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Fungal infections are difficult to prevent and treat in large part due to strain heterogeneity. However, the genetic mechanisms driving pathogen variation remain poorly understood. Here, we determined the extent to which Starships -giant transposons capable of mobilizing numerous fungal genes-generate genetic and phenotypic variability in the human pathogen Aspergillus fumigatus . We analyzed 519 diverse strains, including 12 newly sequenced with long-read technology, to reveal 20 distinct Starships that are generating genomic heterogeneity over timescales potentially relevant for experimental reproducibility. Starship -mobilized genes encode diverse functions, including biofilm-related virulence factors and biosynthetic gene clusters, and many are differentially expressed during infection and antifungal exposure in a strain-specific manner. These findings support a new model of fungal evolution wherein Starships help generate variation in gene content and expression among fungal strains. Together, our results demonstrate that Starships are a previously hidden mechanism generating genotypic and, in turn, phenotypic heterogeneity in a major human fungal pathogen.

The naturally selected fungal crop (Leucoagaricus gongylophorus) farmed by leafcutter ants shows striking parallels with artificially selected plant crops domesticated by humans (e.g. polyploidy, engorged nutritional rewards, and dependence on cultivation). To date, poorly resolved L. gongylophorus genome assemblies based on short-read sequencing have constrained hypotheses about how millions of years under cultivation by ants shaped the fungal crop genome and potentially drove domestication. We use PacBio HiFi sequencing of L. gongylophorus from the leafcutter ant Atta colombica to identify 18 putatively novel biosynthetic gene clusters that likely cemented life as a cultivar (e.g. plant fragment degradation, ant-farmer communication, and antimicrobial defense). Comparative analyses with cultivated and free-living fungi showed genomic signatures of stepwise domestication transitions: (i) free-living to ant-cultivated: loss of genes conferring stress response and detoxification; (ii) hyphal food to engorged nutritional rewards: expansions of genes governing cellular homeostasis, carbohydrate metabolism, and siderophore biosynthesis; and (iii) detrital provisioning to freshly cut plant fragments: gene expansions promoting cell wall biosynthesis, fatty acid metabolism, and DNA repair. Comparisons across L. gongylophorus fungi farmed by 3 leafcutter ant species highlight genomic signatures of exclusively vertical clonal propagation and widespread transposable element activity. These results show how natural selection can shape domesticated cultivar genomes toward long-term ecological resilience of farming systems that have thrived across millennia., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The rapid adaptive evolution of microbes is driven by strong selection pressure acting on genetic variation. How adaptive genetic variation is generated within species and how such variation influences phenotypic trait expression is often not well understood though. We focused on the recent activity of transposable elements (TEs) using deep population genomics and transcriptomics analyses of a fungal plant pathogen with a highly active content of TEs in the genome. Zymoseptoria tritici causes one of the most damaging diseases on wheat, with recent adaptation to the host and environment being facilitated by TE-associated mutations. We obtained genomic and RNA-sequencing data from 146 isolates collected from a single wheat field. We established a genome-wide map of TE insertion polymorphisms in the population by analyzing recent TE insertions among individuals. We quantified the locus-specific transcription of individual TE copies and found considerable population variation at individual TE loci in the population. About 20% of all TE copies show transcription in the genome suggesting that genomic defenses such as repressive epigenetic marks and repeat-induced polymorphisms are at least partially ineffective at preventing the proliferation of TEs in the genome. A quarter of recent TE insertions are associated with expression variation of neighboring genes providing broad potential to influence trait expression. We indeed found that TE insertions are likely responsible for variation in virulence on the host and potentially diverse components of secondary metabolite production. Our large-scale transcriptomics study emphasizes how TE-derived polymorphisms segregate even in individual microbial populations and can broadly underpin trait variation in pathogens.IMPORTANCEPathogens can rapidly adapt to new hosts, antimicrobials, or changes in the environment. Adaptation arises often from mutations in the genome; however, how such variation is generated remains poorly understood. We investigated the most dynamic regions of the genome of Zymoseptoria tritici, a major fungal pathogen of wheat. We focused on the transcription of transposable elements. A large proportion of the transposable elements not only show signatures of potential activity but are also variable within a single population of the pathogen. We find that this variation in activity is likely influencing many important traits of the pathogen. Hence, our work provides insights into how a microbial species can adapt over the shortest time periods based on the activity of transposable elements., Competing Interests: The authors declare no conflict of interest.

Transposable elements (TEs) are key drivers of genomic variation contributing to recent adaptation in most species. Yet, the evolutionary origins and insertion dynamics within species remain poorly understood. We recapitulate the spread of the pathogenicity-associated Styx element across five species that last diverged ∼11 000 years ago. We show that the element likely originated in the Zymoseptoria fungal pathogen genus and underwent multiple independent reactivation events. Using a global 900-genome panel of the wheat pathogen Zymoseptoria tritici, we assess Styx copy number variation and identify renewed transposition activity in Oceania and South America. We show that the element can mobilize to create additional Styx copies in a four-generation pedigree. Importantly, we find that new copies of the element are not affected by genomic defenses suggesting minimal control against the element. Styx copies are preferentially located in recombination breakpoints and likely triggered multiple types of large chromosomal rearrangements. Taken together, we establish the origin, diversification and reactivation of a highly active TE with likely major consequences for chromosomal integrity and the expression of disease., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: In agricultural ecosystems, outbreaks of diseases are frequent and pose a significant threat to food security. A successful pathogen undergoes a complex and well-timed sequence of regulatory changes to avoid detection by the host immune system; hence, well-tuned gene regulation is essential for survival. However, the extent to which the regulatory polymorphisms in a pathogen population provide an adaptive advantage is poorly understood., Results: We used Zymoseptoria tritici, one of the most important pathogens of wheat, to generate a genome-wide map of regulatory polymorphism governing gene expression. We investigated genome-wide transcription levels of 146 strains grown under nutrient starvation and performed expression quantitative trait loci (eQTL) mapping. We identified cis-eQTLs for 65.3% of all genes and the majority of all eQTL loci are within 2kb upstream and downstream of the transcription start site (TSS). We also show that polymorphism in different gene elements contributes disproportionally to gene expression variation. Investigating regulatory polymorphism in gene categories, we found an enrichment of regulatory variants for genes predicted to be important for fungal pathogenesis but with comparatively low effect size, suggesting a separate layer of gene regulation involving epigenetics. We also show that previously reported trait-associated SNPs in pathogen populations are frequently cis-regulatory variants of neighboring genes with implications for the trait architecture., Conclusions: Overall, our study provides extensive evidence that single populations segregate large-scale regulatory variation and are likely to fuel rapid adaptation to resistant hosts and environmental change., (© 2023. The Author(s).)

Managing pathogen damage in wheat production is important for sustaining yields. Fungal plant pathogen genomes encode many small secreted proteins acting as effectors that play key roles in the successful colonization of host tissue and triggering host defenses. AvrStb6 is the first described Zymoseptoria tritici avirulence effector, which triggers Stb6 -mediated immunity in the wheat host in a gene-for-gene manner. Evasion of major resistance factors such as Stb6 challenges deployment decisions on wheat cultivars. In this study, we analyzed the evolution of the AvrStb6 effector in Iranian isolates of Z. tritici . In total, 78 isolates were isolated and purified from 30 infected wheat specimens collected from the East Azerbaijan and Ardabil provinces of Iran. The pathogenicity of all isolates was evaluated on the susceptible wheat cultivar 'Tajan'. A subset of 40 isolates were also tested for pathogenicity on the resistant cultivar 'Shafir' carrying Stb6 . Genetic diversity at the AvrStb6 locus was analyzed for 14 isolates covering the breadth of the observed disease severity. The AvrStb6 sequence variation was high, with virulent isolates carrying highly diverse AvrStb6 haplotypes. In an analysis including more than 1,000 additional AvrStb6 sequences from a global set of isolates, we found that virulent isolates carried AvrStb6 haplotypes either clustering with known virulent haplotypes on different continents or constituting previously unknown haplotypes. Furthermore, we found that AvrStb6 variants from avirulent isolates clustered with known avirulent genotypes from Europe. Our study highlights the relevance of AvrStb6 for Z. tritici virulence and the exceptional global diversity patterns of this effector., Competing Interests: The author(s) declare no conflict of interest.

Zymoseptoria tritici is the fungal pathogen responsible for Septoria tritici blotch on wheat. Disease outcome in this pathosystem is partly determined by isolate-specific resistance, where wheat resistance genes recognize specific fungal factors triggering an immune response. Despite the large number of known wheat resistance genes, fungal molecular determinants involved in such cultivar-specific resistance remain largely unknown. We identified the avirulence factor AvrStb9 using association mapping and functional validation approaches. Pathotyping AvrStb9 transgenic strains on Stb9 cultivars, near isogenic lines and wheat mapping populations, showed that AvrStb9 interacts with Stb9 resistance gene, triggering an immune response. AvrStb9 encodes an unusually large avirulence gene with a predicted secretion signal and a protease domain. It belongs to a S41 protease family conserved across different filamentous fungi in the Ascomycota class and may constitute a core effector. AvrStb9 is also conserved among a global Z. tritici population and carries multiple amino acid substitutions caused by strong positive diversifying selection. These results demonstrate the contribution of an 'atypical' conserved effector protein to fungal avirulence and the role of sequence diversification in the escape of host recognition, adding to our understanding of host-pathogen interactions and the evolutionary processes underlying pathogen adaptation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Amezrou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Microbial pathogens can adapt rapidly to changing environments such as the application of pesticides or host resistance. Copy number variations (CNVs) are a major source of adaptive genetic variation for recent adaptation. Here, we analyse how a major fungal pathogen of barley, Rhynchosporium commune, has adapted to the host environment and fungicide applications. We screen the genomes of 125 isolates sampled across a worldwide set of populations and identify a total of 7,879 gene duplications and 116 gene deletions. Most gene duplications result from segmental chromosomal duplications. Although CNVs are generally under negative selection, we find that genes affected by CNVs are enriched in functions related to host exploitation (i.e., effectors and cell-wall-degrading enzymes). We perform genome-wide association studies (GWAS) and identify a large segmental duplication of CYP51A that has contributed to the emergence of azole resistance and a duplication encompassing an effector gene affecting virulence. We show that the adaptive CNVs were probably created by recently active transposable element families. Moreover, we find that specific transposable element families are important drivers of recent gene CNV. Finally, we use a genome-wide single nucleotide polymorphism data set to replicate the GWAS and contrast it with the CNV-focused analysis. Together, our findings show how extensive segmental duplications create the raw material for recent adaptation in global populations of a fungal pathogen., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Human activity impacts the evolutionary trajectories of many species worldwide. Global trade of agricultural goods contributes to the dispersal of pathogens reshaping their genetic makeup and providing opportunities for virulence gains. Understanding how pathogens surmount control strategies and cope with new climates is crucial to predicting the future impact of crop pathogens. Here, we address this by assembling a global thousand-genome panel of Zymoseptoria tritici, a major fungal pathogen of wheat reported in all production areas worldwide. We identify the global invasion routes and ongoing genetic exchange of the pathogen among wheat-growing regions. We find that the global expansion was accompanied by increased activity of transposable elements and weakened genomic defenses. Finally, we find significant standing variation for adaptation to new climates encountered during the global spread. Our work shows how large population genomic panels enable deep insights into the evolutionary trajectory of a major crop pathogen., (© 2023. The Author(s).)

The activity of transposable elements (TEs) contributes significantly to pathogen genome evolution. TEs often destabilize genome integrity but may also confer adaptive variation in pathogenicity or resistance traits. De-repression of epigenetically silenced TEs often initiates bursts of transposition activity that may be counteracted by purifying selection and genome defenses. However, how these forces interact to determine the expansion routes of TEs within a pathogen species remains largely unknown. Here, we analyzed a set of 19 telomere-to-telomere genomes of the fungal wheat pathogen Zymoseptoria tritici. Phylogenetic reconstruction and ancestral state estimates of individual TE families revealed that TEs have undergone distinct activation and repression periods resulting in highly uneven copy numbers between genomes of the same species. Most TEs are clustered in gene poor niches, indicating strong purifying selection against insertions near coding sequences, or as a consequence of insertion site preferences. TE families with high copy numbers have low sequence divergence and strong signatures of defense mechanisms (i.e., RIP). In contrast, small non-autonomous TEs (i.e., MITEs) are less impacted by defense mechanisms and are often located in close proximity to genes. Individual TE families have experienced multiple distinct burst events that generated many nearly identical copies. We found that a Copia element burst was initiated from recent copies inserted substantially closer to genes compared to older copies. Overall, TE bursts tended to initiate from copies in GC-rich niches that escaped inactivation by genomic defenses. Our work shows how specific genomic environments features provide triggers for TE proliferation in pathogen genomes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Oggenfuss, Croll. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Plant diseases are often caused by co-infections of multiple pathogens with the potential to aggravate disease severity. In genetically diverse pathogen species, co-infections can also be caused by multiple strains of the same species. However, the outcome of such mixed infections by different conspecific genotypes is poorly understood. The interaction among pathogen strains with complex lifestyles outside and inside of the host are likely shaped by diverse traits, including metabolic capacity and the ability to overcome host immune responses. To disentangle competitive outcomes among pathogen strains, we investigated the fungal wheat pathogen Zymoseptoria tritici. The pathogen infects wheat leaves in complex strain assemblies, and highly diverse populations persist between growing seasons. We investigated a set of 14 genetically different strains collected from the same field to assess both competitive outcomes under culture conditions and on the host. Growth kinetics of cocultured strains (~100 pairs) significantly deviated from single strain expectations, indicating competitive exclusion depending on the strain genotype. We found similarly complex outcomes of lesion development on plant leaves following co-infections by the same pairs of strains. While some pairings suppressed overall damage to the host, other combinations exceeded expectations of lesion development based on single strain outcomes. Strain competition outcomes in the absence of the host were poor predictors of outcomes on the host, suggesting that the interaction with the plant immune system adds significant complexity. Intraspecific co-infection dynamics likely make important contributions to disease outcomes in the wild. IMPORTANCE Plants are often attacked by a multitude of pathogens simultaneously, and different species can facilitate or constrain the colonization by others. To what extent simultaneous colonization by different strains of the same species matters, remains unclear. We focused on intra-specific interactions between strains of the major fungal wheat pathogen Zymoseptoria tritici. The pathogen persists in the environment before infecting plant leaves early in the growing season. Leaves are typically colonized by a multitude of strains. Strains cultured in pairs without host were growing differently compared to strains cultured alone. Wheat leaves infected either with single or pairs of strains, we found also highly variable outcomes. Interactions between strains outside of the host were only poorly explaining how strains would interact when on the host, suggesting that pathogen strains engage in complex interactions dependent on the environment. Better understanding within-species interactions will improve our ability to manage crop infections.

Crop pathogens pose severe risks to global food production due to the rapid rise of resistance to pesticides and host resistance breakdowns. Predicting future risks requires monitoring tools to identify changes in the genetic composition of pathogen populations. Here we report the design of a microfluidics-based amplicon sequencing assay to multiplex 798 loci targeting virulence and fungicide resistance genes, and randomly selected genome-wide markers for the fungal pathogen Zymoseptoria tritici. The fungus causes one of the most devastating diseases on wheat showing rapid adaptation to fungicides and host resistance. We optimized the primer design by integrating polymorphism data from 632 genomes of the same species. To test the performance of the assay, we genotyped 192 samples in two replicates. Analysis of the short-read sequence data generated by the assay showed a fairly stable success rate across samples to amplify a large number of loci. The performance was consistent between samples originating from pure genomic DNA as well as material extracted directly from infected wheat leaves. In samples with mixed genotypes, we found that the assay recovers variations in allele frequencies. We also explored the potential of the amplicon assay to recover transposable element insertion polymorphism relevant for fungicide resistance. As a proof-of-concept, we show that the assay recovers the pathogen population structure across French wheat fields. Genomic monitoring of crop pathogens contributes to more sustainable crop protection and yields., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bellah et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The evolution of sequencing technology and multiplexing has rapidly expanded our ability to characterize fungal diversity in the environment. However, obtaining an unbiased assessment of the fungal community using ribosomal markers remains challenging. Longer amplicons were shown to improve taxonomic resolution and resolve ambiguities by reducing the risk of spurious operational taxonomic units. We examined the implications of barcoding strategies by amplifying and sequencing two ribosomal DNA fragments. We analyzed the performance of the full internal transcribed spacer (ITS) and a longer fragment including also a part of the 28S ribosomal subunit replicated on 60 grapevine trunk core samples. Grapevine trunks harbor highly diverse fungal communities with implications for disease development. Using identical handling, amplification, and sequencing procedures, we obtained higher sequencing depths for the shorter ITS amplicon. Despite the more limited access to polymorphism, the overall diversity in amplified sequence variants was higher for the shorter ITS amplicon. We detected no meaningful bias in the phylogenetic composition due to the amplicon choice across analyzed samples. Despite the increased resolution of the longer ITS-28S amplicon, the higher and more consistent yields of the shorter amplicons produced a clearer resolution of the fungal community of grapevine stem samples. Our study highlights that the choice of ribosomal amplicons should be carefully evaluated and adjusted according to specific goals. IMPORTANCE Surveying fungal communities is key to our understanding of ecological functions of diverse habitats. Fungal communities can inform about the resilience of agricultural ecosystems, risks to human health, and impacts of pathogens. Community compositions are typically analyzed using ribosomal DNA sequences. Due to technical limitations, most fungal community surveys were based on amplifying a short but highly variable fragment. Advances in sequencing technology enabled the use of longer fragments that can address some limitations of species identification. In this study, we examined the implications of choosing either a short or long ribosomal sequence fragment by replicating the analyses on 60 grapevine wood core samples. Using highly accurate long-read sequencing, we found that the shorter fragment produced substantially higher yields. The shorter fragment also revealed more sequence and species diversity. Our study highlights that the choice of ribosomal amplicons should be carefully evaluated and adjusted according to specific goals.

Background: The Aldabra giant tortoise (Aldabrachelys gigantea) is one of only two giant tortoise species left in the world. The species is endemic to Aldabra Atoll in Seychelles and is listed as Vulnerable on the International Union for Conservation of Nature Red List (v2.3) due to its limited distribution and threats posed by climate change. Genomic resources for A. gigantea are lacking, hampering conservation efforts for both wild and ex situpopulations. A high-quality genome would also open avenues to investigate the genetic basis of the species' exceptionally long life span., Findings: We produced the first chromosome-level de novo genome assembly of A. gigantea using PacBio High-Fidelity sequencing and high-throughput chromosome conformation capture. We produced a 2.37-Gbp assembly with a scaffold N50 of 148.6 Mbp and a resolution into 26 chromosomes. RNA sequencing-assisted gene model prediction identified 23,953 protein-coding genes and 1.1 Gbp of repetitive sequences. Synteny analyses among turtle genomes revealed high levels of chromosomal collinearity even among distantly related taxa. To assess the utility of the high-quality assembly for species conservation, we performed a low-coverage resequencing of 30 individuals from wild populations and two zoo individuals. Our genome-wide population structure analyses detected genetic population structure in the wild and identified the most likely origin of the zoo-housed individuals. We further identified putatively deleterious mutations to be monitored., Conclusions: We establish a high-quality chromosome-level reference genome for A. gigantea and one of the most complete turtle genomes available. We show that low-coverage whole-genome resequencing, for which alignment to the reference genome is a necessity, is a powerful tool to assess the population structure of the wild population and reveal the geographic origins of ex situ individuals relevant for genetic diversity management and rewilding efforts., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)

Background: Fungi produce a wide range of specialized metabolites (SMs) involved in biotic interactions. Pathways for the production of SMs are often encoded in clusters of tightly arranged genes identified as biosynthetic gene clusters. Such gene clusters can undergo horizontal gene transfers between species and rapid evolutionary change within species. The acquisition, rearrangement, and deletion of gene clusters can generate significant metabolome diversity. However, the genetic basis underlying variation in SM production remains poorly understood., Results: Here, we analyzed the metabolite production of a large population of the fungal pathogen of wheat, Zymoseptoria tritici. The pathogen causes major yield losses and shows variation in gene clusters. We performed untargeted ultra-high performance liquid chromatography-high resolution mass spectrometry to profile the metabolite diversity among 102 isolates of the same species. We found substantial variation in the abundance of the detected metabolites among isolates. Integrating whole-genome sequencing data, we performed metabolite genome-wide association mapping to identify loci underlying variation in metabolite production (i.e., metabolite-GWAS). We found that significantly associated SNPs reside mostly in coding and gene regulatory regions. Associated genes encode mainly transport and catalytic activities. The metabolite-GWAS identified also a polymorphism in the 3'UTR region of a virulence gene related to metabolite production and showing expression variation., Conclusions: Taken together, our study provides a significant resource to unravel polymorphism underlying metabolome diversity within a species. Integrating metabolome screens should be feasible for a range of different plant pathogens and help prioritize molecular studies., (© 2022. The Author(s).)

Malassezia is a fungal genus found on the skin of humans and warm-blooded animals, with 18 species reported to date. In this study, we sequenced and annotated the genome of Malassezia arunalokei, which is the most recently identified Malassezia species, and compared it with Malassezia restricta, the predominant isolate from human skin. Additionally, we reanalyzed previously reported mycobiome data sets with a species-level resolution to investigate M. arunalokei distribution within the mycobiota of human facial skin. We discovered that the M. arunalokei genome is 7.24 Mbp in size and encodes 4,117 protein-coding genes, all of which were clustered with M. restricta. We also found that the average nucleotide identity value of the M. arunalokei genome was 93.5, compared with the genomes of three M. restricta strains, including M. restricta KCTC 27527. Our findings demonstrate that they indeed belong to different species and that M. arunalokei may have experienced specific gene loss events during speciation. Furthermore, our study showed that M. arunalokei was diverged from M. restricta approximately 7.1 million years ago and indicated that M. arunalokei is the most recently diverged species in the Malassezia lineage to date. Finally, our analysis of the facial mycobiome of previously recruited cohorts revealed that M. arunalokei abundance is not associated with seborrheic dermatitis/dandruff or acne, but was revealed to be more abundant on the forehead and cheek than on the scalp. IMPORTANCE Malassezia is the fungus predominantly residing on the human skin and causes various skin diseases, including seborrheic dermatitis and dandruff. To date, 18 species have been reported, and among them, M. restricta is the most predominant on human skin, especially on the scalp. In this study, we sequenced and analyzed the genome of M. arunalokei , which is the most recently identified Malassezia species, and compared it with M. restricta . Moreover, we analyzed the fungal microbiome to investigate the M. arunalokei distribution on human facial skin. We found that M. arunalokei may have experienced specific gene loss events during speciation. Our study also showed that M. arunalokei was diverged from M. restricta approximately 7.1 million years ago and indicated that M. arunalokei is the most recently diverged species in the Malassezia lineage. Finally, our analysis of the facial mycobiome revealed that M. arunalokei has higher relative abundance on the forehead and cheek than the scalp.

Many plant-associated fungi are obligate biotrophs that depend on living hosts to proliferate. However, little is known about the molecular basis of the biotrophic lifestyle, despite the impact of fungi on the environment and food security. In this work, we show that combinations of organic acids and glucose trigger phenotypes that are associated with the late stage of biotrophy for the maize pathogen Ustilago maydis . These phenotypes include the expression of a set of effectors normally observed only during biotrophic development, as well as the formation of melanin associated with sporulation in plant tumors. U. maydis and other hemibiotrophic fungi also respond to a combination of carbon sources with enhanced proliferation. Thus, the response to combinations of nutrients from the host may be a conserved feature of fungal biotrophy.

Hyperparasites can affect the evolution of pathosystems by influencing the stability of both pathogen and host populations. However, how pathogens of perennial hosts evolve in the presence of a hyperparasite has rarely been studied. Here, we investigated temporal changes in genetic diversity of the invasive chestnut blight pathogen Cryphonectria parasitica in the presence of its parasitic mycovirus Cryphonectria hypovirus 1 (CHV1). The virus reduces fungal virulence and represents an effective natural biocontrol agent against chestnut blight in Europe. We analysed genome-wide diversity and CHV1 prevalence in C. parasitica populations in southern Switzerland that were sampled twice at an interval of about 30 years. Overall, we found that both pathogen population structure and CHV1 prevalence were retained over time. The results suggest that recent bottlenecks have influenced the structure of C. parasitica populations in southern Switzerland. Strong balancing selection signals were found at a single vegetative incompatibility (vic) locus, consistent with negative frequency-dependent selection imposed by the vegetative incompatibility system. High levels of mating among related individuals (i.e., inbreeding) and genetic drift are probably at the origin of imbalanced allele ratios at vic loci and subsequently low vc type diversity. Virus infection rates were stable at ~30% over the study period and we found no significant impact of the virus on fungal population diversity. Consequently, the efficacy of CHV1-mediated biocontrol was probably retained., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Epigenetic modifications are key regulators of gene expression and underpin genome integrity. Yet, how epigenetic changes affect the evolution and transcriptional robustness of genes remains largely unknown. Here, we show how the repressive histone mark H3K27me3 underpins the trajectory of highly conserved genes in fungi. We first performed transcriptomic profiling on closely related species of the plant pathogen Fusarium graminearum species complex. We determined transcriptional responsiveness of genes across environmental conditions to determine expression robustness. To infer evolutionary conservation, we used a framework of 23 species across the Fusarium genus including three species covered with histone methylation data. Gene expression variation is negatively correlated with gene conservation confirming that highly conserved genes show higher expression robustness. In contrast, genes marked by H3K27me3 do not show such associations. Furthermore, highly conserved genes marked by H3K27me3 encode smaller proteins, exhibit weaker codon usage bias, higher levels of hydrophobicity, show lower intrinsically disordered regions, and are enriched for functions related to regulation and membrane transport. The evolutionary age of conserved genes with H3K27me3 histone marks falls typically within the origins of the Fusarium genus. We show that highly conserved genes marked by H3K27me3 are more likely to be dispensable for survival during host infection. Lastly, we show that conserved genes exposed to repressive H3K27me3 marks across distantly related Fusarium fungi are associated with transcriptional perturbation at the microevolutionary scale. In conclusion, we show how repressive histone marks are entangled in the evolutionary fate of highly conserved genes across evolutionary timescales., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The fungus Parastagonospora nodorum uses proteinaceous necrotrophic effectors (NEs) to induce tissue necrosis on wheat leaves during infection, leading to the symptoms of septoria nodorum blotch (SNB). The NEs Tox1 and Tox3 induce necrosis on wheat possessing the dominant susceptibility genes Snn1 and Snn3B1/Snn3D1, respectively. We previously observed that Tox1 is epistatic to the expression of Tox3 and a quantitative trait locus (QTL) on chromosome 2A that contributes to SNB resistance/susceptibility. The expression of Tox1 is significantly higher in the Australian strain SN15 compared to the American strain SN4. Inspection of the Tox1 promoter region revealed a 401 bp promoter genetic element in SN4 positioned 267 bp upstream of the start codon that is absent in SN15, called PE401. Analysis of the world-wide P. nodorum population revealed that a high proportion of Northern Hemisphere isolates possess PE401 whereas the opposite was observed in representative P. nodorum isolates from Australia and South Africa. The presence of PE401 removed the epistatic effect of Tox1 on the contribution of the SNB 2A QTL but not Tox3. PE401 was introduced into the Tox1 promoter regulatory region in SN15 to test for direct regulatory roles. Tox1 expression was markedly reduced in the presence of PE401. This suggests a repressor molecule(s) binds PE401 and inhibits Tox1 transcription. Infection assays also demonstrated that P. nodorum which lacks PE401 is more pathogenic on Snn1 wheat varieties than P. nodorum carrying PE401. An infection competition assay between P. nodorum isogenic strains with and without PE401 indicated that the higher Tox1-expressing strain rescued the reduced virulence of the lower Tox1-expressing strain on Snn1 wheat. Our study demonstrated that Tox1 exhibits both 'selfish' and 'altruistic' characteristics. This offers an insight into a complex NE-NE interaction that is occurring within the P. nodorum population. The importance of PE401 in breeding for SNB resistance in wheat is discussed., Competing Interests: The authors have declared that no competing interests exist.

Plant-soil feedbacks refer to effects on plants that are mediated by soil modifications caused by the previous plant generation. Maize conditions the surrounding soil by secretion of root exudates including benzoxazinoids (BXs), a class of bioactive secondary metabolites. Previous work found that a BX-conditioned soil microbiota enhances insect resistance while reducing biomass in the next generation of maize plants. Whether these BX-mediated and microbially driven feedbacks are conserved across different soils and response species is unknown. We found the BX-feedbacks on maize growth and insect resistance conserved between two arable soils, but absent in a more fertile grassland soil, suggesting a soil-type dependence of BX feedbacks. We demonstrated that wheat also responded to BX-feedbacks. While the negative growth response to BX-conditioning was conserved in both cereals, insect resistance showed opposite patterns, with an increase in maize and a decrease in wheat. Wheat pathogen resistance was not affected. Finally and consistent with maize, we found the BX-feedbacks to be cultivar-specific. Taken together, BX-feedbacks affected cereal growth and resistance in a soil and genotype-dependent manner. Cultivar-specificity of BX-feedbacks is a key finding, as it hides the potential to optimize crops that avoid negative plant-soil feedbacks in rotations., (© 2021 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Convergent evolution leads to identical phenotypic traits in different species or populations. Convergence can be driven by standing variation allowing selection to favour identical alleles in parallel or the same mutations can arise independently. However, the molecular basis of such convergent adaptation remains often poorly resolved. Pesticide resistance in agricultural ecosystems is a hallmark of convergence in phenotypic traits. Here, we analyse the major fungal pathogen Zymoseptoria tritici causing serious losses on wheat and with fungicide resistance emergence across several continents. We sampled three population pairs each from a different continent spanning periods early and late in the application of fungicides. To identify causal loci for resistance, we combined knowledge from molecular genetics work and performed genome-wide association studies (GWAS) on a global set of isolates. We discovered yet unknown factors in azole resistance including a gene encoding membrane associated functions. We found strong support for the "hotspot" model of resistance evolution with convergent changes in a small set of loci but additional loci showed more population-specific allele frequency changes. Genome-wide scans of selection showed that half of all known resistance loci were overlapping a selective sweep region. Hence, the application of fungicides was one of the major selective agents acting on the pathogen over the past decades. Furthermore, loci identified through GWAS showed the highest overlap with selective sweep regions underlining the importance to map phenotypic trait variation in evolving populations. Our population genomic analyses highlighted that both de novo mutations and gene flow contributed to convergent pesticide adaptation., (© 2020 John Wiley & Sons Ltd.)

Ectomycorrhizal (ECM) fungi associated with plants constitute one of the most successful symbiotic interactions in forest ecosystems. ECM support trophic exchanges with host plants and are important factors for the survival and stress resilience of trees. However, ECM clades often harbour morpho-species and cryptic lineages, with weak morphological differentiation. How this relates to intraspecific genome variability and ecological functioning is poorly known. Here, we analysed 16 European isolates of the ascomycete Cenococcum geophilum, an extremely ubiquitous forest symbiotic fungus with no known sexual or asexual spore-forming structures but with a massively enlarged genome. We carried out whole-genome sequencing to identify single-nucleotide polymorphisms. We found no geographic structure at the European scale but divergent lineages within sampling sites. Evidence for recombination was restricted to specific cryptic lineages. Lineage differentiation was supported by extensive copy-number variation. Finally, we confirmed heterothallism with a single MAT1 idiomorph per genome. Synteny analyses of the MAT1 locus revealed substantial rearrangements and a pseudogene of the opposite MAT1 idiomorph. Our study provides the first evidence for substantial genome-wide structural variation, lineage-specific recombination and low continent-wide genetic differentiation in C. geophilum. Our study provides a foundation for targeted analyses of intra-specific functional variation in this major symbiosis., (© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Genome evolution is driven by the activity of transposable elements (TEs). The spread of TEs can have deleterious effects including the destabilization of genome integrity and expansions. However, the precise triggers of genome expansions remain poorly understood because genome size evolution is typically investigated only among deeply divergent lineages. Here, we use a large population genomics dataset of 284 individuals from populations across the globe of Zymoseptoria tritici , a major fungal wheat pathogen. We built a robust map of genome-wide TE insertions and deletions to track a total of 2456 polymorphic loci within the species. We show that purifying selection substantially depressed TE frequencies in most populations, but some rare TEs have recently risen in frequency and likely confer benefits. We found that specific TE families have undergone a substantial genome-wide expansion from the pathogen's center of origin to more recently founded populations. The most dramatic increase in TE insertions occurred between a pair of North American populations collected in the same field at an interval of 25 years. We find that both genome-wide counts of TE insertions and genome size have increased with colonization bottlenecks. Hence, the demographic history likely played a major role in shaping genome evolution within the species. We show that both the activation of specific TEs and relaxed purifying selection underpin this incipient expansion of the genome. Our study establishes a model to recapitulate TE-driven genome evolution over deeper evolutionary timescales., Competing Interests: UO, TB, TW, FH, LA, PK, TV, CM, BM, DC none, NS None, (© 2021, Oggenfuss et al.)

Invasive microbial species constitute a major threat to biodiversity, agricultural production and human health. Invasions are often dominated by one or a small number of genotypes, yet the underlying factors driving invasions are poorly understood. The chestnut blight fungus Cryphonectria parasitica first decimated the North American chestnut, and a more recent outbreak threatens European chestnut stands. To unravel the chestnut blight invasion of southeastern Europe, we sequenced 230 genomes of predominantly European strains. Genotypes outside of the invasion zone showed high levels of diversity with evidence for frequent and ongoing recombination. The invasive lineage emerged from the highly diverse European genotype pool rather than a secondary introduction from Asia or North America. The expansion across southeastern Europe was mostly clonal and is dominated by a single mating type, suggesting a fitness advantage of asexual reproduction. Our findings show how an intermediary, highly diverse bridgehead population gave rise to an invasive, largely clonally expanding pathogen., Competing Interests: LS, TB, AF, SP, DC No competing interests declared, (© 2021, Stauber et al.)

Emerging fungal pathogens are a threat to forest and agroecosystems, as well as animal and human health. How pathogens evolve from nonpathogenic ancestors is still poorly understood, making the prediction of future outbreaks challenging. Most pathogens have evolved lifestyle adaptations, which were enabled by specific changes in the gene content of the species. Hence, understanding transitions in the functions encoded by genomes gives valuable insight into the evolution of pathogenicity. Here, we studied lifestyle evolution in the genus Cryphonectria , including the prominent invasive pathogen Cryphonectria parasitica , the causal agent of chestnut blight on Castanea species. We assembled and compared the genomes of pathogenic and putatively nonpathogenic Cryphonectria species, as well as sister group pathogens in the family Cryphonectriaceae (Diaporthales, Ascomycetes), to investigate the evolution of genome size and gene content. We found a striking loss of genes associated with carbohydrate metabolism (CAZymes) in C. parasitica compared to other Cryphonectriaceae. Despite substantial CAZyme gene loss, experimental data suggest that C. parasitica has retained wood colonization abilities shared with other Cryphonectria species. Putative effectors substantially varied in number, cysteine content, and protein length among species. In contrast, secondary metabolite gene clusters show a high degree of conservation within the genus. Overall, our results underpin the recent lifestyle transition of C. parasitica toward a more pathogenic lifestyle. Our findings suggest that a CAZyme loss may have promoted pathogenicity of C. parasitica on Castanea species. Analyzing gene complements underlying key nutrition modes can facilitate the detection of species with the potential to emerge as pathogens. IMPORTANCE Forest and agroecosystems, as well as animal and human health, are threatened by emerging pathogens. Following decimation of chestnuts in the United States, the fungal pathogen Cryphonectria parasitica colonized Europe. After establishment, the pathogen population gave rise to a highly successful lineage that spread rapidly across the continent. Core to our understanding of what makes a successful pathogen is the genetic repertoire enabling the colonization and exploitation of host species. Here, we have assembled >100 genomes across two related genera to identify key genomic determinants leading to the emergence of chestnut blight. We found subtle yet highly specific changes in the transition from saprotrophy to latent pathogenicity mostly determined by enzymes involved in carbohydrate metabolism. Large-scale genomic analyses of genes underlying key nutrition modes can facilitate the detection of species with the potential to emerge as pathogens., (Copyright © 2020 Stauber et al.)

Plant pathogens cause widespread yield losses in agriculture. Understanding the drivers of plant-pathogen interactions requires decoding the molecular dialog leading to either resistance or disease. However, progress in deciphering pathogenicity genes has been severely hampered by suitable model systems and incomplete fungal genome assemblies. Here, we report a significant improvement of the assembly and annotation of the genome of the Fusarium oxysporum ( Fo ) strain Fo5176. Fo comprises a large number of serious plant pathogens on dozens of plant species with largely unresolved pathogenicity factors. The strain Fo5176 infects Arabidopsis thaliana and, hence, constitutes a highly promising model system. We use high-coverage Pacific Biosciences Sequel long-read and Hi-C sequencing data to assemble the genome into 19 chromosomes and a total genome size of 67.98 Mb. The genome has a N50 of 4 Mb and a 99.1% complete BUSCO score. Phylogenomic analyses based on single-copy orthologs clearly place the Fo5176 strain in the Fo f sp. conglutinans clade as expected. We generated RNAseq data from culture medium and plant infections to train gene predictions and identified ∼18,000 genes including ten effector genes known from other Fo clades. We show that Fo5176 is able to infect cabbage and Brussel sprouts of the Brassica oleracea , expanding the usefulness of the Fo5176 model pathosystem. Finally, we performed large-scale comparative genomics analyses comparing the Fo5176 to 103 additional Fo genomes to define core and accessory genomic regions. In conjunction with the molecular tool sets available for A. thaliana , the Fo5176 genome and annotation provides a crucial step toward the establishment of a highly promising pathosystem., (Copyright © 2020 Fokkens et al.)

Plants trigger immune responses upon recognition of fungal cell wall chitin, followed by the release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host through scavenging of chitin oligomers. We previously showed that intrachain LysM dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding groove that competitively sequesters chitin oligomers from host immune receptors. Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase hydrolysis during host colonization. However, the molecular basis for the protection of fungal cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin-dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin-independent Mg1LysM homodimers, a property that confers protection to fungal cell walls against host chitinases., Competing Interests: The authors declare no conflict of interest exists.

Recombination shapes the evolutionary trajectory of populations and plays an important role in the faithful transmission of chromosomes during meiosis. Levels of sexual reproduction and recombination are important properties of host-pathogen interactions because the speed of antagonistic co-evolution depends on the ability of hosts and pathogens to generate genetic variation. However, our understanding of the importance of recombination is limited because large taxonomic groups remain poorly investigated. Here, we analyze recombination rate variation in the basidiomycete fungus Armillaria ostoyae, which is an aggressive pathogen on a broad range of conifers and other trees. We analyzed a previously constructed, dense genetic map based on 198 single basidiospore progeny from a cross. Progeny were genotyped at a genome-wide set of single-nucleotide polymorphism (SNP) markers using double digest restriction site associated DNA sequencing. Based on a linkage map of on 11,700 SNPs spanning 1007.5 cM, we assembled genomic scaffolds into 11 putative chromosomes of a total genome size of 56.6 Mb. We identified 1984 crossover events among all progeny and found that recombination rates were highly variable along chromosomes. Recombination hotspots tended to be in regions close to the telomeres and were more gene-poor than the genomic background. Genes in proximity to recombination hotspots were encoding on average shorter proteins and were enriched for pectin degrading enzymes. Our analyses enable more powerful population and genome-scale studies of a major tree pathogen.

Background: The gene content of a species largely governs its ecological interactions and adaptive potential. A species is therefore defined by both core genes shared between all individuals and accessory genes segregating presence-absence variation. There is growing evidence that eukaryotes, similar to bacteria, show intra-specific variability in gene content. However, it remains largely unknown how functionally relevant such a pangenome structure is for eukaryotes and what mechanisms underlie the emergence of highly polymorphic genome structures., Results: Here, we establish a reference-quality pangenome of a fungal pathogen of wheat based on 19 complete genomes from isolates sampled across six continents. Zymoseptoria tritici causes substantial worldwide losses to wheat production due to rapidly evolved tolerance to fungicides and evasion of host resistance. We performed transcriptome-assisted annotations of each genome to construct a global pangenome. Major chromosomal rearrangements are segregating within the species and underlie extensive gene presence-absence variation. Conserved orthogroups account for only ~ 60% of the species pangenome. Investigating gene functions, we find that the accessory genome is enriched for pathogenesis-related functions and encodes genes involved in metabolite production, host tissue degradation and manipulation of the immune system. De novo transposon annotation of the 19 complete genomes shows that the highly diverse chromosomal structure is tightly associated with transposable element content. Furthermore, transposable element expansions likely underlie recent genome expansions within the species., Conclusions: Taken together, our work establishes a highly complex eukaryotic pangenome providing an unprecedented toolbox to study how pangenome structure impacts crop-pathogen interactions.

Plant pathogens secrete effector proteins to manipulate the host and facilitate infection. Cognate hosts trigger strong defence responses upon detection of these effectors. Consequently, pathogens and hosts undergo rapid coevolutionary arms races driven by adaptive evolution of effectors and receptors. Because of their high rate of turnover, most effectors are thought to be species-specific and the evolutionary trajectories are poorly understood. Here, we investigate the necrosis-inducing protein 1 (NIP1) effector in the multihost pathogen genus Rhynchosporium. We retraced the evolutionary history of the NIP1 locus using whole-genome assemblies of 146 strains covering four closely related species. NIP1 orthologues were present in all species but the locus consistently segregated presence-absence polymorphisms suggesting long-term balancing selection. We also identified previously unknown paralogues of NIP1 that were shared among multiple species and showed substantial copy-number variation within R. commune. The NIP1A paralogue was under significant positive selection suggesting that NIP1A is the dominant effector variant coevolving with host immune receptors. Consistent with this prediction, we found that copy number variation at NIP1A had a stronger effect on virulence than NIP1B. Our analyses unravelled the origins and diversification mechanisms of a pathogen effector family shedding light on how pathogens gain adaptive genetic variation., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Wheat blast was first reported in Brazil in 1985. It spread rapidly across the wheat cropping areas of Brazil to become the most important biotic constraint on wheat production in the region. The alarming appearance of wheat blast in Bangladesh in 2016 greatly increased the urgency to understand this disease, including its causes and consequences. Here, we summarize the current state of knowledge of wheat blast and aim to identify the most important gaps in our understanding of the disease. We also propose a research agenda that aims to improve the management of wheat blast and limit its threat to global wheat production., (© 2018 The Authors. Molecular Plant Pathology published by BSPP and John Wiley & Sons Ltd.)

Host specialization is a key process in ecological divergence and speciation of plant-associated fungi. The underlying determinants of host specialization are generally poorly understood, especially in endophytes, which constitute one of the most abundant components of the plant microbiome. We addressed the genetic basis of host specialization in two sympatric subspecies of grass-endophytic fungi from the Epichloë typhina complex: subsp. typhina and clarkii. The life cycle of these fungi entails unrestricted dispersal of gametes and sexual reproduction before infection of a new host, implying that the host imposes a selective barrier on viability of the progeny. We aimed to detect genes under divergent selection between subspecies, experiencing restricted gene flow due to adaptation to different hosts. Using pooled whole-genome sequencing data, we combined F ST and D XY population statistics in genome scans and detected 57 outlier genes showing strong differentiation between the two subspecies. Genomewide analyses of nucleotide diversity (π), Tajima's D and dN/dS ratios indicated that these genes have evolved under positive selection. Genes encoding secreted proteins were enriched among the genes showing evidence of positive selection, suggesting that molecular plant-fungus interactions are strong drivers of endophyte divergence. We focused on five genes encoding secreted proteins, which were further sequenced in 28 additional isolates collected across Europe to assess genetic variation in a larger sample size. Signature of positive selection in these isolates and putative identification of pathogenic function supports our findings that these genes represent strong candidates for host specialization determinants in Epichloë endophytes. Our results highlight the role of secreted proteins as key determinants of host specialization., (© 2018 John Wiley & Sons Ltd.)

The genetic and environmental homogeneity in agricultural ecosystems is thought to impose strong and uniform selection pressures. However, the impact of this selection on plant pathogen genomes remains largely unknown. We aimed to identify the proportion of the genome and the specific gene functions under positive selection in populations of the fungal wheat pathogen Zymoseptoria tritici. First, we performed genome scans in four field populations that were sampled from different continents and on distinct wheat cultivars to test which genomic regions are under recent selection. Based on extended haplotype homozygosity and composite likelihood ratio tests, we identified 384 and 81 selective sweeps affecting 4% and 0.5% of the 35 Mb core genome, respectively. We found differences both in the number and the position of selective sweeps across the genome between populations. Using a XtX-based outlier detection approach, we identified 51 extremely divergent genomic regions between the allopatric populations, suggesting that divergent selection led to locally adapted pathogen populations. We performed an outlier detection analysis between two sympatric populations infecting two different wheat cultivars to identify evidence for host-driven selection. Selective sweep regions harboured genes that are likely to play a role in successfully establishing host infections. We also identified secondary metabolite gene clusters and an enrichment in genes encoding transporter and protein localization functions. The latter gene functions mediate responses to environmental stress, including interactions with the host. The distinct gene functions under selection indicate that both local host genotypes and abiotic factors contributed to local adaptation., (© 2018 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.)

Background: Structural variation contributes substantially to polymorphism within species. Chromosomal rearrangements that impact genes can lead to functional variation among individuals and influence the expression of phenotypic traits. Genomes of fungal pathogens show substantial chromosomal polymorphism that can drive virulence evolution on host plants. Assessing the adaptive significance of structural variation is challenging, because most studies rely on inferences based on a single reference genome sequence., Results: We constructed and analyzed the pangenome of Zymoseptoria tritici, a major pathogen of wheat that evolved host specialization by chromosomal rearrangements and gene deletions. We used single-molecule real-time sequencing and high-density genetic maps to assemble multiple genomes. We annotated the gene space based on transcriptomics data that covered the infection life cycle of each strain. Based on a total of five telomere-to-telomere genomes, we constructed a pangenome for the species and identified a core set of 9149 genes. However, an additional 6600 genes were exclusive to a subset of the isolates. The substantial accessory genome encoded on average fewer expressed genes but a larger fraction of the candidate effector genes that may interact with the host during infection. We expanded our analyses of the pangenome to a worldwide collection of 123 isolates of the same species. We confirmed that accessory genes were indeed more likely to show deletion polymorphisms and loss-of-function mutations compared to core genes., Conclusions: The pangenome construction of a highly polymorphic eukaryotic pathogen showed that a single reference genome significantly underestimates the gene space of a species. The substantial accessory genome provides a cradle for adaptive evolution.

Fungal plant pathogens rapidly evolve virulence on resistant hosts through mutations in genes encoding proteins that modulate the host immune responses. The mutational spectrum likely includes chromosomal rearrangements responsible for gains or losses of entire genes. However, the mechanisms creating adaptive structural variation in fungal pathogen populations are poorly understood. We used complete genome assemblies to quantify structural variants segregating in the highly polymorphic fungal wheat pathogen Zymoseptoria tritici The genetic basis of virulence in Z. tritici is complex, and populations harbor significant genetic variation for virulence; hence, we aimed to identify whether structural variation led to functional differences. We combined single-molecule real-time sequencing, genetic maps, and transcriptomics data to generate a fully assembled and annotated genome of the highly virulent field isolate 3D7. Comparative genomics analyses against the complete reference genome IPO323 identified large chromosomal inversions and the complete gain or loss of transposable-element clusters, explaining the extensive chromosomal-length polymorphisms found in this species. Both the 3D7 and IPO323 genomes harbored long tracts of sequences exclusive to one of the two genomes. These orphan regions contained 296 genes unique to the 3D7 genome and not previously known for this species. These orphan genes tended to be organized in clusters and showed evidence of mutational decay. Moreover, the orphan genes were enriched in genes encoding putative effectors and included a gene that is one of the most upregulated putative effector genes during wheat infection. Our study showed that this pathogen species harbored extensive chromosomal structure polymorphism that may drive the evolution of virulence., Importance: Pathogen outbreak populations often harbor previously unknown genes conferring virulence. Hence, a key puzzle of rapid pathogen evolution is the origin of such evolutionary novelty in genomes. Chromosomal rearrangements and structural variation in pathogen populations likely play a key role. However, identifying such polymorphism is challenging, as most genome-sequencing approaches only yield information about point mutations. We combined long-read technology and genetic maps to assemble the complete genome of a strain of a highly polymorphic fungal pathogen of wheat. Comparisons against the reference genome of the species showed substantial variation in the chromosome structure and revealed large regions unique to each assembled genome. These regions were enriched in genes encoding likely effector proteins, which are important components of pathogenicity. Our study showed that pathogen populations harbor extensive polymorphism at the chromosome level and that this polymorphism can be a source of adaptive genetic variation in pathogen evolution., (Copyright © 2016 Plissonneau et al.)