Background: In fungal plant pathogens, genome rearrangements followed by selection pressure for adaptive traits have facilitated the co-evolutionary arms race between hosts and their pathogens.Pyrenophora tritici-repentis(Ptr) has emerged recently as a foliar pathogen of wheat worldwide and its populations consist of isolates that vary in their ability to produce combinations of different necrotrophic effectors. These effectors play vital roles in disease development. Here, we sequenced the genomes of a global collection (40 isolates) of Ptr to gain insights into its gene content and genome rearrangements.Results: A comparative genome analysis revealed an open pangenome, with an abundance of accessory genes (~ 57%) reflecting Ptr’s adaptability. A clear distinction between pathogenic and non-pathogenic genomes was observed in size, gene content, and phylogenetic relatedness. Chromosomal rearrangements and structural organization, specifically around effector coding genes, were detailed using long-read assemblies (PacBio RS II) generated in this work in addition to previously assembled genomes. We also discovered the involvement of large mobile elements associated with Ptr’s effectors:ToxA, the gene encoding for the necrosis effector, was found as a single copy within a 143-kb ‘Starship’ transposon (dubbed ‘Horizon’) with a clearly defined target site and target site duplications. ‘Horizon’ was located on different chromosomes in different isolates, indicating mobility, and the previously described ToxhAT transposon (responsible for horizontal transfer ofToxA) was nested within this newly identified Starship. Additionally,ToxB, the gene encoding the chlorosis effector, was clustered as three copies on a 294-kb element, which is likely a different putative ‘Starship’ (dubbed ‘Icarus’) in a ToxB-producing isolate.ToxBand its putative transposon were missing from theToxBnon-coding reference isolate, but the homologtoxband ‘Icarus’ were both present in a different non-coding isolate. This suggests thatToxBmay have been mobile at some point during the evolution of the Ptr genome which is contradictory to the current assumption ofToxBvertical inheritance. Finally, the genome architecture of Ptr was defined as ‘one-compartment’ based on calculated gene distances and evolutionary rates.Conclusions: These findings together reflect on the highly plastic nature of the Ptr genome which has likely helped to drive its worldwide adaptation and has illuminated the involvement of giant transposons in facilitating the evolution of virulence in Ptr.

Fusarium head blight (FHB) and Fusarium crown and root rot (FCRR) are major wheat diseases. Populations of FHB and FCRR pathogens are highly dynamic, and shifts in these populations in different regions is reported. Analyzing fungal populations associated with wheat node and grain tissues collected from different regions can provide useful information and predict diseases that might affect subsequent crops and effective disease management practices. In this study, wheat node and grain samples were collected from four representative sites across the western Canadian prairies in the 2018 growing season to characterize the major Fusarium spp. and other mycobiota associated with wheat in these regions. In total, 994 fungal isolates were recovered, and based on culture and molecular diagnostic methods, three genera constituted over 90% of all fungal isolates, namely Alternaria (39.6%), Fusarium (27.8%), and Parastagonospora (23.9%). A quantitative PCR (qPCR) diagnostic toolkit was developed to quantify the most frequently isolated Fusarium spp. in infected wheat tissues: Fusarium avenaceum, F. culmorum, F. graminearum, and F. poae. This qPCR specificity was validated in silico, in vitro, and in planta and proved specific to the target species. The qPCR results showed that F. graminearum was not detected frequently from wheat node and grain samples collected from four locations in this study. F. poae was the most abundant Fusarium species in grain samples in all tested locations. However, in node samples, F. culmorum (Beaverlodge and Scott) and F. avenaceum (Lacombe and Lethbridge) were the most abundant species. Trichothecene genotyping showed that the 3ADON is the most dominant trichothecene genotype (68%), followed by type-A trichothecenes (29.5%), whereas the 15ADON trichothecene genotype was least dominant (2.5%) and the NIV genotype was not detected. Moreover, a total of 129 translation elongation factor 1-alpha (TEF1α) sequences from nine Fusarium spp. were compared at the haplotype level to evaluate genetic variability and distribution. F. avenaceum and F. poae exhibited higher diversity as reflected by higher number of haplotypes present in these two species compared with the rest.

ToxA is one of the most studied proteinaceous necrotrophic effectors produced by plant pathogens. It has been identified in four pathogens ( Pyrenophora tritici-repentis, Parastagonospora nodorum, Parastagonospora pseudonodorum [formerly Parastagonospora avenaria f. sp. tritici], and Bipolaris sorokiniana) causing leaf spot diseases on cereals worldwide. To date, 24 different ToxA haplotypes have been identified. Some P. tritici-repentis and related species also express ToxB, another small protein necrotrophic effector. We present here a revised and standardized nomenclature for these effectors, which could be extended to other poly-haplotypic genes found across multiple species.

Pyrenophora tritici-repentis (tan spot) is a destructive foliar pathogen of wheat with global impact. This ascomycete fungus possesses a highly plastic open pangenome shaped by the gain and loss of effector genes. This study investigated the allelic variations in the chlorosis-encoding gene ToxB across 422 isolates representing all identified pathotypes and worldwide origins. To gain better insights into ToxB evolution, we examined its presence and variability in other Pyrenophora spp. A ToxB haplotype network was constructed, revealing the evolutionary relationships of this gene (20 haplotypes) across four Pyrenophora species. Notably, toxb, the homolog of ToxB, was detected for the first time in the barley pathogen Pyrenophora teres. The ToxB/ toxb genes display evidence of selection that is characterized by loss of function, duplication, and diverse mutations. Within the ToxB/ toxb open reading frame, 72 mutations were identified, including 14 synonymous, 55 nonsynonymous, and 3 indel mutations. Remarkably, a, ∼5.6-kb Copia-like retrotransposon, named Copia-1_Ptr, was found inserted in the toxb gene of a race 3 isolate. This insert disrupted the ToxB gene's function, a first case of effector gene disruption by a transposable element in P. tritici-repentis. Additionally, a microsatellite with 25 nucleotide repeats (0 to 10) in the upstream region of ToxB suggested a potential mechanism influencing ToxB expression and regulation. Exploring ToxB-like protein distribution in other ascomycetes revealed the presence of ToxB-like proteins in 19 additional species, including the Leotiomycetes class for the first time. The presence/absence pattern of ToxB-like proteins defied species relatedness compared with a phylogenetic tree, suggesting a past horizontal gene transfer event during the evolution of the ToxB gene. [ ] Copyright © 2024 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food. This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

We sequenced the genome of a global collection (40 isolates) of the fungus Pyrenophora tritici-repentis (Ptr), a major foliar pathogen of wheat and model for the evolution of necrotrophic pathogens. Ptr exhibited an open-pangenome, with 43% of genes in the core set and 57% defined as accessory (present in only a subset of isolates), of which 56% were singleton genes (present in only one isolate). A clear distinction between pathogenic and non-pathogenic genomes was observed in size, gene content, and phylogenetic relatedness. Chromosomal rearrangements and structural organization, specifically around the effector coding genes, were explored further using the annotated genomes of two isolates sequenced by PacBio RS II and Illumina HiSeq. The Ptr genome exhibited major chromosomal rearrangements, including chromosomal fusion, translocation, and segment duplications. An intraspecies translocation of ToxA , the necrosis-inducing effector-coding gene, was facilitated within Ptr via a 143 kb ‘ Starship’ transposon (dubbed ‘Horizon’). Additionally, ToxB , the gene encoding the chlorosis-inducing effector, was clustered as three copies on a 294 kb transposable element in a ToxB-producing isolate. ToxB and its carrying transposon were missing from the ToxB non-coding reference isolate, but the homolog toxb and the transposon were both present in another non-coding isolate. The Ptr genome also appears to exhibit a ‘one-compartment’ organization, but may still possess a ‘two-speed genome’ that is facilitated by copy-number variation as reported in other fungal pathosystems. IMPORTANCE: Ptr is one of the most destructive wheat pathogens worldwide. Its genome is a mosaic of present and absent effectors, and serves as a model for examining the evolutionary processes behind the acquisition of virulence in necrotrophs and disease emergence. In this work, we took advantage of a diverse collection of pathogenic Ptr isolates with different global origins and applied short- and long-read sequencing technologies to dissect the Ptr genome. This study provides comprehensive insights into the Ptr genome and highlights its structural organization as an open pangenome with ‘one-compartment’. In addition, we identified the potential involvement of transposable elements in genome expansion and the movement of virulence factors. The ability of effector-coding genes to shuffle across chromosomes on large transposons was illustrated by the intraspecies translocation of ToxA and the multi-copy ToxB . In terms of gene contents, the Ptr genome exhibits a large percentage of orphan genes, particularly in non-pathogenic or weakly-virulent isolates.