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 transcriptomeassisted 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.

Transposable elements (TEs) are drivers of genome evolution and affect the expression landscape of the host genome. Stress is a major factor inducing TE activity; however, the regulatory mechanisms underlying de-repression are poorly understood. Plant pathogens are excellent models to dissect the impact of stress on TEs. The process of plant infection induces stress for the pathogen, and virulence factors (i.e., effectors) located in TE-rich regions become expressed. To dissect TE de-repression dynamics and contributions to virulence, we analyzed the TE expression landscape of four strains of the major wheat pathogen Zymoseptoria tritici. We experimentally exposed strains to nutrient starvation and host infection stress. Contrary to expectations, we show that the two distinct conditions induce the expression of different sets of TEs. In particular, the most highly expressed TEs, including miniature inverted-repeat transposable element and long terminal repeat-Gypsy element, show highly distinct de-repression across stress conditions. Both the genomic context of TEs and the genetic background stress (i.e., different strains harboring the same TEs) were major predictors of de-repression under stress. Gene expression profiles under stress varied significantly depending on the proximity to the closest TEs and genomic defenses against TEs were largely ineffective to prevent de-repression. Next, we analyzed the locus encoding the Avr3D1 effector. We show that the insertion and subsequent silencing of TEs in close proximity likely contributed to reduced expression and virulence on a specific wheat cultivar. The complexity of TE responsiveness to stress across genetic backgrounds and genomic locations demonstrates substantial intraspecific genetic variation to control TEs with consequences for virulence.

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Transposable elements (TEs) are key drivers of adaptive evolution within species. Yet, the propagation of TEs across the genome can be highly deleterious and ultimately lead to genome expansions. Hence, TE activity is likely under complex selection regimes within species. To address this, we analyzed a large whole-genome sequencing dataset of the fungal wheat pathogen Zymoseptoria tritici harboring TE-mediated adaptations to overcome host defenses and fungicides. We built a robust map of genome-wide TE insertion and deletion loci for six populations and 284 fungal individuals across the world. We identified a total of 2’456 unfixed TE loci within the species and a significant excess of rare insertions indicating strong purifying selection. A subset of TEs recently swept to near complete fixation with at least one locus likely contributing to higher levels of fungicide resistance. TE-driven adaptation was also supported by evidence for selective sweeps. In parallel, we identified a substantial genome-wide expansion of TE families from the pathogen’s center of origin to more recently founded populations, suggesting that population bottlenecks played a major role in shaping TE content of the genome. The most dramatic expansion occurred among a pair of North American populations collected in the same field at an interval of 25 years. We show that both the activation of specific TEs and relaxed purifying selection likely underpin the expansion. Our study disentangles the effects of selection and TE bursts leading to intra-specific genome expansions, providing a model to recapitulate TE-driven genome evolution over deeper evolutionary timescales.

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.