The corn smut fungus Ustilago maydis is a model organism for elucidating host colonization strategies of biotrophic fungi. Here we performed an in depth transcriptional profiling of the entire plant-associated development of U. maydis wild-type strains. In our analysis we focused on fungal metabolism, nutritional strategies, secreted effectors and regulatory networks. Secreted proteins were enriched in three distinct expression modules corresponding to stages on the plant surface, establishment of biotrophy and induction of tumors. These modules are likely the key determinants for U. maydis virulence. With respect to nutrient utilization, we observed that expression of several nutrient transporters was tied to these virulence modules rather than being controlled by nutrient availability. We show that oligopeptide transporters likely involved in nitrogen assimilation are important virulence factors. By measuring the intramodular connectivity of transcription factors, we identified the potential drivers for the virulence modules. While known components of the b-mating type cascade emerged as inducers for the plant surface and biotrophy module, we identified a set of yet uncharacterized transcription factors as likely responsible for expression of the tumor module. We demonstrate a crucial role for leaf tumor formation and effector gene expression for one of these transcription factors.

BackgroundSupernumerary 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.ResultsCentromeres 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.ConclusionsAll 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0033-5) contains supplementary material, which is available to authorized users.

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.

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 hypervirulent phenotype in wheat. Mutant phenotypes of ΔZt80707, ΔZt89160 and ΔZt103264 can be restored by insertion of the wild-type genes. However, the insertion of the Zt80707 and Zt89160 orthologs from Z. pseudotritici and Z. ardabiliae do not restore wild-type levels of virulence, suggesting that positively selected substitutions in Z. tritici may relate to divergent host specialization. Interestingly, the gene Zt80707 encodes also a secretion signal that targets the protein for cell secretion. This secretion signal is however only transcribed in Z. tritici, suggesting that Z. tritici-specific substitutions relate to a new function of the protein in the extracellular space of the wheat-Z. tritici interaction. Together, the results presented here highlight that Zt80707, Zt103264 and Zt89160 represent key genes involved in virulence and host-specific disease development of Z. tritici. Our findings illustrate that evolutionary predictions provide a powerful tool for the identification of novel traits crucial for host adaptation and pathogen evolution.

27Chromosome and genome stability are important for normal cell function as instability often 28 correlates with disease and dysfunction of DNA repair mechanisms. Many organisms maintain 29 supernumerary or accessory chromosomes that deviate from standard chromosomes. The 30 pathogenic fungus Zymoseptoria tritici has as many as eight accessory chromosomes, which are 31 highly unstable during meiosis and mitosis, transcriptionally repressed, show enrichment of 32 repetitive elements, and enrichment with heterochromatic histone methylation marks, e.g., 33trimethylation of H3 lysine 9 or lysine 27 (H3K9me3, H3K27me3). To elucidate the role of 34 heterochromatin on genome stability in Z. tritici, we deleted the genes encoding the 35 methyltransferases responsible for H3K9me3 and H3K27me3, kmt1 and kmt6, respectively, and 36 generated a double mutant. We combined experimental evolution and genomic analyses to 37 determine the impact of these deletions on chromosome and genome stability, both in vitro and 38 in planta. We used whole genome sequencing, ChIP-seq, and RNA-seq to compare changes in 39 genome and chromatin structure, and differences in gene expression between mutant and 40 wildtype strains. Analyses of genome and ChIP-seq data in H3K9me3-deficient strains revealed 41 dramatic chromatin reorganization, where H3K27me3 is mostly relocalized into regions that are 42 enriched with H3K9me3 in wild type. Many genome rearrangements and formation of new 43 chromosomes were found in the absence of H3K9me3, accompanied by activation of transposable 44 elements. In stark contrast, loss of H3K27me3 actually increased the stability of accessory 45 chromosomes under normal growth conditions in vitro, even without large scale changes in gene 46 activity. We conclude that H3K9me3 is important for the maintenance of genome stability 47 because it disallows H3K27me3 in these regions. In this system, H3K27me3 reduces the overall 48 stability of accessory chromosomes, generating a "metastable" state for these quasi-essential 49 regions of the genome. 50 3 Author Summary 51 Genome and chromosome stability are essential to maintain normal cell function and viability. 52However, differences in genome and chromosome structure are frequently found in organisms 53 that undergo rapid adaptation to changing environmental conditions, and in humans are often 54 found in cancer cells. We study genome instability in a fungal pathogen that exhibits a high degree 55 of genetic diversity. Regions that show extraordinary diversity in this pathogen are the 56 transposon-rich accessory chromosomes, which contain few genes that are of unknown benefit 57 to the organism but maintained in the population and thus considered "quasi essential". 58Accessory chromosomes in all fungi studied so far are enriched with markers for 59 heterochromatin, namely trimethylation of H3 lysine 9 and 27 (H3K9me3, H3K27me3). We show 60 that loss of these heterochromatin marks has strong but opposing effects on genome stability. 61While loss of the transposon-associate...