Research Project: Host-Pathogen Interactions Affecting Wheat and Barley

Location: Cereal Crops Improvement Research

2025 Annual Report

Objectives
Objective 1. Functionally characterize the pathogen components of the Parastagonospora nodorum-wheat interaction. Sub-objective 1.A: Functionally characterize the role of SnTox5 in necrosis induction and colonization of the wheat leaf. Sub-objective 1.B: Characterize the role of SnTox267 in virulence using laser confocal microscopy. Objective 2. Characterize the infection strategies of Pyrenophora teres, the net blotch pathogen of barley. Objective 3. Identify, validate, and functionally characterize the Pyrenophora teres genes/proteins important in virulence on barley.

Approach
Fungal diseases pose an economic threat to plant crops throughout the world resulting in billions of dollars in losses annually. A significant amount of work has been done to understand biotrophic host-pathogen interactions. However, less progress has been made to understand the relationship between plants and their necrotrophic pathogens. Here we focus on understanding how the necrotrophic pathogens Pyrenophora teres, causal agent of net blotch of barley, and Parastagonospora nodorum, causal agent of septoria nodorum blotch of wheat, manipulate the host defense to allow pathogen colonization that results in disease. In previous work, we functionally validated several genes/proteins in P. nodorum that contributed to the pathogen’s infection strategy. Here, we will functionally characterize these effectors using modern tools. CRISPR-Cas9-based gene editing will be used to characterize regions of the proteins involved in virulence function, laser confocal microscopy will be used to visualize the role and mode of action of these effector proteins in planta, and comparative transcriptome sequencing of in planta effector infiltrations and inoculations will be used to characterize the host response to these effectors. To characterize the P. teres f. teres-barley interaction, we will identify and validate candidate effector genes conferring virulence on susceptible barley. Candidate genes will be identified and validated using both P. teres biparental and globally collected natural populations that differ in virulence. QTL analysis and genome wide association study (GWAS) analysis will be used to locate candidate genomic regions harboring effector genes. Once effector genes have been validated using CRISPR-based gene disruption, strains with and without the effector genes will be used in inoculation experiments to characterize the mode of action of each effector, ultimately resulting in the understanding of the infection strategy of the pathogen. This work will provide critical knowledge to breeders and other researchers targeting the control of these important diseases.

Progress Report
This report documents progress for Project Number 3060-22000-051-000D, entitled “Host-Pathogen Interactions Affecting Wheat and Barley”. Septoria nodorum blotch (SNB) on wheat, net form net blotch (NFNB) and spot form net blotch (SFNB) on barley, are among the most devastating foliar diseases affecting cereal crops. These pathogens can lead to yield losses of up to 50% in susceptible varieties, both in the United States and globally, unless effective control strategies are implemented. This project focuses on characterizing pathogen virulence for each disease. We have conducted genetic characterization of pathogen virulence, identified and validated key genes contributing to this process, and explored the mode of action of the proteins encoded by these genes. Additionally, we have collaborated with partners investigating the role of host plants in these complex interactions. Progress on Objective 1. Functionally characterize the pathogen components of the Parastagonospora nodorum-wheat interaction. Characterization of the P. nodorum-wheat interaction - P. nodorum is a necrotrophic fungal pathogen that induces plant cell death, but rather than stopping the pathogen, the pathogen exploits this cell death process to provide nutrients for pathogen growth and sporulation. SnTox267 is a necrotrophic effector produced by the pathogen that induces programmed cell death (PCD). We compared wild-type P. nodorum isolate Sn4 to the SnTox267-disrupted strain Sn4 SnTox267. As expected, macroscopically, the Sn4 wild-type isolate had significantly larger lesions. Additionally, we compared the infection process of these same strains using laser confocal microscopy. Microscopy showed that the strain with the SnTox267 gene-disruption rarely colonized past the epidermal layer of the leaf, whereas the wild-type strain harboring SnTox267 colonized the epidermis within 24 hours post inoculation (hpi) and progressively colonized the mesophyll layer starting at 48 hpi and continuing through 120 hpi. This preliminary data indicated that SnTox267 is important for complete colonization of the leaf. 3,3'-Diaminobenzidine (DAB) staining is used to visualize the production of hydrogen peroxide, a reactive oxygen species (ROS) that plants produce to defend against pathogens. Necrotrophic pathogens can modulate and may even exploit the oxidative burst (production of ROS) to induce PCD to gain cellular nutrients. On susceptible wheat lines, hydrogen peroxide production was first visualized at 72 hours post inoculation (hpi) when inoculated with the wild-type isolate, Sn4. Susceptible wheat lines inoculated with the mutant strain, Sn4 Tox267, never showed the presence of hydrogen peroxide. The differential presence of hydrogen peroxide in the Sn4 versus Sn4 tox267 shows that SnTox267 plays a role in inducing plant defense response through ROS, which is a benefit to P. nodorum’s necrotrophic lifestyle. Progress on Objective 2. Characterize the infection strategies of Pyrenophora teres, the net blotch pathogen of barley. Pyrenophora teres f. teres and P. teres f. maculata are pathogens that cause diseases in barley across the globe. While some progress has been made in understanding host resistance, there is a substantial knowledge gap in our understanding of the commonly used differential barley lines and their interactions with these pathogens. Moreover, the virulence mechanisms of P. teres are still poorly understood. This study aimed to elucidate the mechanisms by which the pathogen colonizes barley and to explore the host plant’s defensive responses. To understand this host-pathogen interaction, we must first understand the genetics of host resistance/susceptibility and pathogen virulence. Genetics of resistance/susceptibility to barley net form net blotch – A set of more than 20 differential lines has commonly been used to evaluate P. teres f. teres populations across the globe. We used four of these differential lines, Beecher, Harbin, Atlas, and CI11458, to develop two recombinant inbred line (RIL) mapping populations (Beecher × Harbin and Atlas × CI11458) to genetically characterize these sources of resistance using a globally collected set of P. teres f. teres isolates. These two populations were genotyped using the barley 50K single nucleotide polymorphism (SNP) chip and mapped for use in identifying barley genomic loci associated with resistance/susceptibility. SNP markers saturated all seven barley chromosomes, resulting in high-quality maps for use in quantitative trait loci (QTL) analysis. Preliminary data showed two major loci associated with resistance/susceptibility. Further genetic characterization with a diverse set of isolates is underway. Genetics of resistance/susceptibility to barley spot form net blotch - Two recombinant inbred line (RIL) barley populations, TR326 × PI67381 and PI392501 × PI67381, were developed. TR326 and PI392501 have been used as differential lines for evaluating P. teres f. maculata virulence globally. We previously selected PI67381 from the barley core collection as a consistent source of resistance after testing with a global collection of P. teres f. maculata isolates. The TR326 × PI67381 and PI392501 × PI67381 populations were genotyped using the barley 50K SNP chip and subsequently mapped, generating saturated maps of all seven barley chromosomes. These populations, along with the previously mapped Hocket × PI67381, were phenotyped using isolates collected from barley growing regions on five continents. QTL analysis was conducted to identify barley genomic regions associated with resistance/susceptibility. Associations with a known susceptibility locus on chromosomes (Chr) 2H and 7H were consistently present in the Hocket × PI67381and TR326 × PI67381 populations. Significant loci were also identified on Chr 4H (TR326 × PI67381 and PI392501 × PI67381) and 6H (Hocket × PI67381) for some isolates; however, these loci were relatively minor compared to Chr 2H and 7H. PI392501 harbored a 7H and 4H susceptibility; however, the 2H susceptibility locus was not present in PI392501, indicating resistance (lack of susceptibility) at this locus. Further work is underway to fully characterize these barley populations and their susceptibility loci. Genetics of virulence in the spot form net blotch pathogen – A P. teres f. maculata pathogen mapping population was developed from a cross of a virulent isolate collected from cultivated barley in Montana, U.S., and an avirulent isolate collected from wild barley in California, U.S. This population was previously used to characterize virulence on barley line Hockett, showing that virulence genes localized to Chr 1 and 2 were targeting the susceptibility genes identified on barley Chr 2H and 7H, respectively. In the current work, we evaluated the same P. teres f. maculata population using differential barley lines TR326 and PI392501. Preliminary data showed that P. teres f. maculata Chr 2 was associated with virulence on TR326, but Chr 3 was also significant, indicating that P. teres f. maculata likely targets an additional susceptibility gene, possibly the loci on Chr 4H or 6H identified above. Data collected on PI392501 also did not show an association with Chr 1 but did show an association with the Chr 2 locus as well as an association on Chr 3. Collectively, this work indicates that at least three virulence genes segregate in this population. This work begins to lay a foundation for a complete understanding of this host-pathogen interaction, leading to the SFNB disease on barley. Progress on Objective 3. Identify, validate, and functionally characterize the Pyrenophora teres genes/proteins important in virulence on barley. To fill a gap in our understanding of Pyrenophora teres f. teres and P. teres f. maculata virulence, we have worked toward the cloning and functional characterization of virulence genes underlying QTL using both P. teres f. teres and P. teres f. maculata mapping populations. Characterization of P. teres f. teres virulence genes causing net form net blotch (NFNB) on barley - Barley line CI5791 is a source of resistance for NFNB that has been highly effective and durable worldwide. Recently, we identified several isolates, including one named MorSM40-3 collected in Morocco, that overcame this important source of resistance. A P. teres f. teres population segregating for virulence on CI5791 was developed from a cross between MorSM40-3 and the CI5791-avirulent isolate 0-1. Genome assemblies of P. teres f. teres isolates 0-1 and MorSM40-3, along with Illumina RNA-seq data, were used to produce annotations of novel effector candidates in a MorSM40-3 × 0-1 Chr 1 QTL region associated with virulence. We disrupted a candidate virulence gene on Chr 1 in MorSM40-3. Disruption mutants became avirulent on CI5791. Mutants were then screened on the CI5791 × Tifang barley population that segregated for resistance associated with Chr 1 virulence, and the resulting disease data were used for QTL analysis. An association with resistance was identified on barley Chr 6H coming from CI5791, indicating that this effector gene overcomes the 6H resistance, resulting in disease on CI5791. Genetic characterization of virulence of P. teres f. maculata causal agent of spot form net blotch of barley - To identify virulence factors (effector proteins) used by the pathogen to infect barley, we evaluated a P. teres f. maculata mapping population developed from a virulent isolate crossed with an avirulent isolate. Progeny were inoculated on several barley lines to identify genetic loci associated with virulence on P. teres f. maculata Chr 1, 2, and 3. A candidate gene on Chr 1 was disrupted to characterize its impact on virulence. Preliminary data have shown that this gene is responsible for virulence, targeting a susceptibility gene on barley Chr 2H. Further validation is underway.

Accomplishments
1. Pyrenophora teres f. maculata, a pathogen of barley, is an emerging pathogen of durum wheat. As plant pathogens continue to evolve, they pose growing risks to global food security by infecting new hosts and expanding to new regions. P. teres f. maculata, a foliar pathogen found worldwide, can cause yield losses in barley of up to 40%. Recent findings indicate P. teres f. maculata has jumped to wheat, one of the world’s most essential food crops. To assess this risk, researchers at the USDA Agricultural Research Service (ARS) in Fargo, North Dakota, evaluated a diverse global panel of durum wheat for susceptibility. Researchers also used fungal isolates collected from different barley-producing regions to evaluate a biparental mapping population, created by crossing a widely grown local durum variety that is susceptible to the disease with a resistant line. Both studies identified a major genetic locus on Chr 2A linked to disease susceptibility. These findings confirm P. teres f. maculata as an emerging pathogen of durum wheat and pinpoint key genomic regions that may help in breeding resistant wheat varieties. This knowledge can help stakeholders develop improved wheat varieties, boosting productivity and increasing profitability for growers.

Review Publications
Nelson, A., Kariyawasam, G., Wyatt, N.A., Li, J., Haueisen, J., Stukenbrock, E., Borowicz, P., Friesen, T.L. 2024. Assembly and evaluation of a confocal microscopy image analysis pipeline useful in revealing the secrets of plant-fungal interactions. Molecular Plant-Microbe Interactions. 37(12):804-813. https://doi.org/10.1094/MPMI-08-24-0090-TA.
Klindworth, D.L., Saini Sharma, J., Faris, J.D., Friesen, T.L., Peters Haugrud, A.R., Xu, S.S. 2025. Identification of stem rust resistance genes in monogenic lines derived from wheat cultivar Waldron. Crop Science. https://doi.org/10.1002/csc2.70010.
Seneviratne, S., Shi, G., Szabo-Hever, A., Zhang, Z., Peters Haugrud, A.R., Running, K., Singh, G., Nandety, R.S., Fiedler, J.D., Mcclean, P., Xu, S.S., Friesen, T.L., Faris, J.D. 2024. Evolution, diversity, and function of the disease susceptibility gene Snn1 in wheat. The Plant Journal. 119(4):1720-1736. https://doi.org/10.1111/tpj.16879.
Szabo-Hever, A., Running, K., Seneviratne, S., Singh, G., Zhang, Z., Peters Haugrud, A.R., Maccaferri, M., Tuberosa, R., Friesen, T.L., Xu, S.S., Faris, J.D. 2025. Evaluation of durum and hard red spring wheat panels for sensitivity to necrotrophic effectors produced by Parastagonospora nodorum. Plant Disease. 109(4):851-861. https://doi.org/10.1094/PDIS-05-24-0990-RE.
Zhang, Z., Running, K., Seneviratne, S., Peters Haugrud, A.R., Szabo-Hever, A., Singh, G., Holusova, K., Molnar, I., Dolezel, J., Friesen, T.L., Faris, J.D. 2025. Protein kinase-major sperm protein (PK-MSP) genes mediate recognition of the fungal necrotrophic effector SnTox3 to cause septoria nodorum blotch in wheat. Molecular Plant-Microbe Interactions. 38(2):315-327. https://doi.org/10.1094/MPMI-10-24-0125-FI.
Klindworth, D.L., Rouse, M.N., Olivera, P., Jin, Y., Chu, C.N., Friesen, T.L., Zhong, S., Faris, J.D., Fiedler, J.D., Peters Haugrud, A.R., Gu, Y.Q., Elias, E.M., Liu, S., Cai, X., Xu, S.S. 2024. Registration of four durum wheat lines carrying Sr13 alleles for resistance to stem rust. Journal of Plant Registrations. 19(1). Article e20399. https://doi.org/10.1002/plr2.20399.
Guo, J., Shi, G., Islam, M., Kariyawasam, G., Moolhuijzen, P., See, P., Zhong, S., Aboukhaddour, R., Faris, J.D., Friesen, T.L., Liu, Z. 2025. Identification of a novel genetic locus conferring virulence in the wheat tan spot pathogen Pyrenophora tritici-repentis. Fungal Genetics and Biology. https://doi.org/10.1016/j.fgb.2025.104002.
Wyatt, N.A., Skiba, R.M., Peters Haugrud, A.R., Zhang, Q., Szabo-Hever, A., Xu, S.S., Faris, J.D., Friesen, T.L. 2025. Pyrenophora teres f. maculata, causal agent of spot form net blotch of barley, is an emerging pathogen of durum wheat. Phytopathology. https://doi.org/10.1094/PHYTO-01-25-0002-R.
Lhamo, D., Sun, Q., Friesen, T.L., Karmacharya, A., Li, X., Fiedler, J.D., Faris, J.D., Xia, G., Luo, M., Gu, Y.Q., Liu, Z., Xu, S.S. 2024. Association mapping of tan spot and septoria nodorum blotch resistance in cultivated emmer wheat. Theoretical and Applied Genetics. 137. Article 193. https://doi.org/10.1007/s00122-024-04700-2.