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Abstract 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 of ToxA) 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. ToxB and its putative transposon were missing from the ToxB non-coding reference isolate, but the homolog toxb and ‘Icarus’ were both present in a different non-coding isolate. This suggests that ToxB may have been mobile at some point during the evolution of the Ptr genome which is contradictory to the current assumption of ToxB vertical 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.

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 of ToxA) 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. ToxB and its putative transposon were missing from the ToxB non-coding reference isolate, but the homolog toxb and 'Icarus' were both present in a different non-coding isolate. This suggests that ToxB may have been mobile at some point during the evolution of the Ptr genome which is contradictory to the current assumption of ToxB vertical 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. [ABSTRACT FROM AUTHOR]

ABSTRACT 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.

Development of new fungicides, needed for sustainable control of fungal plant pathogens, requires identification of novel anti-fungal targets. Essential fungal-specific proteins are good candidates, but due to their importance, gene deletion mutants are not viable. Consequently, their cellular role often remains elusive. This hindrance can be overcome by the use of conditional mutants, where expression is controlled by an inducible/repressible promoter. Here, we introduce 5 inducible/repressible promoter systems to study essential genes in the wheat pathogen Zymoseptoria tritici . We fused the gene for enhanced green-fluorescent protein ( egfp ) to the promoter region of Z. tritici nitrate reductase (P nar1 ; induced by nitrogen and repressed by ammonium), 1,4- β -endoxylanase A (P ex1A ; induced by xylose and repressed by maltodextrin), l -arabinofuranosidase B (P laraB ; induced by arabinose and repressed by glucose), galactose-1-phosphate uridylyltransferase 7 (P gal7 ; induced by galactose and repressed by glucose) and isocitrate lyase (P icl1 ; induced by sodium acetate and repressed by glucose). This was followed by quantitative analysis of cytoplasmic reporter fluorescence under induced and repressed conditions. We show that P nar1 , P laraB and P ex1A drive very little or no egfp expression when repressed, but induce moderate protein production when induced. In contrast, P gal7 and P icl1 show considerable egfp expression when repressed, and were strongly induced in the presence of their inducers. Normalising the expression levels of all promoters to that of the α-tubulin promoter P tub2 revealed that P laraB was the weakest promoter (∼20% of P tub2 ), whereas P icl1 strongly expressed the reporter (∼250% of P tub2 ). The use of these tools promises a better understanding of essential genes, which will help developing novel control strategies that protect wheat from Z. tritici . [ABSTRACT FROM AUTHOR]

Background: It has been well established that the Gα subunit of the heterotrimeric G-protein in the wheat pathogen Stagonospora nodorum is required for a variety of phenotypes including pathogenicity, melanisation and asexual differentiation. The roles though of the Gγ and Gβ subunits though were unclear. The objective of this study was to identify and understand the role of these subunits and assess their requirement for pathogenicity and development. Results: G-protein Gγ and Gβ subunits, named Gga1 and Gba1 respectively, were identified in the Stagonospora nodorum genome by comparative analysis with known fungal orthologues. A reverse genetics technique was used to study the role of these and revealed that the mutant strains displayed altered in vitro growth including a differential response to a variety of exogenous carbon sources. Pathogenicity assays showed that Stagonospora nodorum strains lacking Gba1 were essentially non-pathogenic whilst Gga1-impaired strains displayed significantly slower growth in planta. Subsequent sporulation assays showed that like the previously described Gα subunit mutants, both Gba1 and Gga1 were required for asexual sporulation with neither mutant strain being able to differentiate either pycnidia nor pycnidiospores under normal growth conditions. Continued incubation at 4°C was found to complement the mutation in each of the G-protein subunits with nearly wild-type levels of pycnidia recovered. Conclusion: This study provides further evidence on the significance of cAMP-dependent signal transduction for many aspects of fungal development and pathogenicity. The observation that cold temperatures can complement the G-protein sporulation defect now provides an ideal tool by which asexual differentiation can now be dissected. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2019 McDonald et al.)