Emergence and spread of the barley net blotch pathogen coincided with crop domestication and cultivation history.

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.

Evolution, diversity, and function of the disease susceptibility gene Snn1 in wheat.

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.

Assembly and evaluation of a confocal microscopy image analysis pipeline useful in revealing the secrets of plant-fungal interactions.

The ability of laser scanning confocal microscopy to generate high-contrast 2D and 3D images has become essential in studying plant-fungal interactions. Techniques such as visualization of native fluorescence, fluorescent protein tagging of microbes, GFP/RFP-fusion proteins, and fluorescent labelling of plant and fungal proteins have been widely used to aid in these investigations. Use of fluorescent proteins has several pitfalls including variability of expression in planta and the requirement of gene transformation. Here we used the unlabeled pathogens Parastagonospora nodorum, Pyrenophora teres f. teres, and Cercospora beticola infecting wheat, barley, and sugar beet respectively, to show the utility of a staining and imaging pipeline that uses propidium iodide (PI), which stains RNA and DNA, and wheat germ agglutinin labeled with fluorescein isothiocyanate (WGA-FITC), which stains chitin, to visualize fungal colonization of plants. This pipeline relies on the use of KOH to remove the cutin layer of the leaf, increasing its permeability, allowing the different stains to penetrate and effectively bind to their targets, resulting in a consistent visualization of cellular structures. To expand the utility of this pipeline, we used the staining techniques in conjunction with machine learning to analyze fungal biomass through volume analysis, as well as quantifying nuclear breakdown, an early indicator of programmed cell death (PCD). This pipeline is simple to use, robust, consistent across host and fungal species and can be applied to most plant-fungal interactions. Therefore, this pipeline can be used to characterize model systems as well as non-model interactions where transformation is not routine.

Variability in Chromosome 1 of Select Moroccan Pyrenophora teres f. teres Isolates Overcomes a Highly Effective Barley Chromosome 6H Source of Resistance.

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.

Association mapping of tan spot and septoria nodorum blotch resistance in cultivated emmer wheat.

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.

A Moroccan Pyrenophora teres f. teres Population Defeats Rpt5, the Broadly Effective Resistance on Barley Chromosome 6H.

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. F2 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 F2 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.

Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability.

Disease lesion mimic mutants (DLMMs) are characterized by the spontaneous development of necrotic spots with various phenotypes designated as necrotic (nec) mutants in barley. The nec mutants were traditionally considered to have aberrant regulation of programmed cell death (PCD) pathways, which have roles in plant immunity and development. Most barley nec3 mutants express cream to orange necrotic lesions contrasting them from typical spontaneous DLMMs that develop dark pigmented lesions indicative of serotonin/phenolics deposition. Barley nec3 mutants grown under sterile conditions did not exhibit necrotic phenotypes until inoculated with adapted pathogens, suggesting that they are not typical DLMMs. The F2 progeny of a cross between nec3-γ1 and variety Quest segregated as a single recessive susceptibility gene post-inoculation with Bipolaris sorokiniana, the causal agent of the disease spot blotch. Nec3 was genetically delimited to 0.14 cM representing 16.5 megabases of physical sequence containing 149 annotated high confidence genes. RNAseq and comparative analysis of the wild type and five independent nec3 mutants identified a single candidate cytochrome P450 gene (HORVU.MOREX.r2.6HG0460850) that was validated as nec3 by independent mutations that result in predicted nonfunctional proteins. Histology studies determined that nec3 mutants had an unstable cutin layer that disrupted normal Bipolaris sorokiniana germ tube development.

Evaluation of durum and hard red spring wheat panels for sensitivity to necrotrophic effectors produced by Parastagonospora nodorum.

Septoria nodorum blotch is an important disease of both durum and hard red spring wheat (HRSW) worldwide. The disease is caused by the necrotrophic fungal pathogen Parastagonospora nodorum when compatible gene-for-gene interactions occur between pathogen-produced necrotrophic effectors (NEs) and corresponding host sensitivity genes. To date, nine sensitivity gene-NE interactions have been identified, but there is little information available regarding their overall frequency in durum and HRSW. Here, we infiltrated a global HRSW panel (HRSWP) and the Global Durum Panel (GDP) with P. nodorum NEs SnToxA, SnTox1, SnTox267, SnTox3, and SnTox5. Frequencies of sensitivity to SnTox1 and SnTox5 were higher in durum compared to HRSW and vice versa for SnTox267 and SnTox3. Strong associations for the known sensitivity loci Tsn1, Snn1, Snn2, Snn3, Snn5, and Snn7 along with potentially novel sensitivity loci on chromosome arms 7DS and 3BL associated with SnToxA and SnTox267, respectively, were identified in the HRSWP. In the GDP, Snn1, Snn3, and Snn5 were identified along with novel loci associated with sensitivity to SnTox267 on chromosome arms 2AS, 2AL, and 6AS and with SnTox5 sensitivity on 2BS and 7BL. These results reveal additional NE sensitivity loci beyond those previously described demonstrating a higher level of genetic complexity of the wheat-P. nodorum system than previously thought. Knowledge regarding the prevalence and genomic locations of SNB susceptibility genes in HRSW and durum will prove useful for developing efficient breeding strategies and improving varieties for SNB resistance.

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

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

Genetic and physical localization of a major susceptibility gene to Pyrenophora teres f. maculata in barley.

KEY MESSAGE: Genetic characterization of a major spot form net blotch susceptibility locus to using linkage mapping to identify a candidate gene and user-friendly markers in barley. Spot form net blotch (SFNB), caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm), is an economically important foliar diseases in barley. Although various resistance loci have been identified, breeding for SFNB-resistant varieties has been hampered due to the complex virulence profile of Ptm populations. One resistance locus in the host may be effective against one specific isolate, but it may confer susceptibility to other isolates. A major susceptibility QTL on chromosome 7H, named Sptm1, was consistently identified in many studies. In the present study, we conduct fine mapping to localize Sptm1 with high resolution. A segregating population was developed from selected F2 progenies of the cross Tradition (S) × PI 67381 (R), in which the disease phenotype was determined by the Sptm1 locus alone. Disease phenotypes of critical recombinants were confirmed in the following two consecutive generations. Genetic mapping anchored the Sptm1 gene to an ⁓400 kb region on chromosome 7H. Gene prediction and annotation identified six protein-coding genes in the delimited Sptm1 region, and the gene encoding a putative cold-responsive protein kinase was selected as a strong candidate. Therefore, providing fine localization and candidate of Sptm1 for functional validation, our study will facilitate the understanding of susceptibility mechanism underlying the barley-Ptm interaction and offers a potential target for gene editing to develop valuable materials with broad-spectrum resistance to SFNB.

Genome-wide association mapping of resistance to the foliar diseases septoria nodorum blotch and tan spot in a global winter wheat collection.

Septoria nodorum blotch (SNB) and tan spot, caused by the necrotrophic fungal pathogens Parastagonospora nodorum and Pyrenophora tritici-repentis, respectively, often occur together as a leaf spotting disease complex on wheat (Triticum aestivum L.). Both pathogens produce necrotrophic effectors (NEs) that contribute to the development of disease. Here, genome-wide association analysis of a diverse panel of 264 winter wheat lines revealed novel loci on chromosomes 5A and 5B associated with sensitivity to the NEs SnTox3 and SnTox5 in addition to the known sensitivity genes for NEs Ptr/SnToxA, SnTox1, SnTox3, and SnTox5. Sensitivity loci for SnTox267 and Ptr ToxB were not detected. Evaluation of the panel with five P. nodorum isolates for SNB development indicated the Snn3-SnTox3 and Tsn1-SnToxA interactions played significant roles in disease development along with additional QTL on chromosomes 2A and 2D, which may correspond to the Snn7-SnTox267 interaction. For tan spot, the Tsc1-Ptr ToxC interaction was associated with disease caused by two isolates, and a novel QTL on chromosome 7D was associated with a third isolate. The Tsn1-ToxA interaction was associated with SNB but not tan spot. Therefore some, but not all, of the previously characterized host gene-NE interactions in these pathosystems play significant roles in disease development in winter wheat. Based on these results, breeders should prioritize the selection of resistance alleles at the Tsc1, Tsn1, Snn3, and Snn7 loci as well as the 2A and 7D QTL to obtain good levels of resistance to SNB and tan spot in winter wheat. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01400-5.

A Revised Nomenclature for ToxA Haplotypes Across Multiple Fungal Species.

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

Association Mapping of Resistance to Tan Spot in the Global Durum Panel.

Tan spot, caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr), is an important disease of durum and common wheat worldwide. Compared with common wheat, less is known about the genetics and molecular basis of tan spot resistance in durum wheat. We evaluated 510 durum lines from the Global Durum Wheat Panel (GDP) for sensitivity to the necrotrophic effectors (NEs) Ptr ToxA and Ptr ToxB and for reaction to Ptr isolates representing races 1 to 5. Overall, susceptible durum lines were most prevalent in South Asia, the Middle East, and North Africa. Genome-wide association analysis showed that the resistance locus Tsr7 was significantly associated with tan spot caused by races 2 and 3, but not races 1, 4, or 5. The NE sensitivity genes Tsc1 and Tsc2 were associated with susceptibility to Ptr ToxC- and Ptr ToxB-producing isolates, respectively, but Tsn1 was not associated with tan spot caused by Ptr ToxA-producing isolates, which further validates that the Tsn1-Ptr ToxA interaction does not play a significant role in tan spot development in durum. A unique locus on chromosome arm 2AS was associated with tan spot caused by race 4, a race once considered avirulent. A novel trait characterized by expanding chlorosis leading to increased disease severity caused by the Ptr ToxB-producing race 5 isolate DW5 was identified, and this trait was governed by a locus on chromosome 5B. We recommend that durum breeders select resistance alleles at the Tsr7, Tsc1, Tsc2, and the chromosome 2AS loci to obtain broad resistance to tan spot.

A protein kinase-major sperm protein gene hijacked by a necrotrophic fungal pathogen triggers disease susceptibility in wheat.

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.

Function and evolution of allelic variations of Sr13 conferring resistance to stem rust in tetraploid wheat (Triticum turgidum L.).

The resistance gene Sr13 is one of the most important genes in durum wheat for controlling stem rust caused by Puccinia graminis f. sp. tritici (Pgt). The Sr13 functional gene CNL13 has haplotypes R1, R2 and R3. The R1/R3 and R2 haplotypes were originally designated as alleles Sr13a and Sr13b, respectively. To detect additional Sr13 alleles, we developed Kompetitive allele specific PCR (KASP™) marker KASPSr13 and four semi-thermal asymmetric reverse PCR markers, rwgsnp37-rwgsnp40, based on the CNL13 sequence. These markers were shown to detect R1, R2 and R3 haplotypes in a panel of diverse tetraploid wheat accessions. We also observed the presence of Sr13 in durum line CAT-A1, although it lacked any of the known haplotypes. Sequence analysis revealed that CNL13 of CAT-A1 differed from the susceptible haplotype S1 by a single nucleotide (C2200T) in the leucine-rich repeat region and differed from the other three R haplotypes by one or two additional nucleotides, confirming that CAT-A1 carries a new (R4) haplotype. Stem rust tests on the monogenic, transgenic and mutant lines showed that R1 differed from R3 in its susceptibility to races TCMJC and THTSC, whereas R4 differed from all other haplotypes for susceptibility to TTKSK, TPPKC and TCCJC. Based on these differences, we designate the R1, R3 and R4 haplotypes as alleles Sr13a, Sr13c and Sr13d, respectively. This study indicates that Sr13d may be the primitive functional allele originating from the S1 haplotype via a point mutation, with the other three R alleles probably being derived from Sr13d through one or two additional point mutations.

A Conserved Hypothetical Gene Is Required but Not Sufficient for Ptr ToxC Production in Pyrenophora tritici-repentis.

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.

Association mapping reveals a reciprocal virulence/avirulence locus within diverse US Pyrenophora teres f. maculata isolates.

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.

A triple threat: the Parastagonospora nodorum SnTox267 effector exploits three distinct host genetic factors to cause disease in wheat.

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.

The Parastagonospora nodorum necrotrophic effector SnTox5 targets the wheat gene Snn5 and facilitates entry into the leaf mesophyll.

Parastagonospora nodorum is an economically important necrotrophic fungal pathogen of wheat. Parastagonospora nodorum secretes necrotrophic effectors that target wheat susceptibility genes to induce programmed cell death (PCD). In this study, we cloned and functionally validated SnTox5 and characterized its role in pathogenesis. We used whole genome sequencing, genome-wide association study (GWAS) mapping, CRISPR-Cas9-based gene disruption, gain-of-function transformation, quantitative trait locus (QTL) analysis, haplotype and isoform analysis, protein modeling, quantitative PCR, and laser confocal microscopy to validate SnTox5 and functionally characterize SnTox5. SnTox5 is a mature 16.26 kDa protein with high structural similarity to SnTox3. Wild-type and mutant P. nodorum strains and wheat genotypes of SnTox5 and Snn5, respectively, were used to show that SnTox5 not only targets Snn5 to induce PCD but also facilitates the colonization of the mesophyll layer even in the absence of Snn5. Here we show that SnTox5 facilitates the efficient colonization of the mesophyll tissue and elicits PCD specific to host lines carrying Snn5. The homology to SnTox3 and the ability of SnTox5 to facilitate the colonizing of the mesophyll also suggest a role in the suppression of host defense before PCD induction.

Genetics of resistance to septoria nodorum blotch in wheat.

Septoria nodorum blotch (SNB) is a foliar disease of wheat caused by the necrotrophic fungal pathogen Parastagonospora nodorum. Research over the last two decades has shown that the wheat-P. nodorum pathosystem mostly follows an inverse gene-for-gene model. The fungus produces necrotrophic effectors (NEs) that interact with specific host gene products encoded by dominant sensitivity (S) genes. When a compatible interaction occurs, a 'defense response' in the host leads to programmed cell death thereby provided dead/dying cells from which the pathogen, being a necrotroph, can acquire nutrients allowing it to grow and sporulate. To date, nine S gene-NE interactions have been characterized in this pathosystem. Five NE-encoding genes, SnTox1, SnTox3, SnToxA, SnTox5, and SnTox267, have been cloned along with three host S genes, Tsn1, Snn1, and Snn3-D1. Studies have shown that P. nodorum hijacks multiple and diverse host targets to cause disease. SNB resistance is often quantitative in nature because multiple compatible interactions usually occur concomitantly. NE gene expression plays a key role in disease severity, and the effect of each compatible interaction can vary depending on the other existing compatible interactions. Numerous SNB-resistance QTL have been identified in addition to the known S genes, and more research is needed to understand the nature of these resistance loci. Marker-assisted elimination of S genes through conventional breeding practices and disruption of S genes using gene editing techniques are both effective strategies for the development of SNB-resistant wheat cultivars, which will become necessary as the global demand for sustenance grows.