BackgroundPyrenophora tritici-repentis (Ptr) is a necrotrophic fungal pathogen that causes the major wheat disease, tan spot. We set out to provide essential genomics-based resources in order to better understand the pathogenicity mechanisms of this important pathogen.ResultsHere, we present eight new Ptr isolate genomes, assembled and annotated; representing races 1, 2 and 5, and a new race. We report a high quality Ptr reference genome, sequenced by PacBio technology with Illumina paired-end data support and optical mapping. An estimated 98% of the genome coverage was mapped to 10 chromosomal groups, using a two-enzyme hybrid approach. The final reference genome was 40.9 Mb and contained a total of 13,797 annotated genes, supported by transcriptomic and proteogenomics data sets.ConclusionsWhole genome comparative analysis revealed major chromosomal segmental rearrangements and fusions, highlighting intraspecific genome plasticity in this species. Furthermore, the Ptr race classification was not supported at the whole genome level, as phylogenetic analysis did not cluster the ToxA producing isolates. This expansion of available Ptr genomics resources will directly facilitate research aimed at controlling tan spot disease.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4680-3) contains supplementary material, which is available to authorized users.

SUMMARYThe fungus Parastagonospora nodorum is the causal agent of Septoria nodorum blotch of wheat (Triticum aestivum). The interaction is mediated by multiple fungal necrotrophic effector-dominant host sensitivity gene interactions. The three bestcharacterized effector-sensitivity gene systems are SnToxATsn1, SnTox1-Snn1 and SnTox3-Snn3. These effector genes are highly expressed during early infection, but expression decreases as the infection progresses to tissue necrosis and sporulation. However, the mechanism of regulation is unknown. We have identified and functionally characterized a gene, referred to as PnPf2, which encodes a putative zinc finger transcription factor. PnPf2 deletion resulted in the down-regulation of SnToxA and SnTox3 expression. Virulence on Tsn1 and Snn3 wheat cultivars was strongly reduced. The SnTox1-Snn1 interaction remained unaffected. Furthermore, we have also identified and deleted an orthologous PtrPf2 from the tan spot fungus Pyrenophora triticirepentis which possesses a near-identical ToxA that was acquired from P. nodorum via horizontal gene transfer. PtrPf2 deletion also resulted in the down-regulation of PtrToxA expression and a near-complete loss of virulence on Tsn1 wheat. We have demonstrated, for the first time, evidence for a functionally conserved signalling component that plays a role in the regulation of a common/horizontally transferred effector found in two major fungal pathogens of wheat.

The necrotrophic fungal pathogen Pyrenophora tritici‐repentis (Ptr) causes the major wheat disease tan spot, and produces multiple necrotrophic effectors that contribute to virulence. The proteinaceous effector ToxA induces necrosis in wheat genotypes possessing the Tsn1 gene, although the importance of the ToxA–Tsn1 interaction itself in varietal disease development has not been well studied. Here, 40 Australian spring wheat varieties were assessed for ToxA sensitivity and disease response to a race 1 wildtype Ptr isolate and ToxA‐deleted strain at both seedling and tillering growth stages. ToxA sensitivity was generally associated with disease susceptibility, but did not always predict spreading necrotic symptoms. Whilst the majority of Tsn1 varieties exhibited lower disease scores following toxa mutant infection, several exhibited no distinct differences between wildtype and toxa symptoms. This implies that ToxA is not the major determinant in tan spot disease development in some host backgrounds and indicates the presence of additional effectors. Unexpectedly, several tsn1 varieties exhibited a reduction in disease severity following toxa mutant inoculation, which may suggest an indirect role for ToxA in pathogen fitness. Additionally, increased chlorosis was observed following toxa mutant infection in three varieties, and further work is required to determine whether this is likely to be due to ToxA epistasis of ToxC symptoms. Taken together, these observations demonstrate that Ptr interacts with the host in a complex and intricate manner, leading to a variety of disease reactions that are dependent or independent of the ToxA–Tsn1 interaction.

The necrotrophic fungal pathogen Pyrenophora tritici-repentis causes tan spot, a major disease of wheat, throughout the world. The proteinaceous effector ToxA is responsible for foliar necrosis on ToxA-sensitive wheat genotypes. The single copy ToxA gene was deleted from a wild-type race 1 P. tritici-repentis isolate via homologous recombination of a knockout construct. Expression of the ToxA transcript was found to be absent in transformants (toxa), as was ToxA protein production in fungal culture filtrates. Plant bioassays were conducted to test transformant pathogenicity. The toxa strains were unable to induce necrosis on ToxA-sensitive wheat genotypes. To our knowledge, this is the first demonstration of a targeted gene knockout in P. tritici-repentis. The ability to undertake gene deletions will facilitate the characterization of other pathogenicity effectors of this economically significant necrotroph.

Here, we evaluate the expression of the proteinaceous effectors ToxA and ToxB, produced by the necrotrophic fungal pathogen Pyrenophora tritici-repentis , which confer tan spot disease susceptibility on wheat. These necrotrophic effectors were expressed in two heterologous systems: Escherichia coli and Pichia pastoris . The E. coli SHuffle system was demonstrated to be superior to P. pastoris in generating high-levels of recombinant proteins that were soluble and stable. In addition, protein extracts from P. pastoris induced non-specific chlorosis on wheat, postulated to be caused by co-purified glucanases secreted by the host. Up to 79.6 μg/ml of ToxB was obtained using the SHuffle system in the absence of the native signal peptide, whilst the ToxA yield was considerably lower at 3.2 μg/ml. Results indicated that a histidine tag at the ToxA C-terminus interfered with effector functionality. Heterologously expressed ToxA and ToxB were tested on a panel of Australian cereals, including 122 varieties of bread wheat, 16 durum, 20 triticale and 5 barley varieties, as well as common plant model species including tobacco and Arabidopsis thaliana . A varying degree of effector sensitivities was observed, with a higher ToxB sensitivity and prevalence in the durum and triticale varieties. ToxB-induced chlorosis was also detected on barley. The heterologous expression of effectors that are easily scalable, will facilitate effector-assisted selection of varieties in wheat breeding programs as well as the investigation of P. tritici-repentis effectors in host and non-host interactions.

Background Necrotrophic effector proteins secreted by fungal pathogens are important virulence factors that mediate the development of disease in wheat. Pyrenophora tritici-repentis (Ptr), the causal agent of wheat tan spot, has a race structure dependent on the combination of effectors. In Ptr, ToxA and ToxB are known proteinaceous effectors responsible for necrosis and chlorosis respectively. While Ptr ToxA is encoded by the single gene ToxA, ToxB has multiple loci in the Ptr genome, which is postulated to be directly related to the level of ToxB production and leaf chlorosis. Although previous analysis has indicated that the majority of the ToxB loci lie on a single chromosome, the exact number and chromosomal locations for all the ToxB loci have not been fully identified. Results In this study, we have sequenced the genome of a race 5 ToxB-producing isolate (DW5), using PacBio long read technology, and found that ToxB duplications are nested in the complex subtelomeric chromosomal regions. A total of ten identical ToxB gene copies were identified and based on flanking sequence identity, nine loci appeared associated with chromosome 10 and a single copy with chromosome 5. Chromosome 10 multiple ToxB gene loci were separated by large sequence regions between 31 and 66 kb within larger segmental duplications in an alternating pattern related to loci strand, and flanked by transposable elements. Conclusion This work provides for the first time the full accompaniment of ToxB loci and surrounding regions, and identifies the organization and distribution of ten ToxB loci to subtelomeric regions. To our knowledge, this is the first report of an interwoven strand-related duplication pattern event. This study further highlights the importance of resolving the highly complex distal chromosomal regions, that remain difficult to assemble, and can harbour important effectors and virulence factors.

Key message Genetic mapping of sensitivity to the Pyrenophora tritici-repentis effector ToxB allowed development of a diagnostic genetic marker, and investigation of wheat pedigrees allowed transmission of sensitive alleles to be tracked. Abstract Tan spot, caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis, is a major disease of wheat (Triticum aestivum). Secretion of the P. tritici-repentis effector ToxB is thought to play a part in mediating infection, causing chlorosis of plant tissue. Here, genetic analysis using an association mapping panel (n = 480) and a multiparent advanced generation intercross (MAGIC) population (n founders = 8, n progeny = 643) genotyped with a 90,000 feature single nucleotide polymorphism (SNP) array found ToxB sensitivity to be highly heritable (h 2 ≥ 0.9), controlled predominantly by the Tsc2 locus on chromosome 2B. Genetic mapping of Tsc2 delineated a 1921-kb interval containing 104 genes in the reference genome of ToxB-insensitive variety 'Chinese Spring'. This allowed development of a co-dominant genetic marker for Tsc2 allelic state, diagnostic for ToxB sensitivity in the association mapping panel. Phenotypic and genotypic analysis in a panel of wheat varieties post-dated the association mapping panel further supported the diagnostic nature of the marker. Combining ToxB phenotype and genotypic data with wheat pedigree datasets allowed historic sources of ToxB sensitivity to be tracked, finding the variety 'Maris Dove' to likely be the historic source of sensitive Tsc2 alleles in the wheat germplasm surveyed. Exploration of the Tsc2 region gene space in the ToxB-sensitive line 'Synthetic W7984' identified candidate genes for future investigation. Additionally, a minor ToxB sensitivity QTL was identified on chromosome 2A. The resources presented here will be of immediate use for marker-assisted selection for ToxB insensitivity and the development of germplasm with additional genetic recombination within the Tsc2 region.

The wheat variety Mace is currently dominating the southern wheat growing regions of Australia. It is high yielding in most environments and resistant to many diseases including yellow spot (also known as tan spot). However, observations of foliar yellowing of Mace have recently been reported in the field. This has raised concerns over a possible breakdown of resistance to yellow spot, which is caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis. West Australian field samples of yellowing Mace leaves were evaluated for P. tritici-repentis infection, and this pathogen was determined to be absent. Instead, Alternaria spp. were isolated from the wheat leaves. Pathogenicity assays showed that the recovered Alternaria spp. were unable to cause disease symptoms on Mace. Furthermore, spontaneous foliar lesions were observed in Mace grown in the absence of pathogens. It is therefore likely that such yellowing is a physiological trait, which will not respond to fungicide application. A marginal impact on yield cannot be excluded.