Publications by authors named "Rhys A Farrer"

31 Publications

Genomic epidemiology of a case cluster in Glasgow, Scotland, 2018.

Microb Genom 2021 Mar 23;7(3). Epub 2021 Feb 23.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

In 2018, a cluster of two cases of cryptococcosis occurred at the Queen Elizabeth University Hospital (QEUH) in Glasgow, Scotland (UK). It was postulated that these cases may have been linked to pigeon droppings found on the hospital site, given there have been previous reports of associated with pigeon guano. Although some samples of pigeon guano taken from the site yielded culturable yeast from genera related to , they have since been classified as or spp., and no isolates of were recovered from either the guano or subsequent widespread air sampling. In an attempt to further elucidate any possible shared source of the clinical isolates, we used whole-genome sequencing and phylogenetic analysis to examine the relationship of the two isolates from the QEUH cases, along with two isolates from sporadic cases treated at a different Glasgow hospital earlier in 2018. Our work demonstrated that these four clinical isolates were not clonally related; while all isolates were from the VNI global lineage and of the same mating type (MATα), the genotypes of the two QEUH isolates were separated by 1885 base changes and belonged to different sub-lineages, recently described as the intercontinental sub-clades VNIa-93 and VNIa-5. In contrast, one of the two sporadic 2018 clinical isolates was determined to belong to the VNIb sub-lineage and the other classified as a VNIV/VNI hybrid. Our work demonstrated that the two 2018 QEUH isolates and the two prior clinical isolates were all genetically distinct. It was not possible to determine whether the QEUH genotypes stemmed from independent sources or from the same source, i.e. pigeons carrying different genotypes, but it should be noted that whilst members of allied genera within the were isolated from the hospital environment, there were no environmental isolations of .
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http://dx.doi.org/10.1099/mgen.0.000537DOI Listing
March 2021

Pathogen and host genetics underpinning cryptococcal disease.

Adv Genet 2020 18;105:1-66. Epub 2020 Mar 18.

Medical Research Council Centre for Medical Mycology at the University of Exeter, Exeter, United Kingdom. Electronic address:

Cryptococcosis is a severe fungal disease causing 220,000 cases of cryptococcal meningitis yearly. The etiological agents of cryptococcosis are taxonomically grouped into at least two species complexes belonging to the genus Cryptococcus. All of these yeasts are environmentally ubiquitous fungi (often found in soil, leaves and decaying wood, tree hollows, and associated with bird feces especially pigeon guano). Infection in a range of animals including humans begins following inhalation of spores or aerosolized yeasts. Recent advances provide fundamental insights into the factors from both the pathogen and its hosts which influence pathogenesis and disease. The complex interactions leading to disease in mammalian hosts have also updated from the availability of better genomic tools and datasets. In this review, we discuss recent genetic research on Cryptococcus, covering the epidemiology, ecology, and evolution of Cryptococcus pathogenic species. We also discuss the insights into the host immune response obtained from the latest genetic modified host models as well as insights from monogenic disorders in humans. Finally we highlight outstanding questions that can be answered in the near future using bioinformatics and genomic tools.
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http://dx.doi.org/10.1016/bs.adgen.2020.02.001DOI Listing
January 2021

Nine Things Genomics Can Tell Us About .

Front Genet 2020 15;11:351. Epub 2020 Apr 15.

Medical Research Council Centre for Medical Mycology at The University of Exeter, Exeter, United Kingdom.

is a recently emerged multidrug-resistant fungal pathogen causing severe illness in hospitalized patients. is most closely related to a few environmental or rarely observed but cosmopolitan species. However, is unique in the concern it is generating among public health agencies for its rapid emergence, difficulty to treat, and the likelihood for further and more extensive outbreaks and spread. To date, five geographically distributed and genetically divergent lineages have been identified, none of which includes isolates that were collected prior to 1996. Indeed, ' ecological niche(s) and emergence remain enigmatic, although a number of hypotheses have been proposed. Recent genomic and transcriptomic work has also identified a variety of gene and chromosomal features that may have conferred with several important clinical phenotypes including its drug-resistance and growth at high temperatures. In this review we discuss nine major lines of enquiry into that big-data technologies and analytical approaches are beginning to answer.
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http://dx.doi.org/10.3389/fgene.2020.00351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174702PMC
April 2020

A New Lineage of Cryptococcus gattii (VGV) Discovered in the Central Zambezian Miombo Woodlands.

mBio 2019 11 12;10(6). Epub 2019 Nov 12.

Molecular Microbiology Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

We discovered a new lineage of the globally important fungal pathogen on the basis of analysis of six isolates collected from three locations spanning the Central Miombo Woodlands of Zambia, Africa. All isolates were from environments (middens and tree holes) that are associated with a small mammal, the African hyrax. Phylogenetic and population genetic analyses confirmed that these isolates form a distinct, deeply divergent lineage, which we name VGV. VGV comprises two subclades (A and B) that are capable of causing mild lung infection with negligible neurotropism in mice. Comparing the VGV genome to previously identified lineages of revealed a unique suite of genes together with gene loss and inversion events. However, standard restriction fragment length polymorphism (RFLP) analysis could not distinguish between VGV and VGIV isolates. We therefore developed a new RFLP method that can reliably identify the newly described lineage. Our work highlights how sampling understudied ecological regions alongside genomic and functional characterization can broaden our understanding of the evolution and ecology of major global pathogens. is an environmental pathogen that causes severe systemic infection in immunocompetent individuals more often than in immunocompromised humans. Over the past 2 decades, researchers have shown that falls within four genetically distinct major lineages. By combining field work from an understudied ecological region (the Central Miombo Woodlands of Zambia, Africa), genome sequencing and assemblies, phylogenetic and population genetic analyses, and phenotypic characterization (morphology, histopathological, drug-sensitivity, survival experiments), we discovered a hitherto unknown lineage, which we name VGV (variety five). The discovery of a new lineage from an understudied ecological region has far-reaching implications for the study and understanding of fungal pathogens and diseases they cause.
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http://dx.doi.org/10.1128/mBio.02306-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851281PMC
November 2019

Evaluation of High-Resolution Melting for Rapid Differentiation of Hybrids and Their Parental Species.

Plant Dis 2019 Sep 29;103(9):2295-2304. Epub 2019 Jul 29.

Department of Plant Pathology and Emerging Pathogens Institute, University of Florida, Gainesville, FL 32611 U.S.A.

species hybrids have been repeatedly reported as causing damaging diseases to cultivated and wild plants. Two known hybrids, and × , are pathogens of Solanaceae and ornamentals, respectively, although the extent of their host ranges are unknown. emerged from hybridization of and an unidentified related species, whereas × emerged from and . Considering that hybrids and parental species can coexist in the same regions and to distinguish them usually requires cloning or whole genome sequencing, we aimed to develop a rapid tool to distinguish them. Specifically, we used high-resolution melting (HRM) assays to differentiate genotypes based on their amplicon melting profiles. We designed primers for × and parental species based on available sequences of and nuclear genes containing polymorphisms between species. For , heterozygous sites from Illumina short reads were used for the same purpose. We identified multiple amplicons exhibiting differences in melting curves between parental species and hybrids. We propose HRM as a rapid method for differentiation of and × hybrids from parental species that could be employed to advance research on these pathogens.
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http://dx.doi.org/10.1094/PDIS-12-18-2291-REDOI Listing
September 2019

Batrachochytrium salamandrivorans.

Authors:
Rhys A Farrer

Trends Microbiol 2019 10 22;27(10):892-893. Epub 2019 May 22.

Medical Research Council Centre for Medical Mycology at the University of Aberdeen, Aberdeen Fungal Group, Institute of Medical Sciences, Foresterhill, Aberdeen, UK. Electronic address:

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http://dx.doi.org/10.1016/j.tim.2019.04.009DOI Listing
October 2019

Rapid phenotypic evolution in multidrug-resistant Klebsiella pneumoniae hospital outbreak strains.

Microb Genom 2019 04 2;5(4). Epub 2019 Apr 2.

2​Department of Clinical Laboratory, Peking University People's Hospital, Beijing, 100044, PR China.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) increasingly cause high-mortality outbreaks in hospital settings globally. Following a patient fatality at a hospital in Beijing due to a blaKPC-2-positive CRKP infection, close monitoring was put in place over the course of 14 months to characterize all blaKPC-2-positive CRKP in circulation in the hospital. Whole genome sequences were generated for 100 isolates from blaKPC-2-positive isolates from infected patients, carriers and the hospital environment. Phylogenetic analyses identified a closely related cluster of 82 sequence type 11 (ST11) isolates circulating in the hospital for at least a year prior to admission of the index patient. The majority of inferred transmissions for these isolates involved patients in intensive care units. Whilst the 82 ST11 isolates collected during the surveillance effort all had closely related chromosomes, we observed extensive diversity in their antimicrobial resistance (AMR) phenotypes. We were able to reconstruct the major genomic changes underpinning this variation in AMR profiles, including multiple gains and losses of entire plasmids and recombination events between plasmids, including transposition of blaKPC-2. We also identified specific cases where variation in plasmid copy number correlated with the level of phenotypic resistance to drugs, suggesting that the number of resistance elements carried by a strain may play a role in determining the level of AMR. Our findings highlight the epidemiological value of whole genome sequencing for investigating multi-drug-resistant hospital infections and illustrate that standard typing schemes cannot capture the extraordinarily fast genome evolution of CRKP isolates.
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http://dx.doi.org/10.1099/mgen.0.000263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521586PMC
April 2019

Genomic insights into multidrug-resistance, mating and virulence in Candida auris and related emerging species.

Nat Commun 2018 12 17;9(1):5346. Epub 2018 Dec 17.

Broad Institute of MIT and Harvard, Cambridge, MA, 02144, USA.

Candida auris is an emergent multidrug-resistant fungal pathogen causing increasing reports of outbreaks. While distantly related to C. albicans and C. glabrata, C. auris is closely related to rarely observed and often multidrug-resistant species from the C. haemulonii clade. Here, we analyze near complete genome assemblies for the four C. auris clades and three related species, and map intra- and inter-species rearrangements across the seven chromosomes. Using RNA-Seq-guided gene predictions, we find that most mating and meiosis genes are conserved and that clades contain either the MTLa or MTLα mating loci. Comparing the genomes of these emerging species to those of other Candida species identifies genes linked to drug resistance and virulence, including expanded families of transporters and lipases, as well as mutations and copy number variants in ERG11. Gene expression analysis identifies transporters and metabolic regulators specific to C. auris and those conserved with related species which may contribute to differences in drug response in this emerging fungal clade.
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http://dx.doi.org/10.1038/s41467-018-07779-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297351PMC
December 2018

Transcriptional Heterogeneity of VGII Compared with Non-VGII Lineages Underpins Key Pathogenicity Pathways.

mSphere 2018 10 24;3(5). Epub 2018 Oct 24.

Infectious Disease and Microbiome Program, The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

is a pathogenic yeast of humans and other animals which causes disease predominantly in immunocompetent hosts. Infection begins when aerosolized yeast or spores enter the body, triggering an immune response, including engulfment by macrophages. To understand the early transcriptional signals in both the yeast and its mammalian host, we performed a time-course dual-transcriptome sequencing (RNA-seq) experiment for four lineages of (lineages VGI to IV) interacting with mouse macrophages at 1, 3, and 6 h postinfection. Comparisons of to gene expression levels indicated that lineage VGII is transcriptionally divergent from non-VGII lineages, including differential expression of genes involved in capsule synthesis, capsule attachment, and ergosterol production. Several paralogous genes demonstrated subfunctionalization between lineages, including upregulation of capsule biosynthesis-related gene and downregulation of in VGIII. Isolates also compensate for lineage-specific gene losses by overexpression of genetically similar paralogs, including overexpression of capsule gene in VGIV, which have lost the gene. Differential expression of one in five genes was detected following coincubation with mouse macrophages; all isolates showed high induction of oxidative-reduction functions and downregulation of capsule attachment genes. We also found that VGII switches expression of two laccase paralogs (from to ) during coincubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages of by upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This report highlights the evolutionary breadth of expression profiles among the lineages of and the diversity of transcriptional responses at this host-pathogen interface. The transcriptional profiles of related pathogens and their responses to host-induced stresses underpin their pathogenicity. Expression differences between related pathogens during host interaction can indicate when and how these genes contribute to virulence, ultimately informing new and improved treatment strategies for those diseases. In this paper, we compare the transcriptional profiles of five isolates representing four lineages of in rich media. Our analyses identified key processes, including those involving cell capsule, ergosterol production, and melanin, that are differentially expressed between lineages, and we found that VGII has the most distinct profile in terms of numbers of differentially expressed genes. All lineages have also undergone subfunctionalization for several paralogs, including capsule biosynthesis and attachment genes. Most genes appeared downregulated during coincubation with macrophages, with the largest decrease observed for capsule attachment genes, which appeared to be coordinated with a stress response, as all lineages also upregulated oxidative stress response genes. Furthermore, VGII upregulated many genes that are linked to ergosterol biosynthesis and switched from expression of the laccase to expression of Finally, we saw a pronounced increase in the FosB/Jun/Egr1 regulatory proteins at early time points in bone marrow-derived macrophages, marking a role in the host response to This work highlights the dynamic roles of key virulence genes in response to macrophages.
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http://dx.doi.org/10.1128/mSphere.00445-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200987PMC
October 2018

Global analysis of mutations driving microevolution of a heterozygous diploid fungal pathogen.

Proc Natl Acad Sci U S A 2018 09 27;115(37):E8688-E8697. Epub 2018 Aug 27.

Department of Molecular Microbiology and Immunology, Brown University, Providence, RI 02912;

is a heterozygous diploid yeast that is a commensal of the human gastrointestinal tract and a prevalent opportunistic pathogen. Here, whole-genome sequencing was performed on multiple isolates passaged both in vitro and in vivo to characterize the complete spectrum of mutations arising in laboratory culture and in the mammalian host. We establish that, independent of culture niche, microevolution is primarily driven by de novo base substitutions and frequent short-tract loss-of-heterozygosity events. An average base-substitution rate of ∼1.2 × 10 per base pair per generation was observed in vitro, with higher rates inferred during host infection. Large-scale chromosomal changes were relatively rare, although chromosome 7 trisomies frequently emerged during passaging in a gastrointestinal model and was associated with increased fitness for this niche. Multiple chromosomal features impacted mutational patterns, with mutation rates elevated in repetitive regions, subtelomeric regions, and in gene families encoding cell surface proteins involved in host adhesion. Strikingly, de novo mutation rates were more than 800-fold higher in regions immediately adjacent to emergent loss-of-heterozygosity tracts, indicative of recombination-induced mutagenesis. Furthermore, genomes showed biased patterns of mutations suggestive of extensive purifying selection during passaging. These results reveal how both cell-intrinsic and cell-extrinsic factors influence microevolution, and provide a quantitative picture of genome dynamics in this heterozygous diploid species.
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http://dx.doi.org/10.1073/pnas.1806002115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140516PMC
September 2018

MARDy: Mycology Antifungal Resistance Database.

Bioinformatics 2018 09;34(18):3233-3234

MRC Centre for Outbreak Analysis and Modelling, Imperial College London, London, UK.

Summary: The increase of antifungal drug resistance is a major global human health concern and threatens agriculture and food security; in order to tackle these concerns, it is important to understand the mechanisms that cause antifungal resistance. The curated Mycology Antifungal Resistance Database (MARDy) is a web-service of antifungal drug resistance mechanisms, including amino acid substitutions, tandem repeat sequences and genome ploidy. MARDy is implemented on a Linux, Apache, MySQL and PHP web development platform and includes a local installation of BLASTn of the database of curated genes.

Availability And Implementation: MARDy can be accessed at http://www.mardy.net and is free to use. The complete database can be retrieved, ordered by organism, gene and drug. Missing or new mycological antifungal resistance data can be relayed to the development team through a contribute entry form. Updates and news will be publicized via a dedicated Twitter feed: @MARDYfungi.
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http://dx.doi.org/10.1093/bioinformatics/bty321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137992PMC
September 2018

Author Correction: Genomic epidemiology of the UK outbreak of the emerging human fungal pathogen Candida auris.

Emerg Microbes Infect 2018 05 31;7(1):104. Epub 2018 May 31.

National Heart and Lung Institute, Imperial College London, London, SW3 6LR, UK.

Correction to: Emerging Microbes & Infections (2018) 7,43 https://doi.org/10.1038/s41426-018-0045-x ; published online 29 March 2018.
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http://dx.doi.org/10.1038/s41426-018-0098-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5981204PMC
May 2018

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi.

Sci Rep 2018 05 17;8(1):7772. Epub 2018 May 17.

Institute of Zoology, Regent's Park, London, NW1 4RY, UK.

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.
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http://dx.doi.org/10.1038/s41598-018-24472-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5958081PMC
May 2018

Recent Asian origin of chytrid fungi causing global amphibian declines.

Science 2018 05;360(6389):621-627

Department of Infectious Disease Epidemiology and MRC Centre for Global Infectious Disease Analysis, School of Public Health, Imperial College London, London W2 1PG, UK.

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus , a proximate driver of global amphibian declines. We traced the source of to the Korean peninsula, where one lineage, ASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for biodiversity and the original source of these lineages that now parasitize amphibians worldwide.
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http://dx.doi.org/10.1126/science.aar1965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311102PMC
May 2018

Genomic epidemiology of the UK outbreak of the emerging human fungal pathogen Candida auris.

Emerg Microbes Infect 2018 Mar 29;7(1):43. Epub 2018 Mar 29.

National Heart and Lung Institute, Imperial College London, London, SW3 6LR, UK.

Candida auris was first described in 2009, and it has since caused nosocomial outbreaks, invasive infections, and fungaemia across at least 19 countries on five continents. An outbreak of C. auris occurred in a specialized cardiothoracic London hospital between April 2015 and November 2016, which to date has been the largest outbreak in the UK, involving a total of 72 patients. To understand the genetic epidemiology of C. auris infection both within this hospital and within a global context, we sequenced the outbreak isolate genomes using Oxford Nanopore Technologies and Illumina platforms to detect antifungal resistance alleles and reannotate the C. auris genome. Phylogenomic analysis placed the UK outbreak in the India/Pakistan clade, demonstrating an Asian origin; the outbreak showed similar genetic diversity to that of the entire clade, and limited local spatiotemporal clustering was observed. One isolate displayed resistance to both echinocandins and 5-flucytosine; the former was associated with a serine to tyrosine amino acid substitution in the gene FKS1, and the latter was associated with a phenylalanine to isoleucine substitution in the gene FUR1. These mutations add to a growing body of research on multiple antifungal drug targets in this organism. Multiple differential episodic selection of antifungal resistant genotypes has occurred within a genetically heterogenous population across this outbreak, creating a resilient pathogen and making it difficult to define local-scale patterns of transmission and implement outbreak control measures.
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http://dx.doi.org/10.1038/s41426-018-0045-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874254PMC
March 2018

Synima: a Synteny imaging tool for annotated genome assemblies.

Authors:
Rhys A Farrer

BMC Bioinformatics 2017 Nov 21;18(1):507. Epub 2017 Nov 21.

Department of Infectious Disease Epidemiology, Imperial College London, London, W2 1PG, UK.

Background: Ortholog prediction and synteny visualization across whole genomes are valuable methods for detecting and representing a range of evolutionary processes such as genome expansion, chromosomal rearrangement, and chromosomal translocation. Few standalone methods are currently available to visualize synteny across any number of annotated genomes.

Results: Here, I present a Synteny Imaging tool (Synima) written in Perl, which uses the graphical features of R. Synima takes orthologues computed from reciprocal best BLAST hits or OrthoMCL, and DAGchainer, and outputs an overview of genome-wide synteny in PDF. Each of these programs are included with the Synima package, and a pipeline for their use. Synima has a range of graphical parameters including size, colours, order, and labels, which are specified in a config file generated by the first run of Synima - and can be subsequently edited. Synima runs quickly on a command line to generate informative and publication quality figures. Synima is open source and freely available from https://github.com/rhysf/Synima under the MIT License.

Conclusions: Synima should be a valuable tool for visualizing synteny between two or more annotated genome assemblies.
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http://dx.doi.org/10.1186/s12859-017-1939-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697234PMC
November 2017

Describing Genomic and Epigenomic Traits Underpinning Emerging Fungal Pathogens.

Adv Genet 2017 28;100:73-140. Epub 2017 Oct 28.

Imperial College London, London, United Kingdom. Electronic address:

An unprecedented number of pathogenic fungi are emerging and causing disease in animals and plants, putting the resilience of wild and managed ecosystems in jeopardy. While the past decades have seen an increase in the number of pathogenic fungi, they have also seen the birth of new big data technologies and analytical approaches to tackle these emerging pathogens. We review how the linked fields of genomics and epigenomics are transforming our ability to address the challenge of emerging fungal pathogens. We explore the methodologies and bioinformatic toolkits that currently exist to rapidly analyze the genomes of unknown fungi, then discuss how these data can be used to address key questions that shed light on their epidemiology. We show how genomic approaches are leading a revolution into our understanding of emerging fungal diseases and speculate on future approaches that will transform our ability to tackle this increasingly important class of emerging pathogens.
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http://dx.doi.org/10.1016/bs.adgen.2017.09.009DOI Listing
July 2018

Genomic innovations linked to infection strategies across emerging pathogenic chytrid fungi.

Nat Commun 2017 03 21;8:14742. Epub 2017 Mar 21.

Genome Sequencing and Analysis Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.

To understand the evolutionary pathways that lead to emerging infections of vertebrates, here we explore the genomic innovations that allow free-living chytrid fungi to adapt to and colonize amphibian hosts. Sequencing and comparing the genomes of two pathogenic species of Batrachochytrium to those of close saprophytic relatives reveals that pathogenicity is associated with remarkable expansions of protease and cell wall gene families, while divergent infection strategies are linked to radiations of lineage-specific gene families. By comparing the host-pathogen response to infection for both pathogens, we illuminate the traits that underpin a strikingly different immune response within a shared host species. Our results show that, despite commonalities that promote infection, specific gene-family radiations contribute to distinct infection strategies. The breadth and evolutionary novelty of candidate virulence factors that we discover underscores the urgent need to halt the advance of pathogenic chytrids and prevent incipient loss of biodiversity.
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http://dx.doi.org/10.1038/ncomms14742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364385PMC
March 2017

Microevolution of Serial Clinical Isolates of var. and .

mBio 2017 03 7;8(2). Epub 2017 Mar 7.

Division of Infectious Diseases, Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA

The pathogenic species of are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in sub-Saharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification of at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution of species under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis. Opportunistic infections caused by species of the pathogenic yeast lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease.
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http://dx.doi.org/10.1128/mBio.00166-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5340869PMC
March 2017

Microevolutionary traits and comparative population genomics of the emerging pathogenic fungus Cryptococcus gattii.

Philos Trans R Soc Lond B Biol Sci 2016 12;371(1709)

Genome Sequencing and Analysis Program, The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

Emerging fungal pathogens cause an expanding burden of disease across the animal kingdom, including a rise in morbidity and mortality in humans. Yet, we currently have only a limited repertoire of available therapeutic interventions. A greater understanding of the mechanisms of fungal virulence and of the emergence of hypervirulence within species is therefore needed for new treatments and mitigation efforts. For example, over the past decade, an unusual lineage of Cryptococcus gattii, which was first detected on Vancouver Island, has spread to the Canadian mainland and the Pacific Northwest infecting otherwise healthy individuals. The molecular changes that led to the development of this hypervirulent cryptococcal lineage remain unclear. To explore this, we traced the history of similar microevolutionary events that can lead to changes in host range and pathogenicity. Here, we detail fine-resolution mapping of genetic differences between two highly related Cryptococcus gattii VGIIc isolates that differ in their virulence traits (phagocytosis, vomocytosis, macrophage death, mitochondrial tubularization and intracellular proliferation). We identified a small number of single site variants within coding regions that potentially contribute to variations in virulence. We then extended our methods across multiple lineages of C. gattii to study how selection is acting on key virulence genes within different lineages.This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience'.
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http://dx.doi.org/10.1098/rstb.2016.0021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095545PMC
December 2016

Zinc Cluster Transcription Factors Alter Virulence in Candida albicans.

Genetics 2017 02 7;205(2):559-576. Epub 2016 Dec 7.

Worcester Polytechnic Institute, Massachusetts 01609

Almost all humans are colonized with Candida albicans However, in immunocompromised individuals, this benign commensal organism becomes a serious, life-threatening pathogen. Here, we describe and analyze the regulatory networks that modulate innate responses in the host niches. We identified Zcf15 and Zcf29, two Zinc Cluster transcription Factors (ZCF) that are required for C. albicans virulence. Previous sequence analysis of clinical C. albicans isolates from immunocompromised patients indicates that both ZCF genes diverged during clonal evolution. Using in vivo animal models, ex vivo cell culture methods, and in vitro sensitivity assays, we demonstrate that knockout mutants of both ZCF15 and ZCF29 are hypersensitive to reactive oxygen species (ROS), suggesting they help neutralize the host-derived ROS produced by phagocytes, as well as establish a sustained infection in vivo Transcriptomic analysis of mutants under resting conditions where cells were not experiencing oxidative stress revealed a large network that control macro and micronutrient homeostasis, which likely contributes to overall pathogen fitness in host niches. Under oxidative stress, both transcription factors regulate a separate set of genes involved in detoxification of ROS and down-regulating ribosome biogenesis. ChIP-seq analysis, which reveals vastly different binding partners for each transcription factor (TF) before and after oxidative stress, further confirms these results. Furthermore, the absence of a dominant binding motif likely facilitates their mobility, and supports the notion that they represent a recent expansion of the ZCF family in the pathogenic Candida species. Our analyses provide a framework for understanding new aspects of the interface between C. albicans and host defense response, and extends our understanding of how complex cell behaviors are linked to the evolution of TFs.
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http://dx.doi.org/10.1534/genetics.116.195024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289837PMC
February 2017

Genome Diversity, Recombination, and Virulence across the Major Lineages of .

mSphere 2016 Sep-Oct;1(5). Epub 2016 Sep 28.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

The genus includes two species of thermally dimorphic fungi that cause paracoccidioidomycosis, a neglected health-threatening human systemic mycosis endemic to Latin America. To examine the genome evolution and the diversity of spp., we conducted whole-genome sequencing of 31 isolates representing the phylogenetic, geographic, and ecological breadth of the genus. These samples included clinical, environmental and laboratory reference strains of the S1, PS2, PS3, and PS4 lineages of and also isolates of species We completed the first annotated genome assemblies for the PS3 and PS4 lineages and found that gene order was highly conserved across the major lineages, with only a few chromosomal rearrangements Comparing whole-genome assemblies of the major lineages with single-nucleotide polymorphisms (SNPs) predicted from the remaining 26 isolates, we identified a deep split of the S1 lineage into two clades we named S1a and S1b. We found evidence for greater genetic exchange between the S1b lineage and all other lineages; this may reflect the broad geographic range of S1b, which is often sympatric with the remaining, largely geographically isolated lineages. In addition, we found evidence of positive selection for the and antigen genes and genes coding for other secreted proteins and proteases and lineage-specific loss-of-function mutations in cell wall and protease genes; these together may contribute to virulence and host immune response variation among natural isolates of spp. These insights into the recent evolutionary events highlight important differences between the lineages that could impact the distribution, pathogenicity, and ecology of . Characterization of genetic differences between lineages of the dimorphic human-pathogenic fungus can identify changes linked to important phenotypes and guide the development of new diagnostics and treatments. In this article, we compared genomes of 31 diverse isolates representing the major lineages of spp. and completed the first annotated genome sequences for the PS3 and PS4 lineages. We analyzed the population structure and characterized the genetic diversity among the lineages of , including a deep split of S1 into two lineages (S1a and S1b), and differentiated S1b, associated with most clinical cases, as the more highly recombining and diverse lineage. In addition, we found patterns of positive selection in surface proteins and secreted enzymes among the lineages, suggesting diversifying mechanisms of pathogenicity and adaptation across this species complex. These genetic differences suggest associations with the geographic range, pathogenicity, and ecological niches of lineages.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040785PMC
http://dx.doi.org/10.1128/mSphere.00213-16DOI Listing
September 2016

Hsf1 and Hsp90 orchestrate temperature-dependent global transcriptional remodelling and chromatin architecture in Candida albicans.

Nat Commun 2016 05 26;7:11704. Epub 2016 May 26.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.

Fever is a universal response to infection, and opportunistic pathogens such as Candida albicans have evolved complex circuitry to sense and respond to heat. Here we harness RNA-seq and ChIP-seq to discover that the heat shock transcription factor, Hsf1, binds distinct motifs in nucleosome-depleted promoter regions to regulate heat shock genes and genes involved in virulence in C. albicans. Consequently, heat shock increases C. albicans host cell adhesion, damage and virulence. Hsf1 activation depends upon the molecular chaperone Hsp90 under basal and heat shock conditions, but the effects are opposite and in part controlled at the level of Hsf1 expression and DNA binding. Finally, we demonstrate that Hsp90 regulates global transcription programs by modulating nucleosome levels at promoters of stress-responsive genes. Thus, we describe a mechanism by which C. albicans responds to temperature via Hsf1 and Hsp90 to orchestrate gene expression and chromatin architecture, thereby enabling thermal adaptation and virulence.
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http://dx.doi.org/10.1038/ncomms11704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894976PMC
May 2016

Genome Evolution and Innovation across the Four Major Lineages of Cryptococcus gattii.

mBio 2015 Sep 1;6(5):e00868-15. Epub 2015 Sep 1.

Genome Sequencing and Analysis Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

Unlabelled: Cryptococcus gattii is a fungal pathogen of humans, causing pulmonary infections in otherwise healthy hosts. To characterize genomic variation among the four major lineages of C. gattii (VGI, -II, -III, and -IV), we generated, annotated, and compared 16 de novo genome assemblies, including the first for the rarely isolated lineages VGIII and VGIV. By identifying syntenic regions across assemblies, we found 15 structural rearrangements, which were almost exclusive to the VGI-III-IV lineages. Using synteny to inform orthology prediction, we identified a core set of 87% of C. gattii genes present as single copies in all four lineages. Remarkably, 737 genes are variably inherited across lineages and are overrepresented for response to oxidative stress, mitochondrial import, and metal binding and transport. Specifically, VGI has an expanded set of iron-binding genes thought to be important to the virulence of Cryptococcus, while VGII has expansions in the stress-related heat shock proteins relative to the other lineages. We also characterized genes uniquely absent in each lineage, including a copper transporter absent from VGIV, which influences Cryptococcus survival during pulmonary infection and the onset of meningoencephalitis. Through inclusion of population-level data for an additional 37 isolates, we identified a new transcontinental clonal group that we name VGIIx, mitochondrial recombination between VGII and VGIII, and positive selection of multidrug transporters and the iron-sulfur protein aconitase along multiple branches of the phylogenetic tree. Our results suggest that gene expansion or contraction and positive selection have introduced substantial variation with links to mechanisms of pathogenicity across this species complex.

Importance: The genetic differences between phenotypically different pathogens provide clues to the underlying mechanisms of those traits and can lead to new drug targets and improved treatments for those diseases. In this paper, we compare 16 genomes belonging to four highly differentiated lineages of Cryptococcus gattii, which cause pulmonary infections in otherwise healthy humans and other animals. Half of these lineages have not had their genomes previously assembled and annotated. We identified 15 ancestral rearrangements in the genome and over 700 genes that are unique to one or more lineages, many of which are associated with virulence. In addition, we found evidence for recent transcontinental spread, mitochondrial genetic exchange, and positive selection in multidrug transporters. Our results suggest that gene expansion/contraction and positive selection are diversifying the mechanisms of pathogenicity across this species complex.
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http://dx.doi.org/10.1128/mBio.00868-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4556806PMC
September 2015

Transmission of Hypervirulence traits via sexual reproduction within and between lineages of the human fungal pathogen cryptococcus gattii.

PLoS Genet 2013 5;9(9):e1003771. Epub 2013 Sep 5.

Institute of Microbiology and Infection & School of Biosciences, University of Birmingham, Birmingham, United Kingdom ; The National Institute of Health Research Surgical Reconstruction and Microbiology Research Centre, Queen Elizabeth Hospital Birmingham, Birmingham, United Kingdom.

Since 1999 a lineage of the pathogen Cryptococcus gattii has been infecting humans and other animals in Canada and the Pacific Northwest of the USA. It is now the largest outbreak of a life-threatening fungal infection in a healthy population in recorded history. The high virulence of outbreak strains is closely linked to the ability of the pathogen to undergo rapid mitochondrial tubularisation and proliferation following engulfment by host phagocytes. Most outbreaks spread by geographic expansion across suitable niches, but it is known that genetic re-assortment and hybridisation can also lead to rapid range and host expansion. In the context of C. gattii, however, the likelihood of virulence traits associated with the outbreak lineages spreading to other lineages via genetic exchange is currently unknown. Here we address this question by conducting outgroup crosses between distantly related C. gattii lineages (VGII and VGIII) and ingroup crosses between isolates from the same molecular type (VGII). Systematic phenotypic characterisation shows that virulence traits are transmitted to outgroups infrequently, but readily inherited during ingroup crosses. In addition, we observed higher levels of biparental (as opposed to uniparental) mitochondrial inheritance during VGII ingroup sexual mating in this species and provide evidence for mitochondrial recombination following mating. Taken together, our data suggest that hypervirulence can spread among the C. gattii lineages VGII and VGIII, potentially creating novel hypervirulent genotypes, and that current models of uniparental mitochondrial inheritance in the Cryptococcus genus may not be universal.
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http://dx.doi.org/10.1371/journal.pgen.1003771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3764205PMC
March 2014

Chromosomal copy number variation, selection and uneven rates of recombination reveal cryptic genome diversity linked to pathogenicity.

PLoS Genet 2013 15;9(8):e1003703. Epub 2013 Aug 15.

The Department of Infectious Disease Epidemiology, Imperial College London, London, United Kingdom.

Pathogenic fungi constitute a growing threat to both plant and animal species on a global scale. Despite a clonal mode of reproduction dominating the population genetic structure of many fungi, putatively asexual species are known to adapt rapidly when confronted by efforts to control their growth and transmission. However, the mechanisms by which adaptive diversity is generated across a clonal background are often poorly understood. We sequenced a global panel of the emergent amphibian pathogen, Batrachochytrium dendrobatidis (Bd), to high depth and characterized rapidly changing features of its genome that we believe hold the key to the worldwide success of this organism. Our analyses show three processes that contribute to the generation of de novo diversity. Firstly, we show that the majority of wild isolates manifest chromosomal copy number variation that changes over short timescales. Secondly, we show that cryptic recombination occurs within all lineages of Bd, leading to large regions of the genome being in linkage equilibrium, and is preferentially associated with classes of genes of known importance for virulence in other pathosystems. Finally, we show that these classes of genes are under directional selection, and that this has predominantly targeted the Global Panzootic Lineage (BdGPL). Our analyses show that Bd manifests an unusually dynamic genome that may have been shaped by its association with the amphibian host. The rates of variation that we document likely explain the high levels of phenotypic variability that have been reported for Bd, and suggests that the dynamic genome of this pathogen has contributed to its success across multiple biomes and host-species.
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http://dx.doi.org/10.1371/journal.pgen.1003703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3744429PMC
March 2014

Using false discovery rates to benchmark SNP-callers in next-generation sequencing projects.

Sci Rep 2013 ;3:1512

Department of Infectious Disease Epidemiology, St Mary's Hospital, Imperial College London, London, UK.

Sequence alignments form the basis for many comparative and population genomic studies. Alignment tools provide a range of accuracies dependent on the divergence between the sequences and the alignment methods. Despite widespread use, there is no standard method for assessing the accuracy of a dataset and alignment strategy after resequencing. We present a framework and tool for determining the overall accuracies of an input read dataset, alignment and SNP-calling method providing an isolate in that dataset has a corresponding, or closely related reference sequence available. In addition to this tool for comparing False Discovery Rates (FDR), we include a method for determining homozygous and heterozygous positions from an alignment using binomial probabilities for an expected error rate. We benchmark this method against other SNP callers using our FDR method with three fungal genomes, finding that it was able achieve a high level of accuracy. These tools are available at http://cfdr.sourceforge.net/.
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http://dx.doi.org/10.1038/srep01512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3604800PMC
September 2013

Genome analyses of an aggressive and invasive lineage of the Irish potato famine pathogen.

PLoS Pathog 2012 4;8(10):e1002940. Epub 2012 Oct 4.

The James Hutton Institute, Invergowrie, Dundee, United Kingdom.

Pest and pathogen losses jeopardise global food security and ever since the 19(th) century Irish famine, potato late blight has exemplified this threat. The causal oomycete pathogen, Phytophthora infestans, undergoes major population shifts in agricultural systems via the successive emergence and migration of asexual lineages. The phenotypic and genotypic bases of these selective sweeps are largely unknown but management strategies need to adapt to reflect the changing pathogen population. Here, we used molecular markers to document the emergence of a lineage, termed 13_A2, in the European P. infestans population, and its rapid displacement of other lineages to exceed 75% of the pathogen population across Great Britain in less than three years. We show that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance. Genome analyses of a 13_A2 isolate revealed extensive genetic and expression polymorphisms particularly in effector genes. Copy number variations, gene gains and losses, amino-acid replacements and changes in expression patterns of disease effector genes within the 13_A2 isolate likely contribute to enhanced virulence and aggressiveness to drive this population displacement. Importantly, 13_A2 isolates carry intact and in planta induced Avrblb1, Avrblb2 and Avrvnt1 effector genes that trigger resistance in potato lines carrying the corresponding R immune receptor genes Rpi-blb1, Rpi-blb2, and Rpi-vnt1.1. These findings point towards a strategy for deploying genetic resistance to mitigate the impact of the 13_A2 lineage and illustrate how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.
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http://dx.doi.org/10.1371/journal.ppat.1002940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464212PMC
April 2013

Multiple emergences of genetically diverse amphibian-infecting chytrids include a globalized hypervirulent recombinant lineage.

Proc Natl Acad Sci U S A 2011 Nov 7;108(46):18732-6. Epub 2011 Nov 7.

Department Infectious Disease Epidemiology, Imperial College, London W2 1PG, United Kingdom.

Batrachochytrium dendrobatidis (Bd) is a globally ubiquitous fungal infection that has emerged to become a primary driver of amphibian biodiversity loss. Despite widespread effort to understand the emergence of this panzootic, the origins of the infection, its patterns of global spread, and principle mode of evolution remain largely unknown. Using comparative population genomics, we discovered three deeply diverged lineages of Bd associated with amphibians. Two of these lineages were found in multiple continents and are associated with known introductions by the amphibian trade. We found that isolates belonging to one clade, the global panzootic lineage (BdGPL) have emerged across at least five continents during the 20th century and are associated with the onset of epizootics in North America, Central America, the Caribbean, Australia, and Europe. The two newly identified divergent lineages, Cape lineage (BdCAPE) and Swiss lineage (BdCH), were found to differ in morphological traits when compared against one another and BdGPL, and we show that BdGPL is hypervirulent. BdGPL uniquely bears the hallmarks of genomic recombination, manifested as extensive intergenomic phylogenetic conflict and patchily distributed heterozygosity. We postulate that contact between previously genetically isolated allopatric populations of Bd may have allowed recombination to occur, resulting in the generation, spread, and invasion of the hypervirulent BdGPL leading to contemporary disease-driven losses in amphibian biodiversity.
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http://dx.doi.org/10.1073/pnas.1111915108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3219125PMC
November 2011

Genome evolution following host jumps in the Irish potato famine pathogen lineage.

Science 2010 Dec;330(6010):1540-3

The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, UK.

Many plant pathogens, including those in the lineage of the Irish potato famine organism Phytophthora infestans, evolve by host jumps followed by specialization. However, how host jumps affect genome evolution remains largely unknown. To determine the patterns of sequence variation in the P. infestans lineage, we resequenced six genomes of four sister species. This revealed uneven evolutionary rates across genomes with genes in repeat-rich regions showing higher rates of structural polymorphisms and positive selection. These loci are enriched in genes induced in planta, implicating host adaptation in genome evolution. Unexpectedly, genes involved in epigenetic processes formed another class of rapidly evolving residents of the gene-sparse regions. These results demonstrate that dynamic repeat-rich genome compartments underpin accelerated gene evolution following host jumps in this pathogen lineage.
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http://dx.doi.org/10.1126/science.1193070DOI Listing
December 2010