RESUMO
Effector genes, encoding molecules involved in disease establishment, are concertedly expressed throughout the lifecycle of plant-pathogenic fungi. However, little is known about how effector gene expression is regulated. Since many effector genes are located in repeat-rich regions, the role of chromatin remodeling in their regulation was recently investigated, notably establishing that the repressive histone modification H3K9me3, deposited by KMT1, was involved in several fungal species including Leptosphaeria maculans. Nevertheless, previous data suggest that a second regulatory layer, probably involving a specific transcription factor (TF), might be required. In L. maculans, a Dothideomycete causing stem canker of oilseed rape, we identified the ortholog of Pf2, a TF belonging to the Zn2Cys6 fungal-specific family, and described as essential for pathogenicity and effector gene expression. We investigated its role together with KMT1, by inactivating and over-expressing LmPf2 in a wild-type strain and a ∆kmt1 mutant. Functional analyses of the corresponding transformants highlighted an essential role of LmPf2 in the establishment of pathogenesis and we found a major effect of LmPf2 on the induction of effector gene expression once KMT1 repression is lifted. Our results show, for the first time, a dual control of effector gene expression.
Assuntos
Ascomicetos , Brassica napus , Leptosphaeria , Ascomicetos/fisiologia , Brassica napus/genética , Virulência/genética , Expressão Gênica , Doenças das Plantas/microbiologiaRESUMO
Blackleg (also known as Phoma or stem canker) is a major, worldwide disease of Brassica crop species, notably B. napus (rapeseed, canola), caused by the ascomycete fungus Leptosphaeria maculans. The outbreak and severity of this disease depend on environmental conditions and management practices, as well as a complex interaction between the pathogen and its hosts. Genetic resistance is a major method to control the disease (and the only control method in some parts of the world, such as continental Europe), but efficient use of genetic resistance is faced with many difficulties: (i) the scarcity of germplasm/genetic resources available, (ii) the different history of use of resistance genes in different parts of the world and the different populations of the fungus the resistance genes are exposed to, (iii) the complexity of the interactions between the plant and the pathogen that expand beyond typical gene-for-gene interactions, (iv) the incredible evolutionary potential of the pathogen and the importance of knowing the molecular processes set up by the fungus to "breakdown' resistances, so that we may design high-throughput diagnostic tools for population surveys, and (v) the different strategies and options to build up the best resistances and to manage them so that they are durable. In this paper, we aim to provide a comprehensive overview of these different points, stressing the differences between the different continents and the current prospects to generate new and durable resistances to blackleg disease.
Assuntos
Resistência à Doença , Leptosphaeria , Melhoramento Vegetal , Doenças das Plantas , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Resistência à Doença/genética , Leptosphaeria/genética , Brassica napus/genética , Brassica napus/microbiologia , Genes de Plantas , Produtos Agrícolas/genética , Produtos Agrícolas/microbiologia , Brassica/genética , Brassica/microbiologiaRESUMO
BACKGROUND: In France, the overall trend in the incidence of cardiovascular disease is unfavourable, especially in young subjects. This highlights the need to promote cardiovascular health by targeting the main risk factors. Social marketing campaigns to improve cardiovascular health should identify unhealthy behaviour and understand the target audience. The objective of this study was to identify poor cardiovascular health profiles in the French population using a clustering method. METHODS: Subjects aged 18-74 years with no history of cardiovascular disease were included from the Esteban cross-sectional survey (2014-16). To evaluate cardiovascular health, seven items were considered as defined by the American Heart Association: blood glucose, blood cholesterol, blood pressure, body mass index, cigarette smoking, diet and physical activity. Cardiovascular health profiles were identified from these seven items by combining multiple correspondence analysis with hierarchical clustering and partitioning. RESULTS: A total of 1673 subjects were included in the main analysis. Five cardiovascular health profiles were identified: two profiles corresponded to subjects with poor cardiovascular health (mainly older men with a low socioeconomic status), two to subjects with intermediate cardiovascular health (one mainly comprised of young women with a low socioeconomic status and the other of young subjects with a high socioeconomic status) and one to subjects with good cardiovascular health (mainly older women). CONCLUSION: This description of cardiovascular health profiles, which led to the identification and characterization of target audiences for future population-based prevention campaigns, should be the starting point for improving cardiovascular health in the French population.
Assuntos
Doenças Cardiovasculares , Masculino , Estados Unidos , Humanos , Feminino , Idoso , Estudos Transversais , Fatores de Risco , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/prevenção & controle , Doenças Cardiovasculares/etiologia , Dieta , Análise por Conglomerados , PrevalênciaRESUMO
Leptosphaeria maculans 'brassicae' (Lmb) and Leptosphaeria maculans 'lepidii' (Lml) are closely related phytopathogenic species that exhibit a large macrosynteny but contrasting genome structure. Lmb has more than 30% of repeats clustered in large repeat-rich regions, while the Lml genome has only a small amount of evenly distributed repeats. Repeat-rich regions of Lmb are enriched in effector genes, expressed during plant infection. The distinct genome structures of Lmb and Lml provide an excellent model for comparing the organization of pathogenicity genes in relation to the chromatin landscape in two closely related phytopathogenic fungi. Here, we performed chromatin immunoprecipitation (ChIP) during axenic culture, targeting histone modifications typical for heterochromatin or euchromatin, combined with transcriptomic analysis to analyze the influence of chromatin organization on gene expression. In both species, we found that facultative heterochromatin is enriched with genes lacking functional annotation, including numerous effector and species-specific genes. Notably, orthologous genes located in H3K27me3 domains are enriched with effector genes. Compared to other fungal species, including Lml, Lmb is distinct in having large H3K9me3 domains associated with repeat-rich regions that contain numerous species-specific effector genes. Discovery of these two distinctive heterochromatin landscapes now raises questions about their involvement in the regulation of pathogenicity, the dynamics of these domains during plant infection and the selective advantage to the fungus to host effector genes in H3K9me3 or H3K27me3 domains.
Assuntos
Ascomicetos , Brassica napus , Ascomicetos/genética , Brassica napus/genética , Genômica , Código das Histonas , LeptosphaeriaRESUMO
A central feature of meiosis is pairing of homologous chromosomes, which occurs in two stages: coalignment of axes followed by installation of the synaptonemal complex (SC). Concomitantly, recombination complexes reposition from on-axis association to the SC central region. We show here that, in the fungus Sordaria macrospora, this critical transition is mediated by robust interaxis bridges that contain an axis component (Spo76/Pds5), DNA, plus colocalizing Mer3/Msh4 recombination proteins and the Zip2-Zip4 mediator complex. Mer3-Msh4-Zip2-Zip4 colocalizing foci are first released from their tight axis association, dependent on the SC transverse-filament protein Sme4/Zip1, before moving to bridges and thus to a between-axis position. Ensuing shortening of bridges and accompanying juxtaposition of axes to 100 nm enables installation of SC central elements at sites of between-axis Mer3-Msh4-Zip2-Zip4 complexes. We show also that the Zip2-Zip4 complex has an intrinsic affinity for chromosome axes at early leptotene, where it localizes independently of recombination, but is dependent on Mer3. Then, later, Zip2-Zip4 has an intrinsic affinity for the SC central element, where it ultimately localizes to sites of crossover complexes at the end of pachytene. These and other findings suggest that the fundamental role of Zip2-Zip4 is to mediate the recombination/structure interface at all post-double-strand break stages. We propose that Zip2-Zip4 directly mediates a molecular handoff of Mer3-Msh4 complexes, from association with axis components to association with SC central components, at the bridge stage, and then directly mediates central region installation during SC nucleation.
Assuntos
Recombinação Genética , Sordariales/genética , Cromossomos Fúngicos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Complexo Sinaptonêmico/metabolismoRESUMO
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: The fungus Leptosphaeria maculans has an exceptionally long and complex relationship with its host plant, Brassica napus, during which it switches between different lifestyles, including asymptomatic, biotrophic, necrotrophic, and saprotrophic stages. The fungus is also exemplary of "two-speed" genome organisms in the genome of which gene-rich and repeat-rich regions alternate. Except for a few stages of plant infection under controlled conditions, nothing is known about the genes mobilized by the fungus throughout its life cycle, which may last several years in the field. RESULTS: We performed RNA-seq on samples corresponding to all stages of the interaction of L. maculans with its host plant, either alive or dead (stem residues after harvest) in controlled conditions or in field experiments under natural inoculum pressure, over periods of time ranging from a few days to months or years. A total of 102 biological samples corresponding to 37 sets of conditions were analyzed. We show here that about 9% of the genes of this fungus are highly expressed during its interactions with its host plant. These genes are distributed into eight well-defined expression clusters, corresponding to specific infection lifestyles or to tissue-specific genes. All expression clusters are enriched in effector genes, and one cluster is specific to the saprophytic lifestyle on plant residues. One cluster, including genes known to be involved in the first phase of asymptomatic fungal growth in leaves, is re-used at each asymptomatic growth stage, regardless of the type of organ infected. The expression of the genes of this cluster is repeatedly turned on and off during infection. Whatever their expression profile, the genes of these clusters are enriched in heterochromatin regions associated with H3K9me3 or H3K27me3 repressive marks. These findings provide support for the hypothesis that part of the fungal genes involved in niche adaptation is located in heterochromatic regions of the genome, conferring an extreme plasticity of expression. CONCLUSION: This work opens up new avenues for plant disease control, by identifying stage-specific effectors that could be used as targets for the identification of novel durable disease resistance genes, or for the in-depth analysis of chromatin remodeling during plant infection, which could be manipulated to interfere with the global expression of effector genes at crucial stages of plant infection.
Assuntos
Brassica napus/microbiologia , Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno , Leptosphaeria/genética , Transcriptoma/fisiologia , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Leptosphaeria/fisiologia , Doenças das Plantas/microbiologiaRESUMO
Plant pathogens secrete an arsenal of small secreted proteins (SSPs) acting as effectors that modulate host immunity to facilitate infection. SSP-encoding genes are often located in particular genomic environments and show waves of concerted expression at diverse stages of plant infection. To date, little is known about the regulation of their expression. The genome of the Ascomycete Leptosphaeria maculans comprises alternating gene-rich GC-isochores and gene-poor AT-isochores. The AT-isochores harbor mosaics of transposable elements, encompassing one-third of the genome, and are enriched in putative effector genes that present similar expression patterns, namely no expression or low-level expression during axenic cultures compared to strong induction of expression during primary infection of oilseed rape (Brassica napus). Here, we investigated the involvement of one specific histone modification, histone H3 lysine 9 methylation (H3K9me3), in epigenetic regulation of concerted effector gene expression in L. maculans. For this purpose, we silenced the expression of two key players in heterochromatin assembly and maintenance, HP1 and DIM-5 by RNAi. By using HP1-GFP as a heterochromatin marker, we observed that almost no chromatin condensation is visible in strains in which LmDIM5 was silenced by RNAi. By whole genome oligoarrays we observed overexpression of 369 or 390 genes, respectively, in the silenced-LmHP1 and -LmDIM5 transformants during growth in axenic culture, clearly favouring expression of SSP-encoding genes within AT-isochores. The ectopic integration of four effector genes in GC-isochores led to their overexpression during growth in axenic culture. These data strongly suggest that epigenetic control, mediated by HP1 and DIM-5, represses the expression of at least part of the effector genes located in AT-isochores during growth in axenic culture. Our hypothesis is that changes of lifestyle and a switch toward pathogenesis lift chromatin-mediated repression, allowing a rapid response to new environmental conditions.
Assuntos
Ascomicetos/genética , Epigênese Genética/genética , Heterocromatina/genética , Doenças das Plantas/genética , Ascomicetos/patogenicidade , Brassica napus/genética , Brassica napus/microbiologia , Regulação Fúngica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , MetilaçãoRESUMO
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
BACKGROUND: Many plant-pathogenic fungi have a tendency towards genome size expansion, mostly driven by increasing content of transposable elements (TEs). Through comparative and evolutionary genomics, five members of the Leptosphaeria maculans-Leptosphaeria biglobosa species complex (class Dothideomycetes, order Pleosporales), having different host ranges and pathogenic abilities towards cruciferous plants, were studied to infer the role of TEs on genome shaping, speciation, and on the rise of better adapted pathogens. RESULTS: L. maculans 'brassicae', the most damaging species on oilseed rape, is the only member of the species complex to have a TE-invaded genome (32.5%) compared to the other members genomes (<4%). These TEs had an impact at the structural level by creating large TE-rich regions and are suspected to have been instrumental in chromosomal rearrangements possibly leading to speciation. TEs, associated with species-specific genes involved in disease process, also possibly had an incidence on evolution of pathogenicity by promoting translocations of effector genes to highly dynamic regions and thus tuning the regulation of effector gene expression in planta. CONCLUSIONS: Invasion of L. maculans 'brassicae' genome by TEs followed by bursts of TE activity allowed this species to evolve and to better adapt to its host, making this genome species a peculiarity within its own species complex as well as in the Pleosporales lineage.
Assuntos
Adaptação Fisiológica/genética , Ascomicetos/genética , Ascomicetos/fisiologia , Elementos de DNA Transponíveis/genética , Evolução Molecular , Interações Hospedeiro-Patógeno , Plantas/microbiologia , Ascomicetos/metabolismo , Ascomicetos/patogenicidade , Cromossomos Fúngicos/genética , Sequência Conservada/genética , Genes Fúngicos/genética , Genômica , Família Multigênica/genética , Filogenia , Especificidade da Espécie , Sintenia/genéticaRESUMO
Filamentous pathogens, including fungi and oomycetes, pose major threats to global food security. Crop pathogens cause damage by secreting effectors that manipulate the host to the pathogen's advantage. Genes encoding such effectors are among the most rapidly evolving genes in pathogen genomes. Here, we review how the major characteristics of the emergence, function, and regulation of effector genes are tightly linked to the genomic compartments where these genes are located in pathogen genomes. The presence of repetitive elements in these compartments is associated with elevated rates of point mutations and sequence rearrangements with a major impact on effector diversification. The expression of many effectors converges on an epigenetic control mediated by the presence of repetitive elements. Population genomics analyses showed that rapidly evolving pathogens show high rates of turnover at effector loci and display a mosaic in effector presence-absence polymorphism among strains. We conclude that effective pathogen containment strategies require a thorough understanding of the effector genome biology and the pathogen's potential for rapid adaptation.
Assuntos
Evolução Molecular , Fungos/genética , Genoma , Oomicetos/genética , Doenças das Plantas/prevenção & controle , Polimorfismo Genético , Adaptação Biológica , Genes Fúngicos/genética , Genoma Fúngico , Doenças das Plantas/microbiologiaRESUMO
Plant-associated fungi often present in their genome areas enriched in repeat sequences and effector genes, the latter being specifically induced in planta. The location of effector genes in regions enriched in repeats has been shown to have an impact on adaptability of fungi but could also provide for tight control of effector gene expression through chromatin-based regulation. The distribution of two repressive histone marks was shown to be an important regulatory layer in two fungal species with different lifestyles. Chromatin-based control of effector gene expression is likely to provide an evolutionary advantage by preventing the expression of genes not needed during vegetative growth and allow for a massive concerted expression at particular time-points of plant infection.
Assuntos
Cromatina/genética , Plantas/microbiologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Fatores de Transcrição/metabolismoRESUMO
Leptosphaeria maculans causes stem canker of oilseed rape (Brassica napus). The APSES transcription factor StuA is a key developmental regulator of fungi, involved in morphogenesis, conidia production and also more recently described as required for secondary metabolite production and for effector gene expression in phytopathogenic fungi. We investigated the involvement of the orthologue of StuA in L. maculans, LmStuA, in morphogenesis, pathogenicity and effector gene expression. LmStuA is induced during mycelial growth and at 14 days after infection, corresponding to the development of pycnidia on oilseed rape leaves, consistent with the function of StuA described so far. We set up the functional characterization of LmStuA using an RNA interference approach. Silenced LmStuA transformants showed typical phenotypic defects of StuA mutants with altered growth in axenic culture and impaired conidia production and perithecia formation. Silencing of LmStuA abolished the pathogenicity of L. maculans on oilseed rape leaves and also resulted in a drastic decrease in expression of at least three effector genes during in planta infection, suggesting either that LmStuA regulates, directly or indirectly, the expression of several effector genes in L. maculans or that the infection stage in which effectors are expressed is not reached when LmStuA expression is silenced.
Assuntos
Ascomicetos/fisiologia , Regulação Fúngica da Expressão Gênica , Fatores de Transcrição/fisiologia , Ascomicetos/genética , Ascomicetos/patogenicidade , Brassica napus/microbiologia , Esporos Fúngicos/genética , Fatores de Virulência/genéticaRESUMO
BACKGROUND: Supernumerary chromosomes have been found in many organisms. In fungi, these "accessory" or "dispensable" chromosomes are present at different frequencies in populations and are usually characterized by higher repetitive DNA content and lower gene density when compared to the core chromosomes. In the reference strain of the wheat pathogen, Zymoseptoria tritici, eight discrete accessory chromosomes have been found. So far, no functional role has been assigned to these chromosomes; however, they have existed as separate entities in the karyotypes of Zymoseptoria species over evolutionary time. In this study, we addressed what-if anything-distinguishes the chromatin of accessory chromosomes from core chromosomes. We used chromatin immunoprecipitation combined with high-throughput sequencing ("ChIP-seq") of DNA associated with the centromere-specific histone H3, CENP-A (CenH3), to identify centromeric DNA, and ChIP-seq with antibodies against dimethylated H3K4, trimethylated H3K9 and trimethylated H3K27 to determine the relative distribution and proportion of euchromatin, obligate and facultative heterochromatin, respectively. RESULTS: Centromeres of the eight accessory chromosomes have the same sequence composition and structure as centromeres of the 13 core chromosomes and they are of similar length. Unlike those of most other fungi, Z. tritici centromeres are not composed entirely of repetitive DNA; some centromeres contain only unique DNA sequences, and bona fide expressed genes are located in regions enriched with CenH3. By fluorescence microscopy, we showed that centromeres of Z. tritici do not cluster into a single chromocenter during interphase. We found dramatically higher enrichment of H3K9me3 and H3K27me3 on the accessory chromosomes, consistent with the twofold higher proportion of repetitive DNA and poorly transcribed genes. In contrast, no single histone modification tested here correlated with the distribution of centromeric nucleosomes. CONCLUSIONS: All centromeres are similar in length and composed of a mixture of unique and repeat DNA, and most contain actively transcribed genes. Centromeres, subtelomeric regions or telomere repeat length cannot account for the differences in transfer fidelity between core and accessory chromosomes, but accessory chromosomes are greatly enriched in nucleosomes with H3K27 trimethylation. Genes on accessory chromosomes appear to be silenced by trimethylation of H3K9 and H3K27.