Hybridization in fungi has recently been recognized as a major force in the generation of new fun... more Hybridization in fungi has recently been recognized as a major force in the generation of new fungal plant pathogens. These include the grass pathogen Zymoseptoria pseudotritici and the powdery mildew pathogen Blumeria graminis triticale of triticale. Hybridization also plays an important role in the transfer of genetic material between species. This process is termed introgressive hybridization and involves extensive backcrossing between hybrid and the parental species. Introgressive hybridization has contributed substantially to the successful spread of plant pathogens such as Ophiostoma ulmi and O. novo-ulmi, the causal agents of Dutch elm disease, and other tree pathogens such as the rust pathogen Melampsora. Hybridization occurs more readily between species that have previously not coexisted, so-called allopatric species. Reproductive barriers between allopatric species are likely to be more permissive allowing interspecific mating to occur. The bringing together of allopatric ...
Filamentous plant pathogens explore host tissues to obtain nutrients for growth and reproduction.... more 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.
The speciation of pathogens can be driven by divergent host specialization. Specialization to a n... more The speciation of pathogens can be driven by divergent host specialization. Specialization to a new host is possible via the acquisition of advantageous mutations fixed by positive selection. Comparative genome analyses of closely related species allows for the identification of such key substitutions via inference of genome-wide signatures of positive selection. We previously used a comparative genomics framework to identify genes that have evolved under positive selection during speciation of the prominent wheat pathogen Zymoseptoria tritici (synonym Mycosphaerella graminicola). In this study, we conducted functional analyses of four genes exhibiting strong signatures of positive selection in Z. tritici. We deleted the four genes in Z. tritici and confirm a virulence-related role of three of the four genes ΔZt80707, ΔZt89160 and ΔZt103264. The two mutants ΔZt80707 and ΔZt103264 show a significant reduction in virulence during infection of wheat; the ΔZt89160 mutant causes a hyperv...
Plant pathogens can evolve new host specificities and overcome host resistances over surprisingly... more Plant pathogens can evolve new host specificities and overcome host resistances over surprisingly few generations, a process that is greatly accelerated by agricultural practices. A new study provides a striking example in which the rapid emergence of a new pathogen via introgressive hybridization mirrors the evolution of a hybrid cereal crop.
Supernumerary chromosomes have been found in many organisms. In fungi, these "accessory"... more 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 H...
Supernumerary chromosomes have been found in many organisms. In fungi, these &amp... more 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. 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. 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.
The global migration patterns of the fungal wheat pathogen Phaeosphaeria nodorum were analysed us... more The global migration patterns of the fungal wheat pathogen Phaeosphaeria nodorum were analysed using 12 microsatellite loci. Analysis of 693 isolates from nine populations indi- cated that the population structure of P. nodorum is characterized by high levels of genetic diversity and a low degree of subdivision between continents. To determine whether genetic similarity of populations was a result of
The presence or absence of specific transcription factors, chromatin remodeling machineries, chro... more 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 t...
ABSTRACT Fungi have relatively small genomes, and many species are amendable to experimental and ... more ABSTRACT Fungi have relatively small genomes, and many species are amendable to experimental and molecular manipulation making them excellent models to unravel the genetic, molecular and cellular aspects of speciation. Pioneering work has been conducted in the two model species Saccharomyces cerevisiae and Neurospora crassa where integrated studies using classical genetics, genomic resources and experimental approaches have shed light on mechanisms conferring reproductive isolation between diverged lineages. Experimental evolution, genetic mapping and molecular manipulation can be considerably more challenging in species of fungal plant pathogens. Yet, insight into speciation genetics is fundamental to understand the emergence of new pathogen species. Now, several fundamental questions relating to speciation genetics can be addressed using the growing resource of genome data. Population genomics and comparative genomics approaches offer the possibility of not only recapturing the evolutionary history of species but also identifying signatures of selection and genomic rearrangements associated with speciation events. Thereby, both the history of species and the underlying genetics associated with the establishment of reproductive isolation can be recovered.
ABSTRACT The evolutionary interplay of fungal virulence and host resistance imposes strong select... more ABSTRACT The evolutionary interplay of fungal virulence and host resistance imposes strong selective constraints that shape fungal pathogens genome structure. Genomes of pathogenic fungi are highly diverse in architecture and gene content. Moreover, analyses of these genomes unveiled pivotal insights into structural diversity and evolutionary mechanisms of genomes as well as into functional repertoires and speciation processes of pathogens. Here, we highlight key features and evolutionary mechanisms of fungal pathogen genomes.
Large amount of genome data are being generated by second- and now also third-generation sequenci... more Large amount of genome data are being generated by second- and now also third-generation sequencing technologies. The challenge no longer lies in the generation of the data, but in the analyses of it. We present an overview of approaches and methods to compare complete sequences of related fungal genomes. We focus on evolutionary analyses of genome alignments to describe species divergence and to identify footprints of demography and natural selection within and between species.
Hybridization in fungi has recently been recognized as a major force in the generation of new fun... more Hybridization in fungi has recently been recognized as a major force in the generation of new fungal plant pathogens. These include the grass pathogen Zymoseptoria pseudotritici and the powdery mildew pathogen Blumeria graminis triticale of triticale. Hybridization also plays an important role in the transfer of genetic material between species. This process is termed introgressive hybridization and involves extensive backcrossing between hybrid and the parental species. Introgressive hybridization has contributed substantially to the successful spread of plant pathogens such as Ophiostoma ulmi and O. novo-ulmi, the causal agents of Dutch elm disease, and other tree pathogens such as the rust pathogen Melampsora. Hybridization occurs more readily between species that have previously not coexisted, so-called allopatric species. Reproductive barriers between allopatric species are likely to be more permissive allowing interspecific mating to occur. The bringing together of allopatric ...
Filamentous plant pathogens explore host tissues to obtain nutrients for growth and reproduction.... more 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.
The speciation of pathogens can be driven by divergent host specialization. Specialization to a n... more The speciation of pathogens can be driven by divergent host specialization. Specialization to a new host is possible via the acquisition of advantageous mutations fixed by positive selection. Comparative genome analyses of closely related species allows for the identification of such key substitutions via inference of genome-wide signatures of positive selection. We previously used a comparative genomics framework to identify genes that have evolved under positive selection during speciation of the prominent wheat pathogen Zymoseptoria tritici (synonym Mycosphaerella graminicola). In this study, we conducted functional analyses of four genes exhibiting strong signatures of positive selection in Z. tritici. We deleted the four genes in Z. tritici and confirm a virulence-related role of three of the four genes ΔZt80707, ΔZt89160 and ΔZt103264. The two mutants ΔZt80707 and ΔZt103264 show a significant reduction in virulence during infection of wheat; the ΔZt89160 mutant causes a hyperv...
Plant pathogens can evolve new host specificities and overcome host resistances over surprisingly... more Plant pathogens can evolve new host specificities and overcome host resistances over surprisingly few generations, a process that is greatly accelerated by agricultural practices. A new study provides a striking example in which the rapid emergence of a new pathogen via introgressive hybridization mirrors the evolution of a hybrid cereal crop.
Supernumerary chromosomes have been found in many organisms. In fungi, these "accessory"... more 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 H...
Supernumerary chromosomes have been found in many organisms. In fungi, these &amp... more 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. 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. 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.
The global migration patterns of the fungal wheat pathogen Phaeosphaeria nodorum were analysed us... more The global migration patterns of the fungal wheat pathogen Phaeosphaeria nodorum were analysed using 12 microsatellite loci. Analysis of 693 isolates from nine populations indi- cated that the population structure of P. nodorum is characterized by high levels of genetic diversity and a low degree of subdivision between continents. To determine whether genetic similarity of populations was a result of
The presence or absence of specific transcription factors, chromatin remodeling machineries, chro... more 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 t...
ABSTRACT Fungi have relatively small genomes, and many species are amendable to experimental and ... more ABSTRACT Fungi have relatively small genomes, and many species are amendable to experimental and molecular manipulation making them excellent models to unravel the genetic, molecular and cellular aspects of speciation. Pioneering work has been conducted in the two model species Saccharomyces cerevisiae and Neurospora crassa where integrated studies using classical genetics, genomic resources and experimental approaches have shed light on mechanisms conferring reproductive isolation between diverged lineages. Experimental evolution, genetic mapping and molecular manipulation can be considerably more challenging in species of fungal plant pathogens. Yet, insight into speciation genetics is fundamental to understand the emergence of new pathogen species. Now, several fundamental questions relating to speciation genetics can be addressed using the growing resource of genome data. Population genomics and comparative genomics approaches offer the possibility of not only recapturing the evolutionary history of species but also identifying signatures of selection and genomic rearrangements associated with speciation events. Thereby, both the history of species and the underlying genetics associated with the establishment of reproductive isolation can be recovered.
ABSTRACT The evolutionary interplay of fungal virulence and host resistance imposes strong select... more ABSTRACT The evolutionary interplay of fungal virulence and host resistance imposes strong selective constraints that shape fungal pathogens genome structure. Genomes of pathogenic fungi are highly diverse in architecture and gene content. Moreover, analyses of these genomes unveiled pivotal insights into structural diversity and evolutionary mechanisms of genomes as well as into functional repertoires and speciation processes of pathogens. Here, we highlight key features and evolutionary mechanisms of fungal pathogen genomes.
Large amount of genome data are being generated by second- and now also third-generation sequenci... more Large amount of genome data are being generated by second- and now also third-generation sequencing technologies. The challenge no longer lies in the generation of the data, but in the analyses of it. We present an overview of approaches and methods to compare complete sequences of related fungal genomes. We focus on evolutionary analyses of genome alignments to describe species divergence and to identify footprints of demography and natural selection within and between species.
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