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1.
G3 (Bethesda) ; 11(4)2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33724368

RESUMEN

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.


Asunto(s)
Ascomicetos , Elementos Transponibles de ADN , Ascomicetos/genética , Elementos Transponibles de ADN/genética , Evolución Molecular , Genoma Fúngico , Virulencia
2.
PLoS Genet ; 17(3): e1009448, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33750960

RESUMEN

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 (C→T) 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.


Asunto(s)
Ascomicetos/enzimología , Ascomicetos/genética , ADN (Citosina-5-)-Metiltransferasa 1/deficiencia , Evolución Molecular , Tasa de Mutación , Mutación , 5-Metilcitosina/metabolismo , Alelos , Ascomicetos/clasificación , Ascomicetos/aislamiento & purificación , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Geografía , Mitosis , Filogeografía , Sitios de Carácter Cuantitativo
3.
Curr Protoc Microbiol ; 59(1): e128, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33175475

RESUMEN

The fungus Zymoseptoria tritici is one of the most devastating pathogens of wheat. Aside from its importance as a disease-causing agent, this species has emerged as a powerful model system for evolutionary genetic studies of crop-infecting fungal pathogens. Z. tritici exhibits exceptionally high levels of genetic and phenotypic diversity as well as morphological plasticity, which can make experimental studies and comparability of results obtained in different laboratories, e.g., from infection assays, challenging. Therefore, standardized experimental methods are crucial for research on Z. tritici biology and the interaction of this fungus with its wheat host. Here, we describe a suite of well-tested and optimized protocols ranging from isolation of Z. tritici field specimens to analyses of virulence assays under controlled conditions. Several biological and technical aspects of working with Z. tritici under laboratory conditions are considered and carefully described in each protocol. © 2020 The Authors. Basic Protocol 1: Purification of Z. tritici field isolates from leaf material Basic Protocol 2: Molecular identification of Z. tritici isolates Support Protocol 1: Rapid extraction of Z. tritici genomic DNA Support Protocol 2: Extraction of high-quality Z. tritici genomic DNA Basic Protocol 3: In vitro culture and long-term storage of Z. tritici isolates Basic Protocol 4: Analysis of Z. tritici virulence in wheat Support Protocol 3: Preparation of Z. tritici inoculum.


Asunto(s)
Ascomicetos/patogenicidad , Hongos/fisiología , Enfermedades de las Plantas/microbiología , Ascomicetos/genética , ADN de Hongos , Interacciones Huésped-Patógeno , Hojas de la Planta/microbiología , Virulencia , Flujo de Trabajo
4.
BMC Genomics ; 21(1): 588, 2020 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-32842972

RESUMEN

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.


Asunto(s)
Ascomicetos , Enfermedades de las Plantas , Ascomicetos/genética , Genoma Fúngico , Prednisolona
5.
Nat Commun ; 11(1): 1910, 2020 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-32313046

RESUMEN

Yield losses caused by fungal pathogens represent a major threat to global food production. One of the most devastating fungal wheat pathogens is Zymoseptoria tritici. Despite the importance of this fungus, the underlying mechanisms of plant-pathogen interactions are poorly understood. Here we present a conceptual framework based on coinfection assays, comparative metabolomics, and microbiome profiling to study the interaction of Z. tritici in susceptible and resistant wheat. We demonstrate that Z. tritici suppresses the production of immune-related metabolites in a susceptible cultivar. Remarkably, this fungus-induced immune suppression spreads within the leaf and even to other leaves, a phenomenon that we term "systemic induced susceptibility". Using a comparative metabolomics approach, we identify defense-related biosynthetic pathways that are suppressed and induced in susceptible and resistant cultivars, respectively. We show that these fungus-induced changes correlate with changes in the wheat leaf microbiome. Our findings suggest that immune suppression by this hemibiotrophic pathogen impacts specialized plant metabolism, alters its associated microbial communities, and renders wheat vulnerable to further infections.


Asunto(s)
Ascomicetos/metabolismo , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/fisiología , Metaboloma , Microbiota/fisiología , Inmunidad de la Planta/fisiología , Ascomicetos/patogenicidad , Benzoxazinas/metabolismo , Vías Biosintéticas , Coinfección , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Metabolismo Secundario , Triticum/inmunología , Triticum/microbiología
6.
Mol Plant Pathol ; 21(1): 124-138, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31702117

RESUMEN

Zymoseptoria tritici is a filamentous fungus causing Septoria tritici blotch in wheat. The pathogen has a narrow host range and infections of grasses other than susceptible wheat are blocked early after stomatal penetration. During these abortive infections, the fungus shows a markedly different gene expression pattern. However, the underlying mechanisms causing differential gene expression during host and non-host interactions are largely unknown, but likely include transcriptional regulators responsible for the onset of an infection programme in compatible hosts. MoCOD1, a member of the fungal Zn(II)2 Cys6 transcription factor family, has been shown to directly affect pathogenicity in the rice blast pathogen Magnaporthe oryzae. Here, we analyse the role of the putative transcription factor Zt107320, a homologue of MoCOD1, during infection of compatible and incompatible hosts by Z. tritici. We show for the first time that Zt107320 is differentially expressed in host versus non-host infections and that lower expression corresponds to an incompatible infection of non-hosts. Applying reverse genetics approaches, we further show that Zt107320 regulates the dimorphic switch as well as the growth rate of Z. tritici and affects fungal cell wall composition in vitro. Moreover, ∆Zt107320 mutants showed reduced virulence during compatible infections of wheat. We conclude that Zt107320 directly influences pathogen fitness and propose that Zt107320 is involved in the regulation of growth processes and pathogenicity during infection.


Asunto(s)
Ascomicetos/genética , Proteínas Fúngicas/metabolismo , Enfermedades de las Plantas/microbiología , Factores de Transcripción/metabolismo , Triticum/microbiología , Ascomicetos/crecimiento & desarrollo , Ascomicetos/patogenicidad , Núcleo Celular/metabolismo , Pared Celular/metabolismo , Filogenia , Genética Inversa
7.
PLoS Genet ; 15(4): e1008093, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-31009462

RESUMEN

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.


Asunto(s)
Inestabilidad Genómica , Histonas/metabolismo , Lisina/metabolismo , Ascomicetos/genética , Ascomicetos/metabolismo , Cromosomas Fúngicos , Eliminación de Gen , Heterocromatina/genética , Heterocromatina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Histonas/química , Metilación , Secuencias Repetitivas de Ácidos Nucleicos , Activación Transcripcional
8.
Ecol Evol ; 9(1): 275-294, 2019 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30680113

RESUMEN

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.

9.
Curr Opin Microbiol ; 32: 31-37, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27153045

RESUMEN

Filamentous plant pathogens explore host tissues to obtain nutrients for growth and reproduction. Diverse strategies for tissue invasion, defense manipulation, and colonization of inter and intra-cellular spaces have evolved. Most research has focused on effector molecules, which are secreted to manipulate plant immunity and facilitate infection. Effector genes are often found to evolve rapidly in response to the antagonistic host-pathogen co-evolution but other traits are also subject to adaptive evolution during specialization to the anatomy, biochemistry and ecology of different plant hosts. Although not directly related to virulence, these traits are important components of specialization but little is known about them. We present and discuss specific life cycle traits that facilitate exploration of plant tissues and underline the importance of increasing our insight into the biology of plant pathogens.


Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/fisiología , Plantas/inmunología , Plantas/microbiología , Bacterias/patogenicidad , Hongos/patogenicidad , Estadios del Ciclo de Vida , Enfermedades de las Plantas/inmunología , Factores de Virulencia/genética
10.
Mycorrhiza ; 24(4): 293-300, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24249491

RESUMEN

The serious problem of extended tissue thickness in the analysis of plant-fungus associations was overcome using a new method that combines physical and optical sectioning of the resin-embedded sample by microtomy and confocal microscopy. Improved tissue infiltration of the fungal-specific, high molecular weight fluorescent probe wheat germ agglutinin conjugated to Alexa Fluor® 633 resulted in high fungus-specific fluorescence even in deeper tissue sections. If autofluorescence was insufficient, additional counterstaining with Calcofluor White M2R or propidium iodide was applied in order to visualise the host plant tissues. Alternatively, the non-specific fluorochrome acid fuchsine was used for rapid staining of both, the plant and the fungal cells. The intricate spatial arrangements of the plant and fungal cells were preserved by immobilization in the hydrophilic resin Unicryl™. Microtomy was used to section the resin-embedded roots or leaves until the desired plane was reached. The data sets generated by confocal laser scanning microscopy of the remaining resin stubs allowed the precise spatial reconstruction of complex structures in the plant-fungus associations of interest. This approach was successfully tested on tissues from ectomycorrhiza (Betula pendula), arbuscular mycorrhiza (Galium aparine; Polygala paniculata, Polygala rupestris), ericoid mycorrhiza (Calluna vulgaris), orchid mycorrhiza (Limodorum abortivum, Serapias parviflora) and on one leaf-fungus association (Zymoseptoria tritici on Triticum aestivum). The method provides an efficient visualisation protocol applicable with a wide range of plant-fungus symbioses.


Asunto(s)
Microscopía Confocal/métodos , Microtomía/métodos , Micorrizas/fisiología , Plantas/microbiología , Colorantes Fluorescentes/metabolismo , Coloración y Etiquetado/métodos
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