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Facultative heterochromatin controls development and differentiation in many eukaryotes. In metazoans, plants, and many filamentous fungi, facultative heterochromatin is characterized by transcriptional repression and enrichment with nucleosomes that are trimethylated at histone H3 lysine 27 (H3K27me3). While loss of H3K27me3 results in derepression of transcriptional gene silencing in many species, additional up- and downstream layers of regulation are necessary to mediate control of transcription in chromosome regions enriched with H3K27me3. Here, we investigated the effects of one histone mark on histone H4, namely H4K20me3, in the fungus Zymoseptoria tritici, a globally important pathogen of wheat. Deletion of kmt5, the gene encoding the sole methyltransferase responsible for H4K20 methylation, resulted in global derepression of transcription, especially in regions of facultative heterochromatin. Derepression in the absence of H4K20me3 not only affected known genes but also a large number of novel, previously undetected transcripts generated from regions of facultative heterochromatin on accessory chromosomes. Transcriptional activation in kmt5 deletion strains was accompanied by a complete loss of Ash1-mediated H3K36me3 and chromatin reorganization affecting H3K27me3 and H3K4me2 distribution in regions of facultative heterochromatin. Strains with H4K20L, M or Q mutations in the single histone H4 gene of Z. tritici recapitulated these chromatin changes, suggesting that H4K20me3 is important for Ash1-mediated H3K36me3. The ∆kmt5 mutants we obtained were more sensitive to genotoxic stressors than wild type and both, ∆kmt5 and ∆ash1, showed greatly increased rates of accessory chromosome loss. Taken together, our results provide insights into an unsuspected mechanism involved in the assembly and maintenance of facultative heterochromatin.
Assuntos
Heterocromatina , Histonas , Heterocromatina/genética , Histonas/genética , Histonas/metabolismo , Cromatina , Nucleossomos , MetilaçãoRESUMO
DNA methylation is found throughout all domains of life, yet the extent and function of DNA methylation differ among eukaryotes. Strains of the plant pathogenic fungus Zymoseptoria tritici appeared to lack cytosine DNA methylation (5mC) because gene amplification followed by Repeat-Induced Point mutation (RIP) resulted in the inactivation of the dim2 DNA methyltransferase gene. 5mC is, however, present in closely related sister species. We demonstrate that inactivation of dim2 occurred recently as some Z. tritici isolates carry a functional dim2 gene. Moreover, we show that dim2 inactivation occurred by a different path than previously hypothesized. We mapped the genome-wide distribution of 5mC in strains with or without functional dim2 alleles. Presence of functional dim2 correlates with high levels of 5mC in transposable elements (TEs), suggesting a role in genome defense. We identified low levels of 5mC in strains carrying non-functional dim2 alleles, suggesting that 5mC is maintained over time, presumably by an active Dnmt5 DNA methyltransferase. Integration of a functional dim2 allele in strains with mutated dim2 restored normal 5mC levels, demonstrating de novo cytosine methylation activity of Dim2. To assess the importance of 5mC for genome evolution, we performed an evolution experiment, comparing genomes of strains with high levels of 5mC to genomes of strains lacking functional dim2. We found that presence of a functional dim2 allele alters nucleotide composition by promoting C to T transitions (CâT) specifically at CpA (CA) sites during mitosis, likely contributing to TE inactivation. Our results show that 5mC density at TEs is a polymorphic trait in Z. tritici populations that can impact genome evolution.
Assuntos
Ascomicetos/enzimologia , Ascomicetos/genética , DNA (Citosina-5-)-Metiltransferase 1/deficiência , Evolução Molecular , Taxa de Mutação , Mutação , 5-Metilcitosina/metabolismo , Alelos , Ascomicetos/classificação , Ascomicetos/isolamento & purificação , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Geografia , Mitose , Filogeografia , Locos de Características QuantitativasRESUMO
Chromosome and genome stability are important for normal cell function as instability often correlates with disease and dysfunction of DNA repair mechanisms. Many organisms maintain supernumerary or accessory chromosomes that deviate from standard chromosomes. The pathogenic fungus Zymoseptoria tritici has as many as eight accessory chromosomes, which are highly unstable during meiosis and mitosis, transcriptionally repressed, show enrichment of repetitive elements, and enrichment with heterochromatic histone methylation marks, e.g., trimethylation of H3 lysine 9 or lysine 27 (H3K9me3, H3K27me3). To elucidate the role of heterochromatin on genome stability in Z. tritici, we deleted the genes encoding the methyltransferases responsible for H3K9me3 and H3K27me3, kmt1 and kmt6, respectively, and generated a double mutant. We combined experimental evolution and genomic analyses to determine the impact of these deletions on chromosome and genome stability, both in vitro and in planta. We used whole genome sequencing, ChIP-seq, and RNA-seq to compare changes in genome and chromatin structure, and differences in gene expression between mutant and wildtype strains. Analyses of genome and ChIP-seq data in H3K9me3-deficient strains revealed dramatic chromatin reorganization, where H3K27me3 is mostly relocalized into regions that are enriched with H3K9me3 in wild type. Many genome rearrangements and formation of new chromosomes were found in the absence of H3K9me3, accompanied by activation of transposable elements. In stark contrast, loss of H3K27me3 actually increased the stability of accessory chromosomes under normal growth conditions in vitro, even without large scale changes in gene activity. We conclude that H3K9me3 is important for the maintenance of genome stability because it disallows H3K27me3 in regions considered constitutive heterochromatin. In this system, H3K27me3 reduces the overall stability of accessory chromosomes, generating a "metastable" state for these quasi-essential regions of the genome.
Assuntos
Instabilidade Genômica , Histonas/metabolismo , Lisina/metabolismo , Ascomicetos/genética , Ascomicetos/metabolismo , Cromossomos Fúngicos , Deleção de Genes , Heterocromatina/genética , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/química , Metilação , Sequências Repetitivas de Ácido Nucleico , Ativação TranscricionalRESUMO
BACKGROUND: Antagonistic co-evolution can drive rapid adaptation in pathogens and shape genome architecture. Comparative genome analyses of several fungal pathogens revealed highly variable genomes, for many species characterized by specific repeat-rich genome compartments with exceptionally high sequence variability. Dynamic genome structure may enable fast adaptation to host genetics. The wheat pathogen Zymoseptoria tritici with its highly variable genome, has emerged as a model organism to study genome evolution of plant pathogens. Here, we compared genomes of Z. tritici isolates and of sister species infecting wild grasses to address the evolution of genome composition and structure. RESULTS: Using long-read technology, we sequenced and assembled genomes of Z. ardabiliae, Z. brevis, Z. pseudotritici and Z. passerinii, together with two isolates of Z. tritici. We report a high extent of genome collinearity among Zymoseptoria species and high conservation of genomic, transcriptomic and epigenomic signatures of compartmentalization. We identify high gene content variability both within and between species. In addition, such variability is mainly limited to the accessory chromosomes and accessory compartments. Despite strong host specificity and non-overlapping host-range between species, predicted effectors are mainly shared among Zymoseptoria species, yet exhibiting a high level of presence-absence polymorphism within Z. tritici. Using in planta transcriptomic data from Z. tritici, we suggest different roles for the shared orthologs and for the accessory genes during infection of their hosts. CONCLUSION: Despite previous reports of high genomic plasticity in Z. tritici, we describe here a high level of conservation in genomic, epigenomic and transcriptomic composition and structure across the genus Zymoseptoria. The compartmentalized genome allows the maintenance of a functional core genome co-occurring with a highly variable accessory genome.
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Ascomicetos , Doenças das Plantas , Ascomicetos/genética , Genoma Fúngico , PrednisolonaRESUMO
The presence or absence of specific transcription factors, chromatin remodeling machineries, chromatin modification enzymes, post-translational histone modifications and histone variants all play crucial roles in the regulation of pathogenicity genes. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) provides an important tool to study genome-wide protein-DNA interactions to help understand gene regulation in the context of native chromatin. ChIP-seq is a convenient in vivo technique to identify, map and characterize occupancy of specific DNA fragments with proteins against which specific antibodies exist or which can be epitope-tagged in vivo. We optimized existing ChIP protocols for use in the wheat pathogen Zymoseptoria tritici and closely related sister species. Here, we provide a detailed method, underscoring which aspects of the technique are organism-specific. Library preparation for Illumina sequencing is described, as this is currently the most widely used ChIP-seq method. One approach for the analysis and visualization of representative sequence is described; improved tools for these analyses are constantly being developed. Using ChIP-seq with antibodies against H3K4me2, which is considered a mark for euchromatin or H3K9me3 and H3K27me3, which are considered marks for heterochromatin, the overall distribution of euchromatin and heterochromatin in the genome of Z. tritici can be determined. Our ChIP-seq protocol was also successfully applied to Z. tritici strains with high levels of melanization or aberrant colony morphology, and to different species of the genus (Z. ardabiliae and Z. pseudotritici), suggesting that our technique is robust. The methods described here provide a powerful framework to study new aspects of chromatin biology and gene regulation in this prominent wheat pathogen.
Assuntos
Ascomicetos/genética , Imunoprecipitação da Cromatina , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Doenças das Plantas/microbiologia , Triticum/microbiologiaRESUMO
Heart failure (HF) is one of the most common diagnoses on admission to hospital in Germany, and one which incurs high costs. Integrated care in case management programs (CMPs) aims to improve treatment quality in the sense of guideline-driven treatment, while reducing hospital admissions, hospital costs, and mortality. A total of 1,844 patient data records from 11 German statutory health insurance companies enrolled in the CMP (intervention group [IG]) were compared with 1,844 standard-care patients (control group) using propensity score matching. The two groups were assessed over three follow-up observation periods regarding the endpoints' treatment costs, hospitalization rate, indicators for treatment quality (diagnostics, physician contact), and mortality. The evaluation revealed no significant differences regarding overall costs. The IG incurred significantly higher outpatient costs, but the medication costs and inpatient costs were not significantly different. There were also no significant differences in the number of hospital admissions. Patients within the CMP had significantly more frequent contact with a cardiologist, and underwent echocardiographic examination significantly more frequently. Mortality during the first follow-up observation year was considerably more favorable for the IG. There are indications that treatment quality is improved in HF patients.
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Transposable elements (TEs) impact genome plasticity, architecture, and evolution in fungal plant pathogens. The wide range of TE content observed in fungal genomes reflects diverse efficacy of host-genome defense mechanisms that can counter-balance TE expansion and spread. Closely related species can harbor drastically different TE repertoires. The evolution of fungal effectors, which are crucial determinants of pathogenicity, has been linked to the activity of TEs in pathogen genomes. Here, we describe how TEs have shaped genome evolution of the fungal wheat pathogen Zymoseptoria tritici and four closely related species. We compared de novo TE annotations and repeat-induced point mutation signatures in 26 genomes from the Zymoseptoria species-complex. Then, we assessed the relative insertion ages of TEs using a comparative genomics approach. Finally, we explored the impact of TE insertions on genome architecture and plasticity. The 26 genomes of Zymoseptoria species reflect different TE dynamics with a majority of recent insertions. TEs associate with accessory genome compartments, with chromosomal rearrangements, with gene presence/absence variation, and with effectors in all Zymoseptoria species. We find that the extent of RIP-like signatures varies among Z. tritici genomes compared to genomes of the sister species. The detection of a reduction of RIP-like signatures and TE recent insertions in Z. tritici reflects ongoing but still moderate TE mobility.
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Ascomicetos , Elementos de DNA Transponíveis , Ascomicetos/genética , Elementos de DNA Transponíveis/genética , Evolução Molecular , Genoma Fúngico , VirulênciaRESUMO
Genome integrity is essential to maintain cellular function and viability. Consequently, genome instability is frequently associated with dysfunction in cells and associated with plant, animal, and human diseases. One consequence of relaxed genome maintenance that may be less appreciated is an increased potential for rapid adaptation to changing environments in all organisms. Here, we discuss evidence for the control and function of facultative heterochromatin, which is delineated by methylation of histone H3 lysine 27 (H3K27me) in many fungi. Aside from its relatively well understood role in transcriptional repression, accumulating evidence suggests that H3K27 methylation has an important role in controlling the balance between maintenance and generation of novelty in fungal genomes. We present a working model for a minimal repressive network mediated by H3K27 methylation in fungi and outline challenges for future research.
Assuntos
Metilação de DNA/genética , Instabilidade Genômica/genética , Heterocromatina/genética , Neurospora crassa/genética , Genoma Fúngico/genética , Histonas/genética , Complexo Repressor Polycomb 2 , Processamento de Proteína Pós-Traducional/genéticaRESUMO
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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The haploid genome of the pathogenic fungus Zymoseptoria tritici is contained on "core" and "accessory" chromosomes. While 13 core chromosomes are found in all strains, as many as eight accessory chromosomes show presence/absence variation and rearrangements among field isolates. The factors influencing these presence/absence polymorphisms are so far unknown. We investigated chromosome stability using experimental evolution, karyotyping, and genome sequencing. We report extremely high and variable rates of accessory chromosome loss during mitotic propagation in vitro and in planta Spontaneous chromosome loss was observed in 2 to >50% of cells during 4 weeks of incubation. Similar rates of chromosome loss in the closely related Zymoseptoria ardabiliae suggest that this extreme chromosome dynamic is a conserved phenomenon in the genus. Elevating the incubation temperature greatly increases instability of accessory and even core chromosomes, causing severe rearrangements involving telomere fusion and chromosome breakage. Chromosome losses do not affect the fitness of Zymoseptoria tritici in vitro, but some lead to increased virulence, suggesting an adaptive role of this extraordinary chromosome instability.
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Ascomicetos/genética , Aberrações Cromossômicas , Deleção Cromossômica , Instabilidade Genômica , Ascomicetos/patogenicidade , Evolução Molecular , Genoma Fúngico , Virulência/genéticaRESUMO
The fungal kingdom comprises some of the most devastating plant pathogens. Sequencing the genomes of fungal pathogens has shown a remarkable variability in genome size and architecture. Population genomic data enable us to understand the mechanisms and the history of changes in genome size and adaptive evolution in plant pathogens. Although transposable elements predominantly have negative effects on their host, fungal pathogens provide prominent examples of advantageous associations between rapidly evolving transposable elements and virulence genes that cause variation in virulence phenotypes. By providing homogeneous environments at large regional scales, managed ecosystems, such as modern agriculture, can be conducive for the rapid evolution and dispersal of pathogens. In this Review, we summarize key examples from fungal plant pathogen genomics and discuss evolutionary processes in pathogenic fungi in the context of molecular evolution, population genomics and agriculture.
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Evolução Biológica , Fungos/genética , Fungos/fisiologia , Genoma Fúngico , Doenças das Plantas/microbiologia , Plantas/microbiologia , Variação GenéticaRESUMO
This corrects the article DOI: 10.1038/nrmicro.2017.76.