Implications of the three-dimensional chromatin organization for genome evolution in a fungal plant pathogen.

The spatial organization of eukaryotic genomes is linked to their biological functions, although it is not clear how this impacts the overall evolution of a genome. Here, we uncover the three-dimensional (3D) genome organization of the phytopathogen Verticillium dahliae, known to possess distinct genomic regions, designated adaptive genomic regions (AGRs), enriched in transposable elements and genes that mediate host infection. Short-range DNA interactions form clear topologically associating domains (TADs) with gene-rich boundaries that show reduced levels of gene expression and reduced genomic variation. Intriguingly, TADs are less clearly insulated in AGRs than in the core genome. At a global scale, the genome contains bipartite long-range interactions, particularly enriched for AGRs and more generally containing segmental duplications. Notably, the patterns observed for V. dahliae are also present in other Verticillium species. Thus, our analysis links 3D genome organization to evolutionary features conserved throughout the Verticillium genus.

Epigenetic regulation of nuclear processes in fungal plant pathogens.

Through the association of protein complexes to DNA, the eukaryotic nuclear genome is broadly organized into open euchromatin that is accessible for enzymes acting on DNA and condensed heterochromatin that is inaccessible. Chemical and physical alterations to chromatin may impact its organization and functionality and are therefore important regulators of nuclear processes. Studies in various fungal plant pathogens have uncovered an association between chromatin organization and expression of in planta-induced genes that are important for pathogenicity. This review discusses chromatin-based regulation mechanisms as determined in the fungal plant pathogen Verticillium dahliae and relates the importance of epigenetic transcriptional regulation and other nuclear processes more broadly in fungal plant pathogens.

The Interspecific Fungal Hybrid Verticillium longisporum Displays Subgenome-Specific Gene Expression.

Hybridization is an important evolutionary mechanism that can enable organisms to adapt to environmental challenges. It has previously been shown that the fungal allodiploid species Verticillium longisporum, the causal agent of verticillium stem striping in rapeseed, originated from at least three independent hybridization events between two haploid Verticillium species. To reveal the impact of genome duplication as a consequence of hybridization, we studied the genome and transcriptome dynamics upon two independent V. longisporum hybridization events, represented by the hybrid lineages "A1/D1" and "A1/D3." We show that V. longisporum genomes are characterized by extensive chromosomal rearrangements, including between parental chromosomal sets. V. longisporum hybrids display signs of evolutionary dynamics that are typically associated with the aftermath of allodiploidization, such as haploidization and more relaxed gene evolution. The expression patterns of the two subgenomes within the two hybrid lineages are more similar than those of the shared A1 parent between the two lineages, showing that the expression patterns of the parental genomes homogenized within a lineage. However, as genes that display differential parental expression in planta do not typically display the same pattern in vitro, we conclude that subgenome-specific responses occur in both lineages. Overall, our study uncovers genomic and transcriptomic plasticity during the evolution of the filamentous fungal hybrid V. longisporum and illustrates its adaptive potential. IMPORTANCEVerticillium is a genus of plant-associated fungi that includes a few plant pathogens that collectively affect a wide range of hosts. On several occasions, haploid Verticillium species hybridized into the stable allodiploid species Verticillium longisporum, which is, in contrast to haploid Verticillium species, a Brassicaceae specialist. Here, we studied the evolutionary genome and transcriptome dynamics of V. longisporum and the impact of the hybridization. V. longisporum genomes display a mosaic structure due to genomic rearrangements between the parental chromosome sets. Similar to other allopolyploid hybrids, V. longisporum displays an ongoing loss of heterozygosity and more relaxed gene evolution. Also, differential parental gene expression is observed, with enrichment for genes that encode secreted proteins. Intriguingly, the majority of these genes display subgenome-specific responses under differential growth conditions. In conclusion, hybridization has incited the genomic and transcriptomic plasticity that enables adaptation to environmental changes in a parental allele-specific fashion.

Three putative DNA methyltransferases of Verticillium dahliae differentially contribute to DNA methylation that is dispensable for growth, development and virulence.

BACKGROUND: DNA methylation is an important epigenetic control mechanism that in many fungi is restricted to genomic regions containing transposable elements (TEs). Two DNA methyltransferases, Dim2 and Dnmt5, are known to perform methylation at cytosines in fungi. While most ascomycete fungi encode both Dim2 and Dnmt5, only few functional studies have been performed in species containing both. METHODS: In this study, we report functional analysis of both Dim2 and Dnmt5 in the plant pathogenic fungus Verticillium dahliae. RESULTS: Our results show that Dim2, but not Dnmt5 or the putative sexual-cycle-related DNA methyltransferase Rid, is responsible for the majority of DNA methylation under the tested conditions. Single or double DNA methyltransferase mutants did not show altered development, virulence, or transcription of genes or TEs. In contrast, Hp1 and Dim5 mutants that are impacted in chromatin-associated processes upstream of DNA methylation are severely affected in development and virulence and display transcriptional reprogramming in specific hypervariable genomic regions (so-called adaptive genomic regions) that contain genes associated with host colonization. As these adaptive genomic regions are largely devoid of DNA methylation and of Hp1- and Dim5-associated heterochromatin, the differential transcription is likely caused by pleiotropic effects rather than by differential DNA methylation. CONCLUSION: Overall, our study suggests that Dim2 is the main DNA methyltransferase in V. dahliae and, in conjunction with work on other fungi, is likely the main active DNMT in ascomycetes, irrespective of Dnmt5 presence. We speculate that Dnmt5 and Rid act under specific, presently enigmatic, conditions or, alternatively, act in DNA-associated processes other than DNA methylation.

Local Rather than Global H3K27me3 Dynamics Are Associated with Differential Gene Expression in Verticillium dahliae.

Differential growth conditions typically trigger global transcriptional responses in filamentous fungi. Such fungal responses to environmental cues involve epigenetic regulation, including chemical histone modifications. It has been proposed that conditionally expressed genes, such as those that encode secondary metabolites but also effectors in pathogenic species, are often associated with a specific histone modification, lysine27 methylation of H3 (H3K27me3). However, thus far, no analyses on the global H3K27me3 profiles have been reported under differential growth conditions in order to assess if H3K27me3 dynamics govern differential transcription. Using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing data from the plant-pathogenic fungus Verticillium dahliae grown in three in vitro cultivation media, we now show that a substantial number of the identified H3K27me3 domains globally display stable profiles among these growth conditions. However, we observe local quantitative differences in H3K27me3 ChIP-seq signals that are associated with a subset of differentially transcribed genes between media. Comparing the in vitro results to expression during plant infection suggests that in planta-induced genes may require chromatin remodeling to achieve expression. Overall, our results demonstrate that some loci display H3K27me3 dynamics associated with concomitant transcriptional variation, but many differentially expressed genes are associated with stable H3K27me3 domains. Thus, we conclude that while H3K27me3 is required for transcriptional repression, it does not appear that transcriptional activation requires the global erasure of H3K27me3. We propose that the H3K27me3 domains that do not undergo dynamic methylation may contribute to transcription through other mechanisms or may serve additional genomic regulatory functions. IMPORTANCE In many organisms, including filamentous fungi, epigenetic mechanisms that involve chemical and physical modifications of DNA without changing the genetic sequence have been implicated in transcriptional responses upon developmental or environmental cues. In fungi, facultative heterochromatin that can decondense to allow transcription in response to developmental changes or environmental stimuli is characterized by the trimethylation of lysine 27 on histone H3 (H3K27me3), and H3K27me3 has been implicated in transcriptional regulation, although the precise mechanisms and functions remain enigmatic. Based on ChIP and RNA sequencing data, we show for the soilborne broad-host-range vascular wilt plant-pathogenic fungus Verticillium dahliae that although some loci display H3K27me3 dynamics that can contribute to transcriptional variation, other loci do not show such a dependence. Thus, although we recognize that H3K27me3 is required for transcriptional repression, we also conclude that this mark is not a conditionally responsive global regulator of differential transcription upon responses to environmental cues.

A unique chromatin profile defines adaptive genomic regions in a fungal plant pathogen.

Genomes store information at scales beyond the linear nucleotide sequence, which impacts genome function at the level of an individual, while influences on populations and long-term genome function remains unclear. Here, we addressed how physical and chemical DNA characteristics influence genome evolution in the plant pathogenic fungus Verticillium dahliae. We identified incomplete DNA methylation of repetitive elements, associated with specific genomic compartments originally defined as Lineage-Specific (LS) regions that contain genes involved in host adaptation. Further chromatin characterization revealed associations with features such as H3 Lys-27 methylated histones (H3K27me3) and accessible DNA. Machine learning trained on chromatin data identified twice as much LS DNA as previously recognized, which was validated through orthogonal analysis, and we propose to refer to this DNA as adaptive genomic regions. Our results provide evidence that specific chromatin profiles define adaptive genomic regions, and highlight how different epigenetic factors contribute to the organization of these regions.

Repetitive Elements Contribute to the Diversity and Evolution of Centromeres in the Fungal Genus Verticillium.

Centromeres are chromosomal regions that are crucial for chromosome segregation during mitosis and meiosis, and failed centromere formation can contribute to chromosomal anomalies. Despite this conserved function, centromeres differ significantly between and even within species. Thus far, systematic studies into the organization and evolution of fungal centromeres remain scarce. In this study, we identified the centromeres in each of the 10 species of the fungal genus Verticillium and characterized their organization and evolution. Chromatin immunoprecipitation of the centromere-specific histone CenH3 (ChIP-seq) and chromatin conformation capture (Hi-C) followed by high-throughput sequencing identified eight conserved, large (∼150-kb), AT-, and repeat-rich regional centromeres that are embedded in heterochromatin in the plant pathogen Verticillium dahliae Using Hi-C, we similarly identified repeat-rich centromeres in the other Verticillium species. Strikingly, a single degenerated long terminal repeat (LTR) retrotransposon is strongly associated with centromeric regions in some but not all Verticillium species. Extensive chromosomal rearrangements occurred during Verticillium evolution, of which some could be linked to centromeres, suggesting that centromeres contributed to chromosomal evolution. The size and organization of centromeres differ considerably between species, and centromere size was found to correlate with the genome-wide repeat content. Overall, our study highlights the contribution of repetitive elements to the diversity and rapid evolution of centromeres within the fungal genus VerticilliumIMPORTANCE The genus Verticillium contains 10 species of plant-associated fungi, some of which are notorious pathogens. Verticillium species evolved by frequent chromosomal rearrangements that contribute to genome plasticity. Centromeres are instrumental for separation of chromosomes during mitosis and meiosis, and failed centromere functionality can lead to chromosomal anomalies. Here, we used a combination of experimental techniques to identify and characterize centromeres in each of the Verticillium species. Intriguingly, we could strongly associate a single repetitive element to the centromeres of some of the Verticillium species. The presence of this element in the centromeres coincides with increased centromere sizes and genome-wide repeat expansions. Collectively, our findings signify a role of repetitive elements in the function, organization, and rapid evolution of centromeres in a set of closely related fungal species.