- Genome compartmentalization predates species divergence in the plant pathogen genus Zymoseptoria. - Full list of author information is available at the end of the article. - The haploid genome of the reference isolate IPO323 comprises thirteen core and eight accessory chromosomes [23]. - Some of these accessory chromosomes may encode traits that impact virulence of the fungus, however no gene encoded on an accessory chromosome has so far been described as a virulence or avirulence deter- minant [24–29]. - In the reference isolate IPO323, the accessory chromo- somes comprise more than 11% of the entire genome assembly. - To which extent such a high amount of accessory DNA is also found in genomes of other mem- bers of the Zymoseptoria genus has so far been unknown due to the lack of high-quality genome assemblies and large-scale population sequencing. - tritici (Zt05 and Zt10) and one isolate of each of the sister species, Z.. - We sequenced and assembled the genome of the reference isolates of Z. - The obtained contigs were filtered based on base-quality confidence and read depth to ensure high quality of the final assemblies (see Methods).. - This filter removed a high number of contigs (between 17 and 58% of the total), but little overall length (between 0.4 and 2.6% of the total assemblies), indicating that most of the excluded contigs were of small size (Table S1 and S5).. - The best assemblies were of the two Z. - The assembly of the Iranian Z. - The assemblies of the Z. - passerinii (Zpa63) genomes included no fully assembled chromosomes, but twelve and ten contigs respectively with telomeres at one of the ends (Table 1. - This range is consistent with the annotation of the reference genome IPO323 reporting 11,839 protein-coding genes [22]. - Based on the whole-genome sequences and the predicted genes we reconstructed the phylogeny of the Zymoseptoria genus using the publicly available genome of Cercospora beticola as an outgroup [38, 39]. - For both trees, the phylogenetic relationship of the Zymoseptoria species is in accordance with previously published phylogeny based on seven loci sequenced in multiples isolates (Fig. - Next we addressed the extent of co-linearity of the Zymoseptoria genomes. - In the reference genome of IPO323, compartments with these genomic and epigenomic hallmarks represent either accessory chromosomes or specific regions of the core chromosomes. - In the genome of the reference Z. - tritici strain, the compartments that exhibit the hallmark of accessory chromosomes includes a particular region of the core chromosome 7 of ~ 0.6 Mb (Fig. - 3 Genome architecture of the reference genome Z. - The segments constituting the first circle represents the chromosomes of IPO323 (a) and contigs of Zp13 (b) ordered according to the synteny with the chromosomes of the reference genome. - The arrows indicate the location of the region on chromosome 7 (and the corresponding syntenic region in Z. - tritici IPO323 and the genome of the Iranian Z. - Each color represents a different chromosome as defined in the reference Z. - like” because the region exhibits clearly two of the three above-mentioned criteria [6, 31]. - Based on synteny plots, we recognize ~ 0.7 Mb of the contig 28 in Z. - pseu- dotritici and ~ 0.6 Mb of the contig 17 for Z. - For the two remaining sister species, the fragmentation of the assembly does not allow the identification of such pattern although we observe a similar tendency with respect to transcrip- tion and species-specific gene enrichment on contig 19. - We observed a region of ~ 0.2 Mb of contig 1 in the Iranian isolate Zt10 corresponding to the core chromosome 3 in the IPO323 genome with high content of isolate-specific genes (Figure S4. - We furthermore identified small segments with species-specific genes on the core chromosomes of the wild-grass infecting sister species including a ~ 0.3 Mb region of the contig 26 in Z. - Overall, secretomes range from 7% of the genes predicted sin Z.. - brevis which has no orthologous cluster detected in any of the other Zymoseptoria genomes (Figure S1). - We identified between 454 and 515 CAZyme genes in the Zymoseptoria species. - Despite the high variability, the predicted effector genes are mostly located on core chro- mosomes and none of the five Zymoseptoria species have more than ten predicted effector genes located on accessory chromosomes (Figure S1).. - We categorized 22,341 gene orthogroups identified in the seven Zymoseptoria genomes and in C. - but not found in the C. - beticola genome, represent 45% of the orthogroups (N = 9955;. - Interestingly, we show that the number of orthogroups detected as isolate-specific is much larger when the comparison includes only members of the same species than when the other species are included and 638 vs 792, 659 and 391 genes for IPO323, Zt05 and Zt10 respectively. - This indicates that a large part of the accessory gene content in Z. - pseudotritici are shared with at least one of the other five Zymoseptoria species (Fig. - However, only 47 (10% of the total. - c The only gene category found to be overrepresented in any of the specificity categories - other than unknown function genes - are predicted effector genes. - Predicted effectors genes are overrepresented in the genus-specific genes and in Z.. - tritici isolates while 68% (N = 240 of 352) show presence-absence polymorphisms in at least one of the three isolates. - Further- more, we distinguished gene expression of the above-defined categories (core genes, genus-specific, species-specific, and isolate-specific). - Overall, we find that gene expression of the species-specific and isolate- specific genes is significantly lower in IPO323 and Zt10, but not in Zt05 (Kruskal-Wallis p-value <. - 5 In planta expression of genes belonging to different specificity levels in the Zymoseptoria pangenome. - In contrast, we observe a significantly higher expression of the species-specific and isolate-specific genes for all three isolates (Table S4. - pseudotritici, and two isolates of the wheat pathgoen Z.. - Here, we con- duct some detailed comparative analyses of genome architec- ture and show a considerable extent of variation in sequence composition during the recent evolution of the Zymoseptoria lineages. - We identify extensive presence-absence variation of protein coding genes in genomes of the five Zymoseptoria species consistent with the variable gene repertoire already reported for one of the species, Z. - In spite of this variation, we observe an overall conserved synteny of the core chromosomes. - In the Zymo- septoria genomes, we observe gene-dense, actively tran- scribed and H3K4me2-enriched compartments associated with most of the core chromosomes. - We hypothesize that these compartments likely represent accessory chromosomes in the different Zymoseptoria species.. - show that the region homologous to chromosome 7 in the other Zymoseptoria species also exhibits accessory compartment hallmarks. - Our results support the occur- rence of a past chromosome fusion, but hereby show that it very likely occurred prior to the divergence of the species (estimated to date tens of thousands of years [21. - Thereby, we show that more than 25% of the genes identified as isolate-specific in a comparison including only Z. - tritici isolates are actually present in the wild-grass infecting sister species. - This observation suggests that a large proportion of the accessory genome of Z. - Our observation opens new perspectives for further analysis to understand how inter-specific gene flow has affected the evolution of the accessory genome of Z.. - Although the lifestyles of the wild-grass infecting Zymoseptoria are poorly understood, the species share major features of their lifestyles. - In Zymoseptoria, most of the predicted effectors are shared among all species, although they show presence-absence variation. - In the Botrytis genus (Dothi- deomycetes), sister species infecting different hosts share effectors with confirmed functions [43]. - genes conserved across the Zymoseptoria genus are likely core pathogenicity factors potentially targeting key plant defense mechanisms common to all of the grass hosts [45]. - Variation in the composition of effector genes in plant pathogen genomes, including the presence of species-specific and isolate-specific may reflect different host specificities and rapid evolution of these genes [45].. - Here we find that only a fraction of the genus-specific predicted effector genes is shared among all Zymosep- toria species. - Differentially expressed genes that are specifically induced during the course of the infection are very likely to have functions essential to pathogenicity of the fungus. - Since these genes show presence-absence polymorphisms in the genus and in the Z. - We conclude that compartmentalization of genomes is an ancestral trait in the Zymoseptoria genus.. - Long read assemblies of the Z. - Sequencing of the isolates Za17, Zb87, and Zp13 was run on a PacBio RS II instrument at the Functional Genomics Center, Zurich, Switzerland. - Sequencing of the Zpa63 isolate was performed at the Max Planck-Genome-Centre, Cologne, Germany.. - We chose the best assemblies generated by comparison of all assembly statistics produced by the software Quast such as the number of finished contigs, the size of the assembly and the N50 [47]. - We used previously published RNA sequencing data to increase the quality of the gene prediction and combined three distinct methodologies . - BRAKER applies GeneMark-ET and Augustus to create the first step of gene predictions based on spliced alignments and to produce a final gene prediction based on the best prediction of the first set [56, 57]. - The predicted gene sequences were the basis for an evaluation of the completeness of the assembly and gene prediction by the program BUSCO v.3 [37]. - To update expression profiles on the new genome assemblies and new gene predictions of the three Z.. - The in planta RNA-seq data was obtained from infected leaves at four different stages corresponding to early and late biotrophic and necro- trophic stages of the three Z. - tritici and the genome of the Z. - pseudotritici and Z.tritici. - B – D) Zoom-in on the contigs highlighted with the arrow in part A, showing the details of the pair-wise synteny of both Z.pseudotritici (Zp13) and Z.. - brevis (Zb87) with the Z.tritici reference (IPO323) chromosomes.. - Contigs are ordered based on synteny with chromosomes of the reference strain IPO323. - Simplified diagram of the pipeline used to predict the functions and subcellular localization of gene model products.. - The PCA plot places bio- logical replicates (four replicates per stage) along the two first PC axes explaining 58 to 65% of the variance (x-axis) and 15 to 25% (y-axis) of the variance within samples, respectively.. - EHS, AF and CL contributed to the design and implementation of the research. - All authors contributed to the writing of the manuscript. - The genome sequence of the reference isolate IPO323 available at: http://genome.jgi.doe.gov/Mycgr3/. - Eva Stukenbrock is a member of the editorial board for the BMC Genomics journal.. - Effector diversification within compartments of the Leptosphaeria. - An analysis of the phylogenetic distribution of the pea pathogenicity genes of Nectria haematococca MPVI supports the hypothesis of their origin by horizontal transfer and uncovers a potentially new pathogen of garden pea: Neocosmospora boniensis. - Origin and domestication of the fungal wheat pathogen Mycosphaerella graminicola via sympatric speciation. - pseudotritici, two progenitor species of the septoria tritici leaf blotch fungus Z. - Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals Dispensome structure, chromosome plasticity, and stealth pathogenesis. - Forward genetics approach reveals host genotype-dependent importance of accessory chromosomes in the fungal wheat pathogen Zymoseptoria tritici. - chromosomes of the wheat pathogen Zymoseptoria tritici. - Pangenome analyses of the wheat pathogen Zymoseptoria tritici reveal the structural basis of a highly plastic eukaryotic genome. - Expression profiling of the wheat pathogen Zymoseptoria tritici reveals genomic patterns of transcription and host-specific regulatory programs.
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