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Barley net form net blotch (NFNB) is a destructive foliar disease caused by Pyrenophora teres f. teres. Barley line CIho5791, which harbors the broadly effective chromosome 6H resistance gene Rpt5 , displays dominant resistance to P. teres f. teres . To genetically characterize P. teres f. teres avirulence/virulence on the barley line CIho5791, we generated a P. teres f. teres mapping population using a cross between the Moroccan CIho5791-virulent isolate MorSM40-3 and the avirulent reference isolate 0-1. Full genome sequences were generated for 103 progenies. Saturated chromosome-level genetic maps were generated, and quantitative trait locus (QTL) mapping identified two major QTL associated with P. teres f. teres avirulence/virulence on CIho5791. The most significant QTL mapped to chromosome (Ch) 1, where the virulent allele was contributed by MorSM40-3. A second QTL mapped to Ch8; however, this virulent allele was contributed by the avirulent parent 0-1. The Ch1 and Ch8 loci accounted for 27 and 15% of the disease variation, respectively, and the avirulent allele at the Ch1 locus was epistatic over the virulent allele at the Ch8 locus. As a validation, we used a natural P. teres f. teres population in a genome-wide association study that identified the same Ch1 and Ch8 loci. We then generated a new reference quality genome assembly of parental isolate MorSM40-3 with annotation supported by deep transcriptome sequencing of infection time points. The annotation identified candidate genes predicted to encode small, secreted proteins, one or more of which are likely responsible for overcoming the CIho5791 resistance. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024., Competing Interests: The author(s) declare no conflict of interest.

Septoria nodorum blotch (SNB), caused by Parastagonospora nodorum, is a disease of durum and common wheat initiated by the recognition of pathogen-produced necrotrophic effectors (NEs) by specific wheat genes. The wheat gene Snn1 was previously cloned, and it encodes a wall-associated kinase that directly interacts with the NE SnTox1 leading to programmed cell death and ultimately the development of SNB. Here, sequence analysis of Snn1 from 114 accessions including diploid, tetraploid, and hexaploid wheat species revealed that some wheat lines possess two copies of Snn1 (designated Snn1-B1 and Snn1-B2) approximately 120 kb apart. Snn1-B2 evolved relatively recently as a paralog of Snn1-B1, and both genes have undergone diversifying selection. Three point mutations associated with the formation of the first SnTox1-sensitive Snn1-B1 allele from a primitive wild wheat were identified. Four subsequent and independent SNPs, three in Snn1-B1 and one in Snn1-B2, converted the sensitive alleles to insensitive forms. Protein modeling indicated these four mutations could abolish Snn1-SnTox1 compatibility either through destabilization of the Snn1 protein or direct disruption of the protein-protein interaction. A high-throughput marker was developed for the absent allele of Snn1, and it was 100% accurate at predicting SnTox1-insensitive lines in both durum and spring wheat. Results of this study increase our understanding of the evolution, diversity, and function of Snn1-B1 and Snn1-B2 genes and will be useful for marker-assisted elimination of these genes for better host resistance., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Key Message: A total of 65 SNPs associated with resistance to tan spot and septoria nodorum blotch were identified in a panel of 180 cultivated emmer accessions through association mapping Tan spot and septoria nodorum blotch (SNB) are foliar diseases caused by the respective fungal pathogens Pyrenophora tritici-repentis and Parastagonospora nodorum that affect global wheat production. To find new sources of resistance, we evaluated a panel of 180 cultivated emmer wheat (Triticum turgidum ssp. dicoccum) accessions for reactions to four P. tritici-repentis isolates Pti2, 86-124, 331-9 and DW5, two P. nodorum isolate, Sn4 and Sn2000, and four necrotrophic effectors (NEs) produced by the pathogens. About 8-36% of the accessions exhibited resistance to the four P. tritici-repentis isolates, with five accessions demonstrating resistance to all isolates. For SNB, 64% accessions showed resistance to Sn4, 43% to Sn2000 and 36% to both isolates, with Spain (11% accessions) as the most common origin of resistance. To understand the genetic basis of resistance, association mapping was performed using SNP (single nucleotide polymorphism) markers generated by genotype-by-sequencing and the 9 K SNP Infinium array. A total of 46 SNPs were significantly associated with tan spot and 19 SNPs with SNB resistance or susceptibility. Six trait loci on chromosome arms 1BL, 3BL, 4AL (2), 6BL and 7AL conferred resistance to two or more isolates. Known NE sensitivity genes for disease development were undetected except Snn5 for Sn2000, suggesting novel genetic factors are controlling host-pathogen interaction in cultivated emmer. The emmer accessions with the highest levels of resistance to the six pathogen isolates (e.g., CItr 14133-1, PI 94634-1 and PI 377672) could serve as donors for tan spot and SNB resistance in wheat breeding programs., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Fungal pathogens cause devastating disease in crops. Understanding the evolutionary origin of pathogens is essential to the prediction of future disease emergence and the potential of pathogens to disperse. The fungus Pyrenophora teres f. teres causes net form net blotch (NFNB), an economically significant disease of barley. In this study, we have used 104 P. teres f. teres genomes from four continents to explore the population structure and demographic history of the fungal pathogen. We showed that P. teres f. teres is structured into populations that tend to be geographically restricted to different regions. Using Multiple Sequentially Markovian Coalescent and machine learning approaches we demonstrated that the demographic history of the pathogen correlates with the history of barley, highlighting the importance of human migration and trade in spreading the pathogen. Exploring signatures of natural selection, we identified several population-specific selective sweeps that colocalized with genomic regions enriched in putative virulence genes, and loci previously identified as determinants of virulence specificities by quantitative trait locus analyses. This reflects rapid adaptation to local hosts and environmental conditions of P. teres f. teres as it spread with barley. Our research highlights how human activities can contribute to the spread of pathogens that significantly impact the productivity of field crops., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Net form net blotch (NFNB), caused by Pyrenophora teres f. teres , is an important barley disease. The centromeric region of barley chromosome 6H has often been associated with resistance or susceptibility to NFNB, including the broadly effective dominant resistance gene Rpt5 derived from barley line CIho 5791. We characterized a population of Moroccan P. teres f. teres isolates that had overcome Rpt5 resistance and identified quantitative trait loci (QTL) that were effective against these isolates. Eight Moroccan P. teres f. teres isolates were phenotyped on barley lines CIho 5791 and Tifang. Six isolates were virulent on CIho 5791, and two were avirulent. A CIho 5791 × Tifang recombinant inbred line (RIL) population was phenotyped with all eight isolates and confirmed the defeat of the 6H resistance locus formerly mapped as Rpt5 in barley line CI9819. A major QTL on chromosome 3H with the resistance allele derived from Tifang, as well as minor QTL, was identified and provided resistance against these isolates. F 2 segregation ratios supported dominant inheritance for both the 3H and 6H resistance. Furthermore, inoculation of progeny isolates derived from a cross of P. teres f. teres isolates 0-1 (virulent on Tifang/avirulent on CIho 5791) and MorSM 40-3 (avirulent on Tifang/virulent on CIho 5791) onto the RIL and F 2 populations determined that recombination between isolates can generate novel genotypes that overcome both resistance genes. Markers linked to the QTL identified in this study can be used to incorporate both resistance loci into elite barley cultivars for durable resistance., Competing Interests: The author(s) declare no conflict of interest.

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., Competing Interests: The author(s) declare no conflict of interest.

Key Message: Pathogen and host genetics were used to uncover an inverse gene-for-gene interaction where virulence genes from the pathogen Pyrenophora teres f. maculata target barley susceptibility genes, resulting in disease. Although models have been proposed to broadly explain how plants and pathogens interact and coevolve, each interaction evolves independently, resulting in various scenarios of host manipulation and plant defense. Spot form net blotch is a foliar disease of barley caused by Pyrenophora teres f. maculata. We developed a barley population (Hockett × PI 67381) segregating for resistance to a diverse set of P. teres f. maculata isolates. Quantitative trait locus analysis identified major loci on barley chromosomes (Chr) 2H and 7H associated with resistance/susceptibility. Subsequently, we used avirulent and virulent P. teres f. maculata isolates to develop a pathogen population, identifying two major virulence loci located on Chr1 and Chr2. To further characterize this host-pathogen interaction, progeny from the pathogen population harboring virulence alleles at either the Chr1 or Chr2 locus was phenotyped on the Hockett × PI 67381 population. Progeny harboring only the Chr1 virulence allele lost the barley Chr7H association but maintained the 2H association. Conversely, isolates harboring only the Chr2 virulence allele lost the barley Chr2H association but maintained the 7H association. Hockett × PI 67381 F 2 individuals showed susceptible/resistant ratios not significantly different than 15:1 and results from F 2 inoculations using the single virulence genotypes were not significantly different from a 3:1 (S:R) ratio, indicating two dominant susceptibility genes. Collectively, this work shows that P. teres f. maculata virulence alleles at the Chr1 and Chr2 loci are targeting the barley 2H and 7H susceptibility alleles in an inverse gene-for-gene manner to facilitate colonization., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Spot form net blotch (SFNB) caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm) is an economically important disease of barley that also infects wheat. Using genetic analysis to characterize loci in Ptm genomes associated with virulence or avirulence is an important step to identify pathogen effectors that determine compatible (virulent) or incompatible (avirulent) interactions with cereal hosts. Association mapping (AM) is a powerful tool for detecting virulence loci utilizing phenotyping and genotyping data generated for natural populations of plant pathogenic fungi., Results: Restriction-site associated DNA genotyping-by-sequencing (RAD-GBS) was used to generate 4,836 single nucleotide polymorphism (SNP) markers for a natural population of 103 Ptm isolates collected from Idaho, Montana and North Dakota. Association mapping analyses were performed utilizing the genotyping and infection type data generated for each isolate when challenged on barley seedlings of thirty SFNB differential barley lines. A total of 39 marker trait associations (MTAs) were detected across the 20 barley lines corresponding to 30 quantitative trait loci (QTL); 26 novel QTL and four that were previously mapped in Ptm biparental populations. These results using diverse US isolates and barley lines showed numerous barley-Ptm genetic interactions with seven of the 30 Ptm virulence/avirulence loci falling on chromosome 3, suggesting that it is a reservoir of diverse virulence effectors. One of the loci exhibited reciprocal virulence/avirulence with one haplotype predominantly present in isolates collected from Idaho increasing virulence on barley line MXB468 and the alternative haplotype predominantly present in isolates collected from North Dakota and Montana increasing virulence on barley line CI9819., Conclusions: Association mapping provided novel insight into the host pathogen genetic interactions occurring in the barley-Ptm pathosystem. The analysis suggests that chromosome 3 of Ptm serves as an effector reservoir in concordance with previous reports for Pyrenophora teres f. teres, the causal agent of the closely related disease net form net blotch. Additionally, these analyses identified the first reported case of a reciprocal pathogen virulence locus. However, further investigation of the pathosystem is required to determine if multiple genes or alleles of the same gene are responsible for this genetic phenomenon., (© 2022. The Author(s).)

The fungus Pyrenophora tritici-repentis causes tan spot, an important foliar disease of wheat worldwide. The fungal pathogen produces three necrotrophic effectors, namely Ptr ToxA, Ptr ToxB, and Ptr ToxC to induce necrosis or chlorosis in wheat. Both Ptr ToxA and Ptr ToxB are proteins, and their encoding genes have been cloned. Ptr ToxC was characterized as a low-molecular weight molecule 20 years ago but the one or more genes controlling its production in P. tritici-repentis are unknown. Here, we report the genetic mapping, molecular cloning, and functional analysis of a fungal gene that is required for Ptr ToxC production. The genetic locus controlling the production of Ptr ToxC, termed ToxC , was mapped to a subtelomeric region using segregating biparental populations, genome sequencing, and association analysis. Additional marker analysis further delimited ToxC to a 173-kb region. The predicted genes in the region were examined for presence/absence polymorphism in different races and isolates leading to the identification of a single candidate gene. Functional validation showed that this gene was required but not sufficient for Ptr ToxC production, thus it is designated as ToxC1 . ToxC1 encoded a conserved hypothetical protein likely located on the vacuole membrane. The gene was highly expressed during infection, and only one haplotype was identified among 120 isolates sequenced. Our work suggests that Ptr ToxC is not a protein and is likely produced through a cascade of biosynthetic pathway. The identification of ToxC1 is a major step toward revealing the Ptr ToxC biosynthetic pathway and studying its molecular interactions with host factors.[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.

Parastagonospora nodorum is a fungal pathogen of wheat. As a necrotrophic specialist, it deploys effector proteins that target dominant host susceptibility genes to elicit programmed cell death (PCD). Here we identify and functionally validate the effector targeting the host susceptibility genes Snn2, Snn6 and Snn7. We utilized whole-genome sequencing, association mapping, gene-disrupted mutants, gain-of-function transformants, virulence assays, bioinformatics and quantitative PCR to characterize these interactions. A single proteinaceous effector, SnTox267, targeted Snn2, Snn6 and Snn7 to trigger PCD. Snn2 and Snn6 functioned cooperatively to trigger PCD in a light-dependent pathway, whereas Snn7-mediated PCD functioned in a light-independent pathway. Isolates harboring 20 SnTox267 protein isoforms quantitatively varied in virulence. The diversity and distribution of isoforms varied between populations, indicating adaptation to local selection pressures. SnTox267 deletion resulted in the upregulation of effector genes SnToxA, SnTox1 and SnTox3. We validated a novel effector operating in an inverse-gene-for-gene manner to target three genetically distinct host susceptibility genes and elicit PCD. The discovery of the complementary gene action of Snn2 and Snn6 indicates their potential function in a guard or decoy model. Additionally, differences in light dependency in the elicited pathways and upregulation of unlinked effectors sheds new light onto a complex fungal necrotroph-host interaction., (© No claim to US Government works New Phytologist © 2021 New Phytologist Foundation.)

Spot form net blotch (SFNB), caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm), is a foliar disease of barley that results in significant yield losses in major growing regions worldwide. Understanding the host-parasite interactions between pathogen virulence/avirulence genes and the corresponding host susceptibility/resistance genes is important for the deployment of genetic resistance against SFNB. Two recombinant inbred mapping populations were developed to characterize genetic resistance/susceptibility to the Ptm isolate 13IM8.3, which was collected from Idaho (ID). An Illumina Infinium array was used to produce a genome-wide marker set. Quantitative trait loci (QTL) analysis identified ten significant resistance/susceptibility loci, with two of the QTL being common to both populations. One of the QTL on 5H appears to be novel, while the remaining loci have been reported previously. Single nucleotide polymorphisms (SNPs) closely linked to or delimiting the significant QTL have been converted to user-friendly markers. Loci and associated molecular markers identified in this study will be useful in genetic mapping and deployment of the genetic resistance to SFNB in barley., (Published by Oxford University Press on behalf of Genetics Society of America 2021.)

The rapid and widespread evolution of fungicide resistance remains a challenge for crop disease management. The demethylation inhibitor (DMI) class of fungicides is a widely used chemistry for managing disease, but there has been a gradual decline in efficacy in many crop pathosystems. Reliance on DMI fungicides has increased resistance in populations of the plant pathogenic fungus Cercospora beticola worldwide. To better understand the genetic and evolutionary basis for DMI resistance in C. beticola, a genome-wide association study (GWAS) and selective sweep analysis were conducted for the first time in this species. We performed whole-genome resequencing of 190 C. beticola isolates infecting sugar beet (Beta vulgaris ssp. vulgaris). All isolates were phenotyped for sensitivity to the DMI tetraconazole. Intragenic markers on chromosomes 1, 4, and 9 were significantly associated with DMI fungicide resistance, including a polyketide synthase gene and the gene encoding the DMI target CbCYP51. Haplotype analysis of CbCYP51 identified a synonymous mutation (E170) and nonsynonymous mutations (L144F, I387M, and Y464S) associated with DMI resistance. Genome-wide scans of selection showed that several of the GWAS mutations for fungicide resistance resided in regions that have recently undergone a selective sweep. Using radial plate growth on selected media as a fitness proxy, we did not find a trade-off associated with DMI fungicide resistance. Taken together, we show that population genomic data from a crop pathogen can allow the identification of mutations conferring fungicide resistance and inform about their origins in the pathogen population., (Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2021.)

Great strides have been made in defining the details of the plant defense response involving biotrophic fungal and bacterial pathogens. The groundwork for the current model was laid by H.H. Flor and others who defined the gene-for-gene hypothesis, which is now known to involve effector-triggered immunity (ETI). PAMP-triggered immunity (PTI) is also a highly effective response to most pathogens because of the recognition of common pathogen molecules by pattern recognition receptors. In this article, we consider the three pathogens that make up the foliar disease complex of wheat, Zymoseptoria tritici , Pyrenophora tritici-repentis , and Parastagonospora nodorum , to review the means by which necrotrophic pathogens circumvent, or outright hijack, the ETI and PTI pathways to cause disease.

Pyrenophora tritici-repentis is an ascomycete fungus that causes tan spot of wheat. The disease has a worldwide distribution and can cause significant yield and quality losses in wheat production. The fungal pathogen is homothallic in nature, which means it can undergo sexual reproduction by selfing to produce pseudothecia on wheat stubble for seasonal survival. Since homothallism precludes the development of bi-parental fungal populations, no genetic linkage map has been developed for P. tritici-repentis for mapping and map-based cloning of fungal virulence genes. In this work, we created two heterothallic strains by deleting one of the mating type genes in each of two parental isolates 86-124 (race 2) and AR CrossB10 (a new race) and developed a bi-parental fungal population between them. The draft genome sequences of the two parental isolates were aligned to the Pt-1C-BFP reference sequence to mine single nucleotide polymorphisms (SNPs). A total of 225 SNP markers were developed for genotyping the entire population. Additionally, 75 simple sequence repeat, and two gene markers were also developed and used in the genotyping. The resulting linkage map consisted of 13 linkage groups spanning 5,075.83 cM in genetic distance. Because the parental isolate AR CrossB10 is a new race and produces Ptr ToxC, it was sequenced using long-read sequencing platforms and de novo assembled into contigs. The majority of the contigs were further anchored into chromosomes with the aid of the linkage maps. The whole genome comparison of AR CrossB10 to the reference genome of M4 revealed a few chromosomal rearrangements. The genetic linkage map and the new AR CrossB10 genome sequence are valuable tools for gene cloning in P. tritici-repentis., (Published by Elsevier Inc.)

Septoria nodorum blotch (SNB), a disease caused by the necrotrophic fungal pathogen Parastagonospora nodorum, is a threat to wheat (Triticum aestivum) production worldwide. Multiple inverse gene-for-gene interactions involving the recognition of necrotrophic effectors (NEs) by wheat sensitivity genes play major roles in causing SNB. One interaction involves the wheat gene Snn3 and the P. nodorum NE SnTox3. Here, we used a map-based strategy to clone the Snn3-D1 gene from Aegilops tauschii, the D-genome progenitor of common wheat. Snn3-D1 contained protein kinase and major sperm protein domains, both of which were essential for function as confirmed by mutagenesis. As opposed to other characterized interactions in this pathosystem, a compatible Snn3-D1-SnTox3 interaction was light-independent, and Snn3-D1 transcriptional expression was downregulated by light and upregulated by darkness. Snn3-D1 likely emerged in Ae. tauschii due to an approximately 218-kb insertion that occurred along the west bank of the Caspian Sea. The identification of this new class of NE sensitivity genes combined with the previously cloned sensitivity genes demonstrates that P. nodorum can take advantage of diverse host targets to trigger SNB susceptibility in wheat., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Pyrenophora teres is the causal agent of net blotch, the most devastating foliar disease of barley. In nature, net blotch is seen in two forms, net form net blotch, caused by P. teres f. teres , and spot form net blotch, caused by P. teres f. maculata . To date, 11 P. teres f. teres genomes have been sequenced and deposited in publicly available repositories, but only one P. teres f. maculata genome has been publicly deposited. Here, we present four additional reference-quality full-genome sequences of P. teres f. maculata isolates with good geographical and phenotypic diversity, with accompanying RNA sequencing-based genome annotations. These additional P. teres f. maculata genomes will aid in the understanding of the genomic complexities of this important barley pathogen.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Parastagonospora nodorum is an important fungal pathogen that causes Septoria nodorum blotch (SNB) in wheat. This pathogen produces several necrotrophic effectors that act as virulence factors; three have been cloned, SnToxA, SnTox1, and SnTox3. In this study, P. nodorum and its sister species P. avenaria f. tritici ( Pat1 ) were isolated from wheat node and grain samples collected from distanced sites in western Canada during 2018. The presence of effector genes and associated haplotypes were determined by PCR and sequence analysis. An internal transcribed spacer-restriction fragment length polymorphism test was developed to distinguish between leaf spotting pathogens ( P. nodorum , Pat1 , Pyrenophora tritici-repentis , and Bipolaris sorokiniana ). P. nodorum was mainly recovered from wheat nodes and to a lesser extent from the grains, while Pat1 was exclusively isolated from grain samples. The effector genes were present in almost all P. nodorum isolates, with the ToxA haplotype 5 (H5) being most prevalent, while a novel ToxA haplotype (denoted here H21) is reported for the first time. In Pat1 , only combinations of SnTox1 and SnTox3 genes were present. A ToxA haplotype network was also constructed to assess the evolutionary relationship among globally found haplotypes to date. Finally, cultivars representing wheat development in Canada for the last century were tested for sensitivity to Sn-effectors and to the presence of Tsn1 , the ToxA sensitivity gene. Of tested cultivars, 32.9 and 56.9% were sensitive to SnTox1 and SnTox3, respectively, and Tsn1 was present in 59% of the cultivars. In conclusion, P. nodorum and Pat1 were prevalent wheat pathogens in Canada with a potential tissue-specific colonization capacity, while producing necrotrophic effectors to which wheat is sensitive.

Pyrenophora teres f. teres causes net form net blotch of barley and is an economically important pathogen throughout the world. However, P. teres f. teres is lacking in the genomic resources necessary to characterize the mechanisms of virulence. Recently a high-quality reference genome was generated for P. teres f. teres isolate 0-1. Here, we present the reference quality sequence and annotation of four new isolates and we use the five available P. teres f. teres genomes for an in-depth comparison, resulting in the generation of hypotheses pertaining to the potential mechanisms and evolution of virulence. Comparative analyses were performed between all five P. teres f. teres genomes, examining genomic organization, structural variations, and core and accessory genomic content, specifically focusing on the genomic characterization of known virulence loci and the localization of genes predicted to encode secreted and effector proteins. We showed that 14 of 15 currently published virulence quantitative trait loci (QTL) span accessory genomic regions, consistent with these accessory regions being important drivers of host adaptation. Additionally, these accessory genomic regions were frequently found in subtelomeric regions of chromosomes, with 10 of the 14 accessory region QTL localizing to subtelomeric regions. Comparative analysis of the subtelomeric regions of P. teres f. teres chromosomes revealed translocation events in which homology was detected between nonhomologous chromosomes at a significantly higher rate than the rest of the genome. These results indicate that the subtelomeric accessory genomic compartments not only harbor most of the known virulence loci but, also, that these regions have the capacity to rapidly evolve.

Pyrenophora teres f. teres and P. teres f. maculata are significant pathogens that cause net blotch of barley. An increased number of loci involved in P. teres resistance or susceptibility responses of barley as well as interacting P. teres virulence effector loci have recently been identified through biparental and association mapping studies of both the pathogen and host. Characterization of the resistance/susceptibility loci in the host and the interacting effector loci in the pathogen will provide a path for targeted gene validation for better-informed release of resistant barley cultivars. This review assembles concise consensus maps for all loci published for both the host and pathogen, providing a useful resource for the community to be used in pathogen characterization and barley breeding for resistance to both forms of P. teres., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Filamentous fungi rapidly evolve in response to environmental selection pressures in part due to their genomic plasticity. Parastagonospora nodorum, a fungal pathogen of wheat and causal agent of septoria nodorum blotch, responds to selection pressure exerted by its host, influencing the gain, loss, or functional diversification of virulence determinants, known as effector genes. Whole genome resequencing of 197 P. nodorum isolates collected from spring, durum, and winter wheat production regions of the United States enabled the examination of effector diversity and genomic regions under selection specific to geographically discrete populations. 1,026,859 SNPs/InDels were used to identify novel loci, as well as SnToxA and SnTox3 as factors in disease. Genes displaying presence/absence variation, predicted effector genes, and genes localized on an accessory chromosome had significantly higher pN/pS ratios, indicating a higher rate of sequence evolution. Population structure analyses indicated two P. nodorum populations corresponding to the Upper Midwest (Population 1) and Southern/Eastern United States (Population 2). Prevalence of SnToxA varied greatly between the two populations which correlated with presence of the host sensitivity gene Tsn1 in the most prevalent cultivars in the corresponding regions. Additionally, 12 and 5 candidate effector genes were observed to be under diversifying selection among isolates from Population 1 and 2, respectively, but under purifying selection or neutrally evolving in the opposite population. Selective sweep analysis revealed 10 and 19 regions that had recently undergone positive selection in Population 1 and 2, respectively, involving 92 genes in total. When comparing genes with and without presence/absence variation, those genes exhibiting this variation were significantly closer to transposable elements. Taken together, these results indicate that P. nodorum is rapidly adapting to distinct selection pressures unique to spring and winter wheat production regions by rapid adaptive evolution and various routes of genomic diversification, potentially facilitated through transposable element activity., Competing Interests: The authors have declared that no competing interests exist.

The wheat- Parastagonospora nodorum pathosystem involves the recognition of pathogen-secreted necrotrophic effectors (NEs) by corresponding wheat NE sensitivity genes. This inverse gene-for-gene recognition leads to necrotrophic effector-triggered susceptibility and ultimately septoria nodorum blotch disease. Here, we used multiple pathogen isolates to individually evaluate the effects of the host gene-NE interactions Tan spot necrosis1 -Stagonospora nodorum ToxinA ( Tsn1 -SnToxA), Stagonospora nodorum necrosis1 -Stagonospora nodorum Toxin1 ( Snn1 -SnTox1), and Stagonospora nodorum necrosis3-B genome homeolog1 -Stagonospora nodorum Toxin3 ( Snn3-B1 -SnTox3), alone and in various combinations, to determine the relative importance of these interactions in causing disease. Genetic analysis of a recombinant inbred wheat population inoculated separately with three P. nodorum isolates, all of which produce all three NEs, indicated that the Tsn1 -SnToxA and Snn3-B1 -SnTox3 interactions contributed to disease caused by all four isolates, but their effects varied and ranged from epistatic to additive. The Snn1 -SnTox1 interaction was associated with increased disease for one isolate, but for other isolates, there was evidence that this interaction inhibited the expression of other host gene-NE interactions. RNA sequencing analysis in planta showed that SnTox1 was differentially expressed between these three isolates after infection. Further analysis of NE gene-knockout isolates showed that the effect of some interactions could be masked or inhibited by other compatible interactions, and the regulation of this occurs at the level of NE gene transcription. Collectively, these results show that the inverse gene-for-gene interactions leading to necrotrophic effector-triggered susceptibility in the wheat- P. nodorum pathosystem vary in their effects depending on the genetic backgrounds of the pathogen and host, and interplay among the interactions is complex and intricately regulated., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

We report a fungal pan-genome study involving Parastagonospora spp., including 21 isolates of the wheat (Triticum aestivum) pathogen Parastagonospora nodorum, 10 of the grass-infecting Parastagonospora avenae, and 2 of a closely related undefined sister species. We observed substantial variation in the distribution of polymorphisms across the pan-genome, including repeat-induced point mutations, diversifying selection and gene gains and losses. We also discovered chromosome-scale inter and intraspecific presence/absence variation of some sequences, suggesting the occurrence of one or more accessory chromosomes or regions that may play a role in host-pathogen interactions. The presence of known pathogenicity effector loci SnToxA, SnTox1, and SnTox3 varied substantially among isolates. Three P. nodorum isolates lacked functional versions for all three loci, whereas three P. avenae isolates carried one or both of the SnTox1 and SnTox3 genes, indicating previously unrecognized potential for discovering additional effectors in the P. nodorum-wheat pathosystem. We utilized the pan-genomic comparative analysis to improve the prediction of pathogenicity effector candidates, recovering the three confirmed effectors among our top-ranked candidates. We propose applying this pan-genomic approach to identify the effector repertoire involved in other host-microbe interactions involving necrotrophic pathogens in the Pezizomycotina.

Parastagonospora nodorum , the causal agent of Septoria nodorum blotch in wheat, has emerged as a model necrotrophic fungal organism for the study of host-microbe interactions. To date, three necrotrophic effectors have been identified and characterized from this pathogen, including SnToxA, SnTox1, and SnTox3. Necrotrophic effector identification was greatly aided by the development of a draft genome of Australian isolate SN15 via Sanger sequencing, yet it remained largely fragmented. This research presents the development of nearly finished genomes of P. nodorum isolates Sn4, Sn2000, and Sn79-1087 using long-read sequencing technology. RNAseq analysis of isolate Sn4, consisting of eight time points covering various developmental and infection stages, mediated the annotation of 13,379 genes. Analysis of these genomes revealed large-scale polymorphism between the three isolates, including the complete absence of contig 23 from isolate Sn79-1087, and a region of genome expansion on contig 10 in isolates Sn4 and Sn2000. Additionally, these genomes exhibit the hallmark characteristics of a "two-speed" genome, being partitioned into two distinct GC-equilibrated and AT-rich compartments. Interestingly, isolate Sn79-1087 contains a lower proportion of AT-rich segments, indicating a potential lack of evolutionary hotspots. These newly sequenced genomes, consisting of telomere-to-telomere assemblies of nearly all 23 P. nodorum chromosomes, provide a robust foundation for the further examination of effector biology and genome evolution., (Copyright © 2018 Richards et al.)

Pyrenophora teres f. teres , the causal agent of net form net blotch (NFNB) of barley, is a destructive pathogen in barley-growing regions throughout the world. Typical yield losses due to NFNB range from 10 to 40%; however, complete loss has been observed on highly susceptible barley lines where environmental conditions favor the pathogen. Currently, genomic resources for this economically important pathogen are limited to a fragmented draft genome assembly and annotation, with limited RNA support of the P. teres f. teres isolate 0-1. This research presents an updated 0-1 reference assembly facilitated by long-read sequencing and scaffolding with the assistance of genetic linkage maps. Additionally, genome annotation was mediated by RNAseq analysis using three infection time points and a pure culture sample, resulting in 11,541 high-confidence gene models. The 0-1 genome assembly and annotation presented here now contains the majority of the repetitive content of the genome. Analysis of the 0-1 genome revealed classic characteristics of a "two-speed" genome, being compartmentalized into GC-equilibrated and AT-rich compartments. The assembly of repetitive AT-rich regions will be important for future investigation of genes known as effectors, which often reside in close proximity to repetitive regions. These effectors are responsible for manipulation of the host defense during infection. This updated P. teres f. teres isolate 0-1 reference genome assembly and annotation provides a robust resource for the examination of the barley- P. teres f. teres host-pathogen coevolution., (Copyright © 2018 Wyatt et al.)

The ascomycete Pyrenophora tritici-repentis (Ptr) is an important fungal pathogen worldwide that causes tan spot of wheat. The fungus is self-fertile because each isolate contains both mating type (MAT) idiomorphs. In this work, we developed knockouts of the MAT genes in Ptr and tested fertility of the knockout strains and outcrossing between the knockout strains carrying the opposite mating type. The fungal strains with deletions of either MAT1-1-1 or MAT1-2-1 did not form mature pseudothecia making them functionally heterothallic. The cross between the heterothallic strains of the same isolate (86-124) was fully fertile with the only difference compared to the homothallic wild type strain being the slightly lower percentage of pseudothecium formation. However, the cross between 86-124 (race 2, ToxA-containing isolate) and DW5 (race 5, ToxB-containing isolate) was partially fertile and had fewer mature pseudothecia. Furthermore, most mature asci produced only two or four instead of eight functional ascospores. A collection of ascospores from this cross was obtained and genotyped for the presence of the ToxA, ToxB and MAT genes as well as simple sequence repeat markers. The segregation of these genes and markers and recombination of different allele types at these loci was observed. This work clearly demonstrates that the fungus requires both MAT genes for sexual production and can undergo outcrossing and sexual recombination. It also establishes a new and practical way for further characterizing fungal virulence in Ptr through the development of segregating fungal populations and subsequent genetic analysis., (Published by Elsevier Inc.)

Pyrenophora teres f. teres is the causal agent of net form net blotch (NFNB) of barley. In order to map the genetics of avirulence/virulence in P. teres f. teres, a fungal population was developed using P. teres f. teres isolates BB25 (Denmark) and FGOH04Ptt-21 (North Dakota, USA) due to these two isolates differing in virulence on several common barley lines. 109 progeny isolates were obtained from the BB25 by FGOH04Ptt-21 cross that were then used for NFNB disease evaluation across eight barley lines, four of which have been used commonly as NFNB differential lines as well as four cultivars commonly used in barley production in the Northern Great Plains. BB25 was virulent on one of the barley lines and avirulent on seven of the barley lines whereas, FGOH04Ptt-21 was virulent on all eight barley lines evaluated. Genetic maps were generated with single nucleotide polymorphism (SNP) markers obtained using a restriction associated DNA genotyping by sequencing (RAD-GBS) approach. Sixteen linkage groups were formed and were used to identify quantitative trait loci (QTL) associated with avirulence/virulence. Nine unique QTL were identified on eight linkage groups out of which three QTL had major effects (R 2 ≥45%) while the remaining six QTL were relatively minor (R 2 <20%). One or two major effect loci were identified for the lines commonly used as differentials. Conversely, variation in virulence on the local barley cultivars was mostly associated with small effect loci that contributed quantitatively to disease., (Published by Elsevier Inc.)

Pyrenophora teres f. maculata is the cause of the foliar disease spot form net blotch (SFNB) on barley. To evaluate pathogen genetics underlying the P. teres f. maculata -barley interaction, we developed a 105-progeny population by crossing two globally diverse isolates, one from North Dakota and the other from Western Australia. Progeny were phenotyped on a set of four barley genotypes showing a differential reaction to the parental isolates, then genotyped using a restriction site-associated-genotype-by-sequencing (RAD-GBS) approach. Genetic maps were developed for use in quantitative trait locus (QTL) analysis to identify virulence-associated QTL. Six QTL were identified on five different linkage groups and individually accounted for 20-37% of the disease variation, with the number of significant QTL ranging from two to four for the barley genotypes evaluated. The data presented demonstrate the complexity of virulence involved in the P. teres f. maculata -barley pathosystem and begins to lay the foundation for understanding this important interaction., (Copyright © 2017 Carlsen et al.)

Key Message: The effect of the SnTox3-Snn3 interaction was documented for the first time under natural infection at the adult plant stage in the field. Co-segregating SNP markers were identified. Parastagonospora nodorum is a necrotrophic pathogen of wheat, causing Septoria nodorum blotch (SNB) affecting both the leaf and glume. P. nodorum is the major leaf blotch pathogen on spring wheat in Norway. Resistance to the disease is quantitative, but several host-specific interactions between necrotrophic effectors (NEs) and host sensitivity (Snn) genes have been identified, playing a major role at the seedling stage. However, the effect of these interactions in the field under natural infection has not been investigated. In the present study, we saturated the genetic map of the recombinant inbred (RI) population SHA3/CBRD × Naxos using the Illumina 90 K SNP chip. The population had previously been evaluated for segregation of SNB susceptibility in field trials. Here, we infiltrated the population with the purified NEs SnToxA, SnTox1 and SnTox3, and mapped the Snn3 locus on 5BS based on sensitivity segregation and SNP marker data. We also conducted inoculation and culture filtrate (CF) infiltration experiments on the population with four selected P. nodorum isolates from Norway and North America. Remapping of quantitative trait loci (QTL) for field resistance showed that the SnTox3-Snn3 interaction could explain 24% of the phenotypic variation in the field, and more than 51% of the variation in seedling inoculations. To our knowledge, this is the first time the effect of this interaction has been documented at the adult plant stage under natural infection in the field.

Necrotrophic pathogens live and feed on dying tissue, but their interactions with plants are not well understood compared to biotrophic pathogens. The wheat Snn1 gene confers susceptibility to strains of the necrotrophic pathogen Parastagonospora nodorum that produce the SnTox1 protein. We report the positional cloning of Snn1 , a member of the wall-associated kinase class of receptors, which are known to drive pathways for biotrophic pathogen resistance. Recognition of SnTox1 by Snn1 activates programmed cell death, which allows this necrotroph to gain nutrients and sporulate. These results demonstrate that necrotrophic pathogens such as P. nodorum hijack host molecular pathways that are typically involved in resistance to biotrophic pathogens, revealing the complex nature of susceptibility and resistance in necrotrophic and biotrophic pathogen interactions with plants.

Parastagonospora nodorum is a necrotrophic fungal pathogen causing Septoria nodorum blotch on wheat. We have identified nine necrotrophic effector-host dominant sensitivity gene interactions, and we have cloned three of the necrotrophic effector genes, including SnToxA, SnTox1, and SnTox3. Because sexual populations of P. nodorum are difficult to develop under lab conditions, genome-wide association study (GWAS) is the best population genomic approach to identify genomic regions associated with traits using natural populations. In this article, we used a global collection of 191 P. nodorum isolates from which we identified 2,983 single-nucleotide polymorphism (SNP) markers and gene markers for SnToxA and SnTox3 to evaluate the power of GWAS on two popular wheat breeding lines that were sensitive to SnToxA and SnTox3. Strong marker trait associations (MTA) with P. nodorum virulence that mapped to SnTox3 and SnToxA were first identified using the marker set described above. A novel locus in the P. nodorum genome associated with virulence was also identified as a result of this analysis. To evaluate whether a sufficient level of marker saturation was available, we designed a set of primers every 1 kb in the genomic regions containing SnToxA and SnTox3. Polymerase chain reaction amplification was performed across the 191 isolates and the presence/absence polymorphism was scored and used as the genotype. The marker proximity necessary to identify MTA flanking SnToxA and SnTox3 ranged from 4 to 5 and 1 to 7 kb, respectively. Similar analysis was performed on the novel locus. Using a 45% missing data threshold, two more SNP were identified spanning a 4.6-kb genomic region at the novel locus. These results showed that the rate of linkage disequilibrium (LD) decay in P. nodorum and, likely, other fungi is high compared with plants and animals. The fast LD decay in P. nodorum is an advantage only if sufficient marker density is attained. Based on our results with the SnToxA and SnTox3 regions, markers are needed every 9 or 8 kb, respectively, or in every gene, to guarantee that genes associated with a quantitative trait such as virulence are not missed.

SnTox1 induces programmed cell death and the up-regulation of pathogenesis-related genes including chitinases. Additionally, SnTox1 has structural homology to several plant chitin-binding proteins. Therefore, we evaluated SnTox1 for chitin binding and localization. We transformed an avirulent strain of Parastagonospora nodorum as well as three nonpathogens of wheat (Triticum aestivum), including a necrotrophic pathogen of barley, a hemibiotrophic pathogen of sugar beet and a saprotroph, to evaluate the role of SnTox1 in infection and in protection from wheat chitinases. SnTox1 bound chitin and an SnTox1-green fluorescent fusion protein localized to the mycelial cell wall. Purified SnTox1 induced necrosis in the absence of the pathogen when sprayed on the leaf surface and appeared to remain on the leaf surface while inducing both epidermal and mesophyll cell death. SnTox1 protected the different fungi from chitinase degradation. SnTox1 was sufficient to change the host range of a necrotrophic pathogen but not a hemibiotroph or saprotroph. Collectively, this work shows that SnTox1 probably interacts with a receptor on the outside of the cell to induce cell death to acquire nutrients, but SnTox1 accomplishes a second role in that it protects against one aspect of the defense response, namely the effects of wheat chitinases., (No claim to original US government works New Phytologist © 2016 New Phytologist Trust.)

Genetic and genomics tools to characterize host-pathogen interactions are disproportionately directed to the host because of the focus on resistance. However, understanding the genetics of pathogen virulence is equally important and has been limited by the high cost of de novo genotyping of species with limited marker data. Non-resource-prohibitive methods that overcome the limitation of genotyping are now available through genotype-by-sequencing (GBS). The use of a two-enzyme restriction-associated DNA (RAD)-GBS method adapted for Ion Torrent sequencing technology provided robust and reproducible high-density genotyping of several fungal species. A total of 5783 and 2373 unique loci, 'sequence tags', containing 16,441 and 9992 single nucleotide polymorphisms (SNPs) were identified and characterized from natural populations of Pyrenophora teres f. maculata and Sphaerulina musiva, respectively. The data generated from the P. teres f. maculata natural population were used in association mapping analysis to map the mating-type gene to high resolution. To further validate the methodology, a biparental population of P. teres f. teres, previously used to develop a genetic map utilizing simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers, was re-analysed using the SNP markers generated from this protocol. A robust genetic map containing 1393 SNPs on 997 sequence tags spread across 15 linkage groups with anchored reference markers was generated from the P. teres f. teres biparental population. The robust high-density markers generated using this protocol will allow positional cloning in biparental fungal populations, association mapping of natural fungal populations and population genetics studies., (© 2014 BSPP AND JOHN WILEY & SONS LTD.)

Culture filtrates (CFs) of the fungal wheat pathogen Zymoseptoria tritici were assayed for necrosis-inducing activity after infiltration in leaves of various wheat cultivars. Active fractions were partially purified and characterized. The necrosis-inducing factors in CFs are proteinaceous, heat stable and their necrosis-inducing activity is temperature and light dependent. The in planta activity of CFs was tested by a time series of proteinase K (PK) co-infiltrations, which was unable to affect activity 30min after CF infiltrations. This suggests that the necrosis inducing proteins (NIPs) are either absent from the apoplast and likely actively transported into mesophyll cells or protected from the protease by association with a receptor. Alternatively, plant cell death signaling pathways might be fully engaged during the first 30min and cannot be reversed even after PK treatment. Further fractionation of the CFs with the highest necrosis-inducing activity involved fast performance liquid chromatography, SDS-PAGE and mass spectrometry. This revealed that most of the proteins present in the fractions have not been described before. The two most prominent ZtNIP encoding candidates were heterologously expressed in Pichia pastoris and subsequent infiltration assays showed their differential activity in a range of wheat cultivars., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Barley net form net blotch (NFNB), caused by the necrotrophic fungus Pyrenophora teres f. teres, is a destructive foliar disease in barley-growing regions worldwide. Little is known about the genetic and molecular basis of this pathosystem. Here, we identified a small secreted proteinaceous necrotrophic effector (NE), designated PttNE1, from intercellular wash fluids of the susceptible barley line Hector after inoculation with P. teres f. teres isolate 0-1. Using a barley recombinant inbred line (RIL) population developed from a cross between the sensitive/susceptible line Hector and the insensitive/resistant line NDB 112 (HN population), sensitivity to PttNE1, which we have named SPN1, mapped to a common resistance/susceptibility region on barley chromosome 6H. PttNE1-SPN1 interaction accounted for 31% of the disease variation when the HN population was inoculated with the 0-1 isolate. Strong accumulation of hydrogen peroxide and increased levels of electrolyte leakage were associated with the susceptible reaction, but not the resistant reaction. In addition, the HN RIL population was evaluated for its reactions to 10 geographically diverse P. teres f. teres isolates. Quantitative trait locus (QTL) mapping led to the identification of at least 10 genomic regions associated with disease, with chromosomes 3H and 6H harbouring major QTLs for resistance/susceptibility. SPN1 was associated with all the 6H QTLs, except one. Collectively, this information indicates that the barley-P. teres f. teres pathosystem follows, at least partially, an NE-triggered susceptibility (NETS) model that has been described in other necrotrophic fungal disease systems, especially in the Dothideomycete class of fungi., (© 2014 BSPP AND JOHN WILEY & SONS LTD.)

The necrotrophic fungal pathogen Pyrenophora teres f. teres causes the foliar disease net form net blotch (NFNB) on barley. To investigate the genetics of virulence in the barley- P. teres f. teres pathosystem, we evaluated 118 progeny derived from a cross between the California isolates 15A and 6A on the barley lines Rika and Kombar, chosen based on their differential reactions to isolates 15A and 6A for NFNB disease. Genetic maps generated with SNP, SSR, and AFLP markers were scanned for quantitative trait loci (QTL) associated with virulence in P. teres f. teres. Loci underlying two major QTL, VR1 and VR2, were associated with virulence on Rika barley, accounting for 35% and 20% of the disease reaction type variation, respectively. Two different loci, VK1 and VK2, were shown to underlie two major QTL associated with virulence on Kombar barley accounting for 26% and 19% of the disease reaction type variation, respectively. Progeny isolates harboring VK1, VK2, or VR2 alone were inoculated onto a Rika×Kombar recombinant inbred line mapping population and the susceptibility induced by each pathogen genotype corresponded to the same region on barley chromosome 6H as that identified for the parental isolates 15A and 6A. The data presented here indicate that the P. teres f. teres - barley interaction can at least partially be explained by pathogen-produced necrotrophic effectors (NEs) that interact with dominant barley susceptibility genes resulting in NE triggered susceptibility (NETS)., (Published by Elsevier Inc.)

Population genetic and phylogenetic studies have shown that Phaeosphaeria nodorum is a member of a species complex that probably shares its center of origin with wheat (Triticum aestivum and Triticum durum). We examined the evolutionary histories of three known necrotrophic effectors (NEs) produced by P. nodorum and compared them with neutral loci. We screened over 1000 individuals for the presence/absence of each effector and assigned each individual to a multi-effector genotype. Diversity at each NE locus was assessed by sequencing c. 200 individuals for each locus. We found significant differences in effector frequency among populations. We propose that these differences reflect the presence/absence of the corresponding susceptibility gene in wheat cultivars. The population harboring the highest sequence diversity was different for each effector locus and never coincided with populations harboring the highest diversity at neutral loci. Coalescent and phylogenetic analyses showed a discontinuous presence of all three NEs among nine closely related Phaeosphaeria species. Only two of the nine species were found to harbor NEs. We present evidence that the three described NEs of P. nodorum were transmitted to its sister species, Phaeosphaeria avenaria tritici 1, via interspecific hybridization., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Stagonospora nodorum is an important wheat (Triticum aestivum) pathogen in many parts of the world, causing major yield losses. It was the first species in the large fungal Dothideomycete class to be genome sequenced. The reference genome sequence (SN15) has been instrumental in the discovery of genes encoding necrotrophic effectors that induce disease symptoms in specific host genotypes. Here we present the genome sequence of two further S. nodorum strains (Sn4 and Sn79) that differ in their effector repertoire from the reference. Sn79 is avirulent on wheat and produces no apparent effectors when infiltrated onto many cultivars and mapping population parents. Sn4 is pathogenic on wheat and has virulences not found in SN15. The new strains, sequenced with short-read Illumina chemistry, are compared with SN15 by a combination of mapping and de novo assembly approaches. Each of the genomes contains a large number of strain-specific genes, many of which have no meaningful similarity to any known gene. Large contiguous sections of the reference genome are absent in the two newly sequenced strains. We refer to these differences as "sectional gene absences." The presence of genes in pathogenic strains and absence in Sn79 is added to computationally predicted properties of known proteins to produce a list of likely effector candidates. Transposon insertion was observed in the mitochondrial genomes of virulent strains where the avirulent strain retained the likely ancestral sequence. The study suggests that short-read enabled comparative genomics is an effective way to both identify new S. nodorum effector candidates and to illuminate evolutionary processes in this species.

The Stagonospora nodorum-wheat interaction involves multiple pathogen-produced necrotrophic effectors that interact directly or indirectly with specific host gene products to induce the disease Stagonospora nodorum blotch (SNB). Here, we used a tetraploid wheat mapping population to identify and characterize a sixth effector-host gene interaction in the wheat-S. nodorum system. Initial characterization of the effector SnTox5 indicated that it is a proteinaceous necrotrophic effector that induces necrosis on host lines harbouring the Snn5 sensitivity gene, which was mapped to the long arm of wheat chromosome 4B. On the basis of ultrafiltration, SnTox5 is probably in the size range 10-30 kDa. Analysis of SNB development in the mapping population indicated that the SnTox5-Snn5 interaction explains 37%-63% of the variation, demonstrating that this interaction plays a significant role in disease development. When the SnTox5-Snn5 and SnToxA-Tsn1 interactions occurred together, the level of SNB was increased significantly. Similar to several other interactions in this system, the SnTox5-Snn5 interaction is light dependent, suggesting that multiple interactions may exploit the same pathways to cause disease., (PUBLISHED 2012. THIS ARTICLE IS A US GOVERNMENT WORK AND IS IN THE PUBLIC DOMAIN IN THE USA.)

The origin of the fungal wheat pathogen Phaeosphaeria nodorum remains unclear despite earlier intensive global population genetic and phylogeographical studies. We sequenced 1683 bp distributed across three loci in 355 globally distributed Phaeosphaeria isolates, including 74 collected in Iran near the center of origin of wheat. We identified nine phylogenetically distinct clades, including two previously unknown species tentatively named P1 and P2 collected in Iran. Coalescent analysis indicates that P1 and P2 are sister species of P. nodorum and the other Phaeosphaeria species identified in our analysis. Two species, P. nodorum and P. avenaria f. sp. tritici 1 (Pat1), comprised ~85% of the sampled isolates, making them the dominant wheat-infecting pathogens within the species complex. We designed a PCR-RFLP assay to distinguish P. nodorum from Pat1. Approximately 4% of P. nodorum and Pat1 isolates showed evidence of hybridization. Measures of private allelic richness at SSR and sequence loci suggest that the center of origin of P. nodorum coincides with its host in the Fertile Crescent. We hypothesize that the origin of this species complex is also in the Fertile Crescent, with four species out of nine found exclusively in the Iranian collections., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The wheat pathogen Stagonospora nodorum, causal organism of the wheat disease Stagonospora nodorum blotch, has emerged as a model for the Dothideomycetes, a large fungal taxon that includes many important plant pathogens. The initial annotation of the genome assembly included 16,586 nuclear gene models. These gene models were used to design a microarray that has been interrogated with labelled transcripts from six cDNA samples: four from infected wheat plants at time points spanning early infection to sporulation, and two time points taken from growth in artificial media. Positive signals of expression were obtained for 12,281 genes. This represents strong corroborative evidence of the validity of these gene models. Significantly differential expression between the various time points was observed. When infected samples were compared with axenic cultures, 2882 genes were expressed at a higher level in planta and 3630 were expressed more highly in vitro. Similar numbers were differentially expressed between different developmental stages. The earliest time points in planta were particularly enriched in differentially expressed genes. A disproportionate number of the early expressed gene products were predicted to be secreted, but otherwise had no obvious sequence homology to functionally characterized genes. These genes are candidate necrotrophic effectors. We have focused attention on genes for carbohydrate metabolism and the specific biosynthetic pathways active during growth in planta. The analysis points to a very dynamic adjustment of metabolism during infection. Functional analysis of a gene in the coenzyme A biosynthetic pathway showed that the enzyme was dispensable for growth, indicating that a precursor is supplied by the plant., (© 2011 The Authors. Molecular Plant Pathology © 2011 BSPP and Blackwell Publishing Ltd.)

In this study, proteomics and metabolomics were used to study the wheat response to exposure to the SnToxA effector protein secreted by the fungal pathogen Stagonospora nodorum during infection. Ninety-one different acidic and basic proteins and 101 metabolites were differentially abundant when comparing SnToxA- and control-treated wheat leaves during a 72-h time course. Proteins involved in photosynthesis were observed to increase marginally initially after exposure, before decreasing rapidly and significantly. Proteins and metabolites associated with the detoxification of reactive oxygen species in the chloroplast were also differentially abundant during SnToxA exposure, implying that the disruption of photosynthesis causes the rapid accumulation of chloroplastic reactive oxygen species. Metabolite profiling revealed major metabolic perturbations in central carbon metabolism, evidenced by significant increases in tricarboxylic acid (TCA) cycle intermediates, suggestive of an attempt by the plant to generate ATP and reducing equivalents in response to the collapse of photosynthesis caused by SnToxA. This was supported by the observation that the TCA cycle enzyme malate dehydrogenase was up-regulated in response to SnToxA. The infiltration of SnToxA also resulted in a significant increase in abundance of many pathogenicity-related proteins, even in the absence of the pathogen or other pathogen-associated molecular patterns. This approach highlights the complementary nature of proteomics and metabolomics in studying effector-host interactions, and provides further support for the hypothesis that necrotrophic pathogens, such as S. nodorum, appear to exploit existing host cell death mechanisms to promote pathogen growth and cause disease., (© 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.)

ToxA is a proteinaceous necrotrophic effector produced by Stagonospora nodorum and Pyrenophora tritici-repentis. In this study, all eight mature isoforms of the ToxA protein were purified and compared. Circular dichroism spectra indicated that all isoforms were structurally intact and had indistinguishable secondary structural features. ToxA isoforms were infiltrated into wheat lines that carry the sensitivity gene Tsn1. It was observed that different wheat lines carrying identical Tsn1 alleles varied in sensitivity to ToxA. All ToxA isoforms induced necrosis when introduced into any Tsn1 wheat line but we observed quantitative variation in effector activity, with the least-active version found in isolates of P. tritici-repentis. Pathogen sporulation increased with higher doses of ToxA. The isoforms that induced the most rapid necrosis also induced the most sporulation, indicating that pathogen fitness is affected by differences in ToxA activity. We show that differences in toxin activity encoded by a single gene can contribute to the quantitative inheritance of necrotrophic virulence. Our findings support the hypothesis that the variation at ToxA results from selection that favors increased toxin activity.

Stagonospora nodorum is a major necrotrophic pathogen of wheat that causes the diseases S. nodorum leaf and glume blotch. A series of tools and resources, including functional genomics, a genome sequence, proteomics and metabolomics, host-mapping populations, and a worldwide collection of isolates, have enabled the dissection of pathogenicity mechanisms. Metabolic and signaling genes required for pathogenicity have been defined. Interaction with the host is dominated by interplay of fungal effectors that induce necrosis on wheat lines carrying specific sensitivity loci. As such, the pathogen has emerged as a model for the Pleosporales group of pathogens.

Recently, great strides have been made in the area of host-pathogen interactions involving necrotrophic fungi. In this article we describe a method to identify, produce, and characterize effectors that are important in host-necrotrophic fungal pathogen interactions, and to genetically characterize the interactions. The main strength of this method is the combined use of pathogen inoculation, a pathogen culture filtrate bioassay, and genetic analysis of susceptibility and sensitivity in segregating host-mapping populations. These methods have been successfully used to identify several Stagonospora nodorum necrotrophic effectors and to characterize the genetic and phenotypic effects of individual host-effector interactions in the wheat-S. nodorum system. S. nodorum isolates that induce a differential response on two lines are used to produce culture filtrates that contain necrotrophic effectors while the wheat lines differing in reaction to the pathogen are used to develop a mapping population. The wheat population is used to develop DNA marker-based genetic linkage maps and culture filtrates are infiltrated across the mapping population. Linkage and quantitative trait loci (QTL) analysis is used to identify regions of the wheat genome harboring genes that govern sensitivity to necrotrophic effectors. The same populations are inoculated with the effector-producing isolate to determine the significance and proportion of disease explained by individual host gene-effector interactions. Additionally, from this information, differential lines that are sensitive to single effectors are developed for further purification and characterization of the effectors, eventually resulting in the identification, molecular cloning, and characterization of the effector genes.

Necrotrophic effectors (also known as host-selective toxins) are important determinants of disease in the wheat-Stagonospora nodorum pathosystem. To date, five necrotrophic effector-host gene interactions have been identified in this system. Most of these interactions have additive effects while some are epistatic. The Snn4-SnTox4 interaction was originally identified in a recombinant-inbred population derived from a cross between the Swiss winter wheat cultivars 'Arina' and 'Forno' using the S. nodorum isolate Sn99CH 1A7a. Here, we used a recombinant-inbred population consisting of 121 lines developed from a cross between the hexaploid land race Salamouni and the hexaploid wheat 'Katepwa' (SK population). The SK population was used for the construction of linkage maps and quantitative trait loci (QTL) detection using the Swiss S. nodorum isolate Sn99CH 1A7a. The linkage maps developed in the SK population spanned 3,228 centimorgans (cM) and consisted of 441 simple-sequence repeats, 9 restriction fragment length polymorphisms, 29 expressed sequence tag sequence-tagged site markers, and 5 phenotypic markers. The average marker density was 6.7 cM/marker. Two QTL, designated QSnb.fcu-1A and QSnb.fcu-7A on chromosome arms 1AS and 7AS, respectively, were associated with disease caused by the S. nodorum isolate Sn99CH 1A7a. The effects of QSnb.fcu-1A were determined by the Snn4-SnTox4 interaction and accounted for 23.5% of the phenotypic variation in this population, whereas QSnb.fcu-7A accounted for 16.4% of the phenotypic variation for disease but was not associated with any known effector sensitivity locus. The effects of both QTL were largely additive and collectively accounted for 35.7% of the total phenotypic variation. The results of this research validate the effects of a compatible Snn4-SnTox4 interaction in a different genetic background, and it provides knowledge regarding genomic regions and molecular markers that can be used to improve Stagonospora nodorum blotch resistance in wheat germplasm.

Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola, is one of the most devastating foliar diseases of wheat. We screened five synthetic hexaploid wheats (SHs), 13 wheat varieties that represent the differential set of cultivars and two susceptible checks with a global set of 20 isolates and discovered exceptionally broad STB resistance in SHs. Subsequent development and analyses of recombinant inbred lines (RILs) from a cross between the SH M3 and the highly susceptible bread wheat cv. Kulm revealed two novel resistance loci on chromosomes 3D and 5A. The 3D resistance was expressed in the seedling and adult plant stages, and it controlled necrosis (N) and pycnidia (P) development as well as the latency periods of these parameters. This locus, which is closely linked to the microsatellite marker Xgwm494, was tentatively designated Stb16q and explained from 41 to 71% of the phenotypic variation at seedling stage and 28-31% in mature plants. The resistance locus on chromosome 5A was specifically expressed in the adult plant stage, associated with SSR marker Xhbg247, explained 12-32% of the variation in disease, was designated Stb17, and is the first unambiguously identified and named QTL for adult plant resistance to M. graminicola. Our results confirm that common wheat progenitors might be a rich source of new Stb resistance genes/QTLs that can be deployed in commercial breeding programs.

Genetic transformation is an essential tool for the modern study of gene function and the genetic improvement of an organism. The genetic transformation of many fungal species is well established and can be carried out by utilizing different transformation methods including electroporation, Agrobacterium, biolistics, or polyethylene glycol (PEG)-mediated transformation. Due to its technical simplicity and common equipment requirements, PEG-mediated transformation is still the most commonly used method for genetic transformation in filamentous fungi. Here, we describe a PEG-based protocol developed for genetic transformation of Stagonospora nodorum, a fungal pathogen of wheat. This protocol is directly applicable to other fungi especially those in the Dothideomycete class of fungi.

The wheat pathogen Stagonospora nodorum produces multiple necrotrophic effectors (also called host-selective toxins) that promote disease by interacting with corresponding host sensitivity gene products. SnTox1 was the first necrotrophic effector identified in S. nodorum, and was shown to induce necrosis on wheat lines carrying Snn1. Here, we report the molecular cloning and validation of SnTox1 as well as the preliminary characterization of the mechanism underlying the SnTox1-Snn1 interaction which leads to susceptibility. SnTox1 was identified using bioinformatics tools and verified by heterologous expression in Pichia pastoris. SnTox1 encodes a 117 amino acid protein with the first 17 amino acids predicted as a signal peptide, and strikingly, the mature protein contains 16 cysteine residues, a common feature for some avirulence effectors. The transformation of SnTox1 into an avirulent S. nodorum isolate was sufficient to make the strain pathogenic. Additionally, the deletion of SnTox1 in virulent isolates rendered the SnTox1 mutated strains avirulent on the Snn1 differential wheat line. SnTox1 was present in 85% of a global collection of S. nodorum isolates. We identified a total of 11 protein isoforms and found evidence for strong diversifying selection operating on SnTox1. The SnTox1-Snn1 interaction results in an oxidative burst, DNA laddering, and pathogenesis related (PR) gene expression, all hallmarks of a defense response. In the absence of light, the development of SnTox1-induced necrosis and disease symptoms were completely blocked. By comparing the infection processes of a GFP-tagged avirulent isolate and the same isolate transformed with SnTox1, we conclude that SnTox1 may play a critical role during fungal penetration. This research further demonstrates that necrotrophic fungal pathogens utilize small effector proteins to exploit plant resistance pathways for their colonization, which provides important insights into the molecular basis of the wheat-S. nodorum interaction, an emerging model for necrotrophic pathosystems.