporter proteins and hypothetical polyol transporter proteins of R. conduplicans and various ascomycetous fungi suggested that the characterized yeast’ polyol transporters form multiple clades with the polyol transporter-like proteins selected from the diverse ascomycetous taxa. Thus, polyol transporter genes are widely conserved among Ascomycota, regardless of lichen-forming status. In addition, the phylogenetic clusters suggested that LFFs belonging to Lecanoromycetes have duplicated proteins in each cluster. Consequently, the number of sequences similar to characterized yeast’ polyol transporters were evaluated using the genomes of 472 species or strains of Ascomycota. Among these, LFFs belonging to Lecanoromycetes had greater numbers of deduced polyol transporter proteins. Thus, various polyol transporters are conserved in Ascomycota and polyol transporter genes appear to have expanded during the evolution of Lecanoromycetes. Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

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April 21, 2020  |  

PacBio full-length cDNA sequencing integrated with RNA-seq reads drastically improves the discovery of splicing transcripts in rice.

In eukaryotes, alternative splicing (AS) greatly expands the diversity of transcripts. However, it is challenging to accurately determine full-length splicing isoforms. Recently, more studies have taken advantage of Pacific Bioscience (PacBio) long-read sequencing to identify full-length transcripts. Nevertheless, the high error rate of PacBio reads seriously offsets the advantages of long reads, especially for accurately identifying splicing junctions. To best capitalize on the features of long reads, we used Illumina RNA-seq reads to improve PacBio circular consensus sequence (CCS) quality and to validate splicing patterns in the rice transcriptome. We evaluated the impact of CCS accuracy on the number and the validation rate of splicing isoforms, and integrated a comprehensive pipeline of splicing transcripts analysis by Iso-Seq and RNA-seq (STAIR) to identify the full-length multi-exon isoforms in rice seedling transcriptome (Oryza sativa L. ssp. japonica). STAIR discovered 11 733 full-length multi-exon isoforms, 6599 more than the SMRT Portal RS_IsoSeq pipeline did. Of these splicing isoforms identified, 4453 (37.9%) were missed in assembled transcripts from RNA-seq reads, and 5204 (44.4%), including 268 multi-exon long non-coding RNAs (lncRNAs), were not reported in the MSU_osa1r7 annotation. Some randomly selected unreported splicing junctions were verified by polymerase chain reaction (PCR) amplification. In addition, we investigated alternative polyadenylation (APA) events in transcripts and identified 829 major polyadenylation [poly(A)] site clusters (PACs). The analysis of splicing isoforms and APA events will facilitate the annotation of the rice genome and studies on the expression and polyadenylation of AS genes in different developmental stages or growth conditions of rice. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

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April 21, 2020  |  

Highly flexible infection programs in a specialized wheat pathogen.

Many filamentous plant pathogens exhibit high levels of genomic variability, yet the impact of this variation on host-pathogen interactions is largely unknown. We have addressed host specialization in the wheat pathogen Zymoseptoria tritici. Our study builds on comparative analyses of infection and gene expression phenotypes of three isolates and reveals the extent to which genomic variation translates into phenotypic variation. The isolates exhibit genetic and genomic variation but are similarly virulent. By combining confocal microscopy, disease monitoring, staining of ROS, and comparative transcriptome analyses, we conducted a detailed comparison of the infection processes of these isolates in a susceptible wheat cultivar. We characterized four core infection stages: establishment, biotrophic growth, lifestyle transition, and necrotrophic growth and asexual reproduction that are shared by the three isolates. However, we demonstrate differentiated temporal and spatial infection development and significant differences in the expression profiles of the three isolates during the infection stages. More than 20% of the genes were differentially expressed and these genes were located significantly closer to transposable elements, suggesting an impact of epigenetic regulation. Further, differentially expressed genes were enriched in effector candidates suggesting that isolate-specific strategies for manipulating host defenses are present in Z. tritici. We demonstrate that individuals of a host-specialized pathogen have highly differentiated infection programs characterized by flexible infection development and functional redundancy. This illustrates how high genetic diversity in pathogen populations results in highly differentiated infection phenotypes, which fact needs to be acknowledged to understand host-pathogen interactions and pathogen evolution.

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April 21, 2020  |  

Toxin and genome evolution in a Drosophila defensive symbiosis.

Defenses conferred by microbial symbionts play a vital role in the health and fitness of their animal hosts. An important outstanding question in the study of defensive symbiosis is what determines long term stability and effectiveness against diverse natural enemies. In this study, we combine genome and transcriptome sequencing, symbiont transfection and parasite protection experiments, and toxin activity assays to examine the evolution of the defensive symbiosis between Drosophila flies and their vertically transmitted Spiroplasma bacterial symbionts, focusing in particular on ribosome-inactivating proteins (RIPs), symbiont-encoded toxins that have been implicated in protection against both parasitic wasps and nematodes. Although many strains of Spiroplasma, including the male-killing symbiont (sMel) of Drosophila melanogaster, protect against parasitic wasps, only the strain (sNeo) that infects the mycophagous fly Drosophila neotestacea appears to protect against parasitic nematodes. We find that RIP repertoire is a major differentiating factor between strains that do and do not offer nematode protection, and that sMel RIPs do not show activity against nematode ribosomes in vivo. We also discovered a strain of Spiroplasma infecting a mycophagous phorid fly, Megaselia nigra. Although both the host and its Spiroplasma are distantly related to D. neotestacea and its symbiont, genome sequencing revealed that the M. nigra symbiont encodes abundant and diverse RIPs, including plasmid-encoded toxins that are closely related to the RIPs in sNeo. Our results suggest that distantly related Spiroplasma RIP toxins may perform specialized functions with regard to parasite specificity and suggest an important role for horizontal gene transfer in the emergence of novel defensive phenotypes.

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