The Necrotrophic Pathogen Parastagonospora nodorum Is a Master Manipulator of Wheat Defense.

Parastagonospora nodorum is a necrotrophic pathogen of wheat that is particularly destructive in major wheat-growing regions of the United States, northern Europe, Australia, and South America. P. nodorum secretes necrotrophic effectors that target wheat susceptibility genes to induce programmed cell death (PCD), resulting in increased colonization of host tissue and, ultimately, sporulation to complete its pathogenic life cycle. Intensive research over the last two decades has led to the functional characterization of five proteinaceous necrotrophic effectors, SnTox1, SnToxA, SnTox267, SnTox3, and SnTox5, and three wheat susceptibility genes, Tsn1, Snn1, and Snn3D-1. Functional characterization has revealed that these effectors, in addition to inducing PCD, have additional roles in pathogenesis, including chitin binding that results in protection from wheat chitinases, blocking defense response signaling, and facilitating plant colonization. There are still large gaps in our understanding of how this necrotrophic pathogen is successfully manipulating wheat defense to complete its life cycle. This review summarizes our current knowledge, identifies knowledge gaps, and provides a summary of well-developed tools and resources currently available to study the P. nodorum-wheat interaction, which has become a model for necrotrophic specialist interactions. Further functional characterization of the effectors involved in this interaction and work toward a complete understanding of how P. nodorum manipulates wheat defense will provide fundamental knowledge about this and other necrotrophic interactions. Additionally, a broader understanding of this interaction will contribute to the successful management of Septoria nodorum blotch disease on wheat. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A thousand-genome panel retraces the global spread and adaptation of a major fungal crop pathogen.

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.

A Revised Nomenclature for ToxA Haplotypes Across Multiple Fungal Species.

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.

Multi-stage resistance to Zymoseptoria tritici revealed by GWAS in an Australian bread wheat diversity panel.

Septoria tritici blotch (STB) has been ranked the third most important wheat disease in the world, threatening a large area of wheat production. Although major genes play an important role in the protection against Zymoseptoria tritici infection, the lifespan of their resistance unfortunately is very short in modern wheat production systems. Combinations of quantitative resistance with minor effects, therefore, are believed to have prolonged and more durable resistance to Z. tritici. In this study, new quantitative trait loci (QTLs) were identified that are responsible for seedling-stage resistance and adult-plant stage resistance (APR). More importantly was the characterisation of a previously unidentified QTL that can provide resistance during different stages of plant growth or multi-stage resistance (MSR). At the seedling stage, we discovered a new isolate-specific QTL, QSt.wai.1A.1. At the adult-plant stage, the new QTL QStb.wai.6A.2 provided stable and consistent APR in multiple sites and years, while the QTL QStb.wai.7A.2 was highlighted to have MSR. The stacking of multiple favourable MSR alleles was found to improve resistance to Z. tritici by up to 40%.

Molecular plant immunity against biotrophic, hemibiotrophic, and necrotrophic fungi.

Pathogenic fungi use diverse infection strategies to obtain nutrients from plants. Biotrophic fungi feed only on living plant tissue, whereas necrotrophic fungi kill host cells to extract nutrients. To prevent disease, plants need to distinguish between pathogens with different life cycles, as a successful defense against a biotroph, which often involves programmed cell-death around the site of infection, is not an appropriate response to some necrotrophs. Plants utilize a vast collection of extracellular and intracellular receptors to detect the signatures of pathogen attack. In turn, pathogens are under strong selection to mask or avoid certain receptor responses while enhancing or manipulating other receptor responses to promote virulence. In this review, we focus on the plant receptors involved in resistance responses to fungal pathogens and highlight, with examples, how the infection strategy of fungal pathogens can determine if recognition responses are effective at preventing disease.

The necrotrophic effector ToxA from Parastagonospora nodorum interacts with wheat NHL proteins to facilitate Tsn1-mediated necrosis.

The plant pathogen Parastagonospora nodorum secretes necrotrophic effectors to promote disease. These effectors induce cell death on wheat cultivars carrying dominant susceptibility genes in an inverse gene-for-gene manner. However, the molecular mechanisms underpinning these interactions and resulting cell death remain unclear. Here, we used a yeast two-hybrid library approach to identify wheat proteins that interact with the necrotrophic effector ToxA. Using this strategy, we identified an interaction between ToxA and a wheat transmembrane NDR/HIN1-like protein (TaNHL10) and confirmed the interaction using in planta co-immunoprecipitation and confocal microscopy co-localization analysis. We showed that the C-terminus of TaNHL10 is extracellular whilst the N-terminus is localized in the cytoplasm. Further analyses using yeast two-hybrid and confocal microscopy co-localization showed that ToxA interacts with the C-terminal LEA2 extracellular domain of TaNHL10. Random mutagenesis was then used to identify a ToxA mutant, ToxAN109D , which was unable to interact with TaNHL10 in yeast two-hybrid assays. Subsequent heterologous expression and purification of ToxAN109D in Nicotiania benthamiana revealed that the mutated protein was unable to induce necrosis on Tsn1-dominant wheat cultivars, confirming that the interaction of ToxA with TaNHL10 is required to induce cell death. Collectively, these data advance our understanding on how ToxA induces cell death during infection and further highlight the importance of host cell surface interactions in necrotrophic pathosystems.

Optimized Production of Disulfide-Bonded Fungal Effectors in Escherichia coli Using CyDisCo and FunCyDisCo Coexpression Approaches.

Effectors are a key part of the arsenal of plant-pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognized by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterize effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a coexpression system within the heterologous host Escherichia coli, termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalized coexpression approach termed FunCyDisCo (Fungi CyDisCo) can significantly improve the production yields of numerous disulfide-bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallization of several of these reported fungal effectors. We suggest this approach could be broadly useful in the investigation of the function and recognition of a broad range of disulfide bond-containing effectors.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Simple and efficient heterologous expression of necrosis-inducing effectors using the model plant Nicotiana benthamiana.

Plant fungal pathogens cause devastating diseases on cereal plants and threaten global food security. During infection, these pathogens secrete proteinaceous effectors that promote disease. Some of these effectors from necrotrophic plant pathogens induce a cell death response (necrosis), which facilitates pathogen growth in planta. Characterization of these effectors typically requires heterologous expression, and microbial expression systems such as bacteria and yeast are the predominantly used. However, microbial expression systems often require optimization for any given effector and are, in general, not suitable for effectors involving cysteine bridges and posttranslational modifications for activity. Here, we describe a simple and efficient method for expressing such effectors in the model plant Nicotiana benthamiana. Briefly, an effector protein is transiently expressed and secreted into the apoplast of N. benthamiana by Agrobacterium-mediated infiltration. Two to three days subsequent to agroinfiltration, the apoplast from the infiltrated leaves is extracted and can be directly used for phenotyping on host plants. The efficacy of this approach was demonstrated by expressing the ToxA, Tox3, and Tox1 necrosis-inducing effectors from Parastagonospora nodorum. All three effectors produced in N. benthamiana were capable of inducing necrosis in wheat lines, and two of three showed visible bands on Coomassie-stained gel. These data suggest that N. benthamiana-agroinfiltration system is a feasible tool to obtain fungal effectors, especially those that require disulfide bonds and posttranslational modifications. Furthermore, due to the low number of proteins typically observed in the apoplast (compared with intracellular), this simple and high-throughput approach circumvents the requirement to lyse cells and further purifies the target proteins that are required in other heterologous systems. Because of its simplicity and potential for high-throughput, this method is highly amenable to the phenotyping of candidate protein effectors on host plants.

The identification of a transposon affecting the asexual reproduction of the wheat pathogen Zymoseptoria tritici.

Zymoseptoria tritici, the causal agent of Septoria tritici blotch, is a fungal wheat pathogen that causes significant global yield losses. Within Z. tritici populations, quantitative differences in virulence among different isolates are commonly observed; however, the genetic components that underpin these differences remain elusive. In this study, intraspecific comparative transcriptomic analysis was used to identify candidate genes that contribute to differences in virulence on the wheat cultivar WW2449. This led to the identification of a multicopy gene that was not expressed in the high-virulence isolate when compared to the medium- and low-virulence isolates. Further investigation suggested this gene resides in a 7.9-kb transposon. Subsequent long-read sequencing of the isolates used in the transcriptomic analysis confirmed that this gene did reside in an active Class II transposon, which is composed of four genes named REP9-1 to -4. Silencing and overexpression of REP9-1 in two distinct genetic backgrounds demonstrated that its expression alone reduces the number of pycnidia produced by Z. tritici during infection. The REP9-1 gene identified within a Class II transposon is the first discovery of a gene in a transposable element that influences the virulence of Z. tritici. This discovery adds further complexity to genetic loci that contribute to quantitative virulence in this important pathogen.

The crystal structure of SnTox3 from the necrotrophic fungus Parastagonospora nodorum reveals a unique effector fold and provides insight into Snn3 recognition and pro-domain protease processing of fungal effectors.

Plant pathogens cause disease through secreted effector proteins, which act to promote infection. Typically, the sequences of effectors provide little functional information and further targeted experimentation is required. Here, we utilized a structure/function approach to study SnTox3, an effector from the necrotrophic fungal pathogen Parastagonospora nodorum, which causes cell death in wheat-lines carrying the sensitivity gene Snn3. We developed a workflow for the production of SnTox3 in a heterologous host that enabled crystal structure determination and functional studies. We show this approach can be successfully applied to study effectors from other pathogenic fungi. The ß-barrel fold of SnTox3 is a novel fold among fungal effectors. Structure-guided mutagenesis enabled the identification of residues required for Snn3 recognition. SnTox3 is a pre-pro-protein, and the pro-domain of SnTox3 can be cleaved in vitro by the protease Kex2. Complementing this, an in silico study uncovered the prevalence of a conserved motif (LxxR) in an expanded set of putative pro-domain-containing fungal effectors, some of which can be cleaved by Kex2 in vitro. Our in vitro and in silico study suggests that Kex2-processed pro-domain (designated here as K2PP) effectors are common in fungi and this may have broad implications for the approaches used to study their functions.

PR1-mediated defence via C-terminal peptide release is targeted by a fungal pathogen effector.

The effector SnTox3 from Parastagonospora nodorum elicits a strong necrotic response in susceptible wheat and also interacts with wheat pathogenesis-related protein 1 (TaPR-1), although the function of this interaction in disease is unclear. Here, we dissect TaPR1 function by studying SnTox3-TaPR1 interaction and demonstrate the dual functionality of SnTox3. We utilized site-directed mutagenesis to identify an SnTox3 variant, SnTox3P173S , that was unable to interact with TaPR1 in yeast-two-hybrid assays. Additionally, using recombinant proteins we established a novel protein-mediated phenotyping assay allowing functional studies to be undertaken in wheat. Wheat leaves infiltrated with TaPR1 proteins showed significantly less disease compared to control leaves, correlating with a strong increase in defence gene expression. This activity was dependent on release of the TaCAPE1 peptide embedded within TaPR1 by an unidentified serine protease. The priming activity of TaPR1 was compromised by SnTox3 but not the noninteracting variant SnTox3P173S , and we demonstrate that SnTox3 prevents TaCAPE1 release from TaPR1 in vitro. SnTox3 independently functions to induce necrosis through recognition by Snn3 and also suppresses host defence through a direct interaction with TaPR1 proteins. Importantly, this study also advances our understanding of the role of PR1 proteins in host-microbe interactions as inducers of host defence signalling.

Extracellular vesicles from the apoplastic fungal wheat pathogen Zymoseptoria tritici.

BACKGROUND: The fungal pathogen Zymoseptoria tritici is a significant constraint to wheat production in temperate cropping regions around the world. Despite its agronomic impacts, the mechanisms allowing the pathogen to asymptomatically invade and grow in the apoplast of wheat leaves before causing extensive host cell death remain elusive. Given recent evidence of extracellular vesicles (EVs)-secreted, membrane-bound nanoparticles containing molecular cargo-being implicated in extracellular communication between plants and fungal pathogen, we have initiated an in vitro investigation of EVs from this apoplastic fungal wheat pathogen. We aimed to isolate EVs from Z. tritici broth cultures and examine their protein composition in relation to the soluble protein in the culture filtrate and to existing fungal EV proteomes. RESULTS: Zymoseptoria tritici EVs were isolated from broth culture filtrates using differential ultracentrifugation (DUC) and examined with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Z. tritici EVs were observed as a heterogeneous population of particles, with most between 50 and 250 nm. These particles were found in abundance in the culture filtrates of viable Z. tritici cultures, but not heat-killed cultures incubated for an equivalent time and of comparable biomass. Bottom-up proteomic analysis using LC-MS/MS, followed by stringent filtering revealed 240 Z. tritici EV proteins. These proteins were distinct from soluble proteins identified in Z. tritici culture filtrates, but were similar to proteins identified in EVs from other fungi, based on sequence similarity analyses. Notably, a putative marker protein recently identified in Candida albicans EVs was also consistently detected in Z. tritici EVs. CONCLUSION: We have shown EVs can be isolated from the devastating fungal wheat pathogen Z. tritici and are similar to protein composition to previously characterised fungal EVs. EVs from human pathogenic fungi are implicated in virulence, but the role of EVs in the interaction of phytopathogenic fungi and their hosts is unknown. These in vitro analyses provide a basis for expanding investigations of Z. tritici EVs in planta, to examine their involvement in the infection process of this apoplastic wheat pathogen and more broadly, advance understanding of noncanonical secretion in filamentous plant pathogens.

Victorin, the host-selective cyclic peptide toxin from the oat pathogen Cochliobolus victoriae, is ribosomally encoded.

The necrotrophic fungal pathogen Cochliobolus victoriae produces victorin, a host-selective toxin (HST) essential for pathogenicity to certain oat cultivars with resistance against crown rust. Victorin is a mixture of highly modified heterodetic cyclic hexapeptides, previously assumed to be synthesized by a nonribosomal peptide synthetase. Herein, we demonstrate that victorin is a member of the ribosomally synthesized and posttranslationally modified peptide (RiPP) family of natural products. Analysis of a newly generated long-read assembly of the C. victoriae genome revealed three copies of precursor peptide genes (vicA1-3) with variable numbers of "GLKLAF" core peptide repeats corresponding to the victorin peptide backbone. vicA1-3 are located in repeat-rich gene-sparse regions of the genome and are loosely clustered with putative victorin biosynthetic genes, which are supported by the discovery of compact gene clusters harboring corresponding homologs in two distantly related plant-associated Sordariomycete fungi. Deletion of at least one copy of vicA resulted in strongly diminished victorin production. Deletion of a gene encoding a DUF3328 protein (VicYb) abolished the production altogether, supporting its predicted role in oxidative cyclization of the core peptide. In addition, we uncovered a copper amine oxidase (CAO) encoded by vicK, in which its deletion led to the accumulation of new glycine-containing victorin derivatives. The role of VicK in oxidative deamination of the N-terminal glycyl moiety of the hexapeptides to the active glyoxylate forms was confirmed in vitro. This study finally unraveled the genetic and molecular bases for biosynthesis of one of the first discovered HSTs and expanded our understanding of underexplored fungal RiPPs.

Assessing the efficacy of CRISPR/Cas9 genome editing in the wheat pathogen Parastagonspora nodorum.

BACKGROUND: The genome-editing tool CRISPR/Cas9 has revolutionized gene manipulation by providing an efficient method to generate targeted mutations. This technique deploys the Cas9 endonuclease and a guide RNA (sgRNA) which interact to form a Cas9-sgRNA complex that initiates gene editing through the introduction of double stranded DNA breaks. We tested the efficacy of the CRISPR/Cas9 approach as a means of facilitating a variety of reverse genetic approaches in the wheat pathogenic fungus Parastagonospora nodorum. RESULTS: Parastagonospora nodorum protoplasts were transformed with the Cas9 protein and sgRNA in the form of a preassembled ribonuclear protein (RNP) complex targeting the Tox3 effector gene. Subsequent screening of the P. nodorum transformants revealed 100% editing of those mutants screened. We further tested the efficacy of RNP complex when co-transformed with a Tox3-Homology Directed Repair cassette harbouring 1 kb of homologous flanking DNA. Subsequent screening of resulting transformants demonstrated homologous recombination efficiencies exceeding 70%. A further transformation with a Tox3-Homology Directed Repair cassette harbouring a selectable marker with 50 bp micro-homology flanks was also achieved with 25% homologous recombination efficiency. The success of these homology directed repair approaches demonstrate that CRISPR/Cas9 is amenable to other in vivo DNA manipulation approaches such as the insertion of DNA and generating point mutations. CONCLUSION: These data highlight the significant potential that CRISPR/Cas9 has in expediting transgene-free gene knockouts in Parastagonospora nodorum and also in facilitating other gene manipulation approaches. Access to these tools will significantly decrease the time required to assess the requirement of gene for disease and to undertake functional studies to determine its role.

Volatile Molecules Secreted by the Wheat Pathogen Parastagonospora nodorum Are Involved in Development and Phytotoxicity.

Septoria nodorum blotch is a major disease of wheat caused by the fungus Parastagonospora nodorum. Recent studies have demonstrated that secondary metabolites, including polyketides and non-ribosomal peptides, produced by the pathogen play important roles in disease and development. However, there is currently no knowledge on the composition or biological activity of the volatile organic compounds (VOCs) secreted by P. nodorum. To address this, we undertook a series of growth and phytotoxicity assays and demonstrated that P. nodorum VOCs inhibited bacterial growth, were phytotoxic and suppressed self-growth. Mass spectrometry analysis revealed that 3-methyl-1-butanol, 2-methyl-1-butanol, 2-methyl-1-propanol, and 2-phenylethanol were dominant in the VOC mixture and phenotypic assays using these short chain alcohols confirmed that they were phytotoxic. Further analysis of the VOCs also identified the presence of multiple sesquiterpenes of which four were identified via mass spectrometry and nuclear magnetic resonance as ß-elemene, α-cyperone, eudesma-4,11-diene and acora-4,9-diene. Subsequent reverse genetics studies were able to link these molecules to corresponding sesquiterpene synthases in the P. nodorum genome. However, despite extensive testing, these molecules were not involved in either of the growth inhibition or phytotoxicity phenotypes previously observed. Plant assays using mutants of the pathogen lacking the synthetic genes revealed that the identified sesquiterpenes were not required for disease formation on wheat leaves. Collectively, these data have significantly extended our knowledge of the VOCs in fungi and provided the basis for further dissecting the roles of sesquiterpenes in plant disease.

Genomics-Driven Discovery of Phytotoxic Cytochalasans Involved in the Virulence of the Wheat Pathogen Parastagonospora nodorum.

The etiology of fungal pathogenesis of grains is critical to global food security. The large number of orphan biosynthetic gene clusters uncovered in fungal plant pathogen genome sequencing projects suggests that we have a significant knowledge gap about the secondary metabolite repertoires of these pathogens and their roles in plant pathogenesis. Cytochalasans are a family of natural products of significant interest due to their ability to bind to actin and interfere with cellular processes that involved actin polymerization; however, our understanding of their biosynthesis and biological roles remains incomplete. Here, we identified a putative polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene cluster (phm) that was upregulated in the pathogen Parastagonospora nodorum during its infection on wheat. Overexpression of the transcription factor gene phmR encoded in the phm gene cluster resulted in the production of two leucine-derived cytochalasans, phomacins D and E (1 and 2, respectively), and an acetonyl adduct phomacin F. Heterologous expression of the PKS-NRPS gene phmA and the trans-enoyl reductase (ER) gene phmE in Aspergillus nidulans resulted in the production of a novel 2-pyrrolidone precursor prephomacin. Reverse genetics and wheat seedling infection assays showed that ΔphmA mutants exhibited significantly reduced virulence compared to the wild type. We further demonstrated that both 1 and 2 showed potent actin polymerization-inhibitory activities and exhibited potentially monocot-specific antigerminative activities. The findings from this study have advanced our knowledge based on the biosynthesis and biological roles of cytochalasans, the latter of which could have significant implications for our understanding of the molecular mechanisms of fungus-plant interactions.

A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat.

The fungus Parastagonospora nodorum infects wheat through the use of necrotrophic effector (NE) proteins that cause host-specific tissue necrosis. The Zn2Cys6 transcription factor PnPf2 positively regulates NE gene expression and is required for virulence on wheat. Little is known about other downstream targets of PnPf2. We compared the transcriptomes of the P. nodorum wildtype and a strain deleted in PnPf2 (pf2-69) during in vitro growth and host infection to further elucidate targets of PnPf2 signalling. Gene ontology enrichment analysis of the differentially expressed (DE) genes revealed that genes associated with plant cell wall degradation and proteolysis were enriched in down-regulated DE gene sets in pf2-69 compared to SN15. In contrast, genes associated with redox control, nutrient and ion transport were up-regulated in the mutant. Further analysis of the DE gene set revealed that PnPf2 positively regulates twelve genes that encode effector-like proteins. Two of these genes encode proteins with homology to previously characterised effectors in other fungal phytopathogens. In addition to modulating effector gene expression, PnPf2 may play a broader role in the establishment of a necrotrophic lifestyle by orchestrating the expression of genes associated with plant cell wall degradation and nutrient assimilation.

Transposon-Mediated Horizontal Transfer of the Host-Specific Virulence Protein ToxA between Three Fungal Wheat Pathogens.

Most known examples of horizontal gene transfer (HGT) between eukaryotes are ancient. These events are identified primarily using phylogenetic methods on coding regions alone. Only rarely are there examples of HGT where noncoding DNA is also reported. The gene encoding the wheat virulence protein ToxA and the surrounding 14 kb is one of these rare examples. ToxA has been horizontally transferred between three fungal wheat pathogens (Parastagonospora nodorum, Pyrenophora tritici-repentis, and Bipolaris sorokiniana) as part of a conserved ∼14 kb element which contains coding and noncoding regions. Here we used long-read sequencing to define the extent of HGT between these three fungal species. Construction of near-chromosomal-level assemblies enabled identification of terminal inverted repeats on either end of the 14 kb region, typical of a type II DNA transposon. This is the first description of ToxA with complete transposon features, which we call ToxhAT. In all three species, ToxhAT resides in a large (140-to-250 kb) transposon-rich genomic island which is absent in isolates that do not carry the gene (annotated here as toxa- ). We demonstrate that the horizontal transfer of ToxhAT between P. tritici-repentis and P. nodorum occurred as part of a large (∼80 kb) HGT which is now undergoing extensive decay. In B. sorokiniana, in contrast, ToxhAT and its resident genomic island are mobile within the genome. Together, these data provide insight into the noncoding regions that facilitate HGT between eukaryotes and into the genomic processes which mask the extent of HGT between these species.IMPORTANCE This work dissects the tripartite horizontal transfer of ToxA, a gene that has a direct negative impact on global wheat yields. Defining the extent of horizontally transferred DNA is important because it can provide clues to the mechanisms that facilitate HGT. Our analysis of ToxA and its surrounding 14 kb suggests that this gene was horizontally transferred in two independent events, with one event likely facilitated by a type II DNA transposon. These horizontal transfer events are now in various processes of decay in each species due to the repeated insertion of new transposons and subsequent rounds of targeted mutation by a fungal genome defense mechanism known as repeat induced point mutation. This work highlights the role that HGT plays in the evolution of host adaptation in eukaryotic pathogens. It also increases the growing body of evidence indicating that transposons facilitate adaptive HGT events between fungi present in similar environments and hosts.