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ARS Home Plains Area Fargo, North Dakota Edward T. Schafer Agricultural Research Center Cereal Crops Improvement Research Research





- Friesen, Timothy
- 2023
- 2022
- Publications
- Plant Diseases (303)

Research Project: Host-Pathogen Interactions Affecting Wheat and Barley **Location:
                            Cereal Crops Improvement Research** 2023 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) (formerly Stagonospora nodorum blotch) on wheat and net blotch on barley and are two of the most destructive leaf diseases of cereals, both in the United States and worldwide. In this project we have focused on elucidating the mechanisms of virulence for each pathogen by genetically characterizing the virulence of the pathogen, identifying and validating the genes contributing to virulence, and investigating the mode of action of the corresponding proteins encoded by these genes. We have worked closely with collaborators focused on the host plant's involvement in these interactions.
Progress on Objective 1. Functionally characterize the pathogen components of the Parastagonospora nodorum-wheat interaction. P. nodorum is a necrotrophic fungal pathogen that induces the host defense response resulting in host cell death, but instead of stopping the pathogen, this process provides nutrients for pathogen growth.  SnTox5 is a necrotrophic effector that is known to induce cell death to the advantage of the pathogen. The mode of action of SnTox5 was evaluated using a gene expression (RNAseq) approach where both host (wheat) and pathogen (P. nodorum) gene expression was compared when a susceptible wheat line was inoculated with an SnTox5 producing isolate side by side with a strain that had a disrupted SnTox5 gene. Gene expression results showed that SnTox5 was involved in the manipulation of the host defense response through the upregulation of several classes of defense response genes that ultimately resulted in cell death. Pathogen gene expression analysis showed several proteins predicted to be involved in virulence were significantly upregulated in the SnTox5 expressing strain relative to the SnTox5 disrupted strain. These effector-like proteins are predicted to be involved in host manipulation as well as protection of the pathogen during colonization of the host.
SnTox267 produced by P. nodorum was previously shown to target at least two host pathways to induce necrosis. A new staining technique was used along with laser confocal microscopy to look at the impact of SnTox267 on colonization. Identifying the impact of SnTox267 was accomplished by inoculating wheat lines sensitive or insensitive to SnTox267 with P. nodorum isolates harboring SnTox267 side by side with SnTox267 disrupted strains. Results showed that SnTox267 strains colonized faster and built significantly more biomass than the SnTox267 disrupted strains. Additionally, SnTox267 disrupted strains failed to colonize the mesophyll (middle) layer of the leaf in both sensitive and insensitive wheat lines, whereas P. nodorum strains expressing SnTox267 were able to colonize the mesophyll layer in wheat lines sensitive or insensitive to SnTox267. This result indicates that SnTox267 is somehow involved in mesophyll colonization, even in the absence of the host sensitivity/susceptibility targets.
Progress on Objective 2. Characterize the infection strategies of Pyrenophora teres, the net blotch pathogen of barley. Using confocal microscopy, we have begun to characterize the infection strategy of P. teres f. teres, causal agent of net form net blotch. A virulent pathogen initially penetrates the epidermal cell and creates invasive hyphae (feeding structure) by 24 hours post-inoculations. Once this penetration takes place, the pathogen exits the epidermal cell and colonizes the mesophyll (middle) layer of the leaf by staying in the intercellular space. The pathogen moves horizontally toward the tip and base of the leaf in front of any cell death (necrosis) followed by lateral branching, surrounding as many mesophyll cells as possible, positioning itself to obtain the cellular nutrients to complete its pathogenic life cycle once programmed cell death (PCD) is initiated. Once the pathogen has colonized a host area (around 72 hours post inoculation), necrosis is induced resulting in cell collapse, making cellular nutrients available for the pathogen. By 96 hours, the pathogen has completely colonized the leaf and cell death continues behind the colonizing pathogen. By 120 hours post inoculation, the pathogen has completely colonized the host, cell death has spread across the leaf and the pathogen is preparing itself for sporulation to move by wind or splash dispersal to new uninfected leaves. This information is useful in designing control strategies for this pathogen.
During this study we have also developed a new staining technique that can be applied to any plant fungal interaction (see Accomplishments). This technique uses multiple stains that target components of the host and pathogen to differentiate the two. These include stains that target chitin in the fungal cell wall, a stain that targets DNA/RNA resulting in the ability to visualize the host nucleus and cytoplasm. Additionally, autofluorescence of plant cell components allow the visualization of the plant cell as the background for infection. This staining technique improves on our previous method of transforming the pathogen with green or red fluorescent proteins to visualize the pathogen because now we can visualize any fungal pathogen without the need for transformation. This method is already being used by other labs working with other plant fungal interactions. 
Progress on Objective 3.  Identify, validate, and functionally characterize the Pyrenophora teres genes/proteins important in virulence on barley. The genes VR1 and VR2 were each identified to confer virulence on the barley like Rika. These genes were validated using gene disruption in the virulent isolate 6A, an isolate carrying the virulent allele of both VR1 and VR2. As an additional validation step, allele swaps were done in an avirulent isolate where the avirulent form of the VR1 or VR2 gene was replaced with the virulent (6A form) to show that addition of the 6A allele at either the VR1 or VR2 locus made the avirulent isolate virulent, proving that VR1 and VR2 independently cause virulence. Additionally, the same avirulent isolate was used to edit both VR1 and VR2 to the virulent form. This strain was even more virulent than the single virulence strains, indicating that VR1 and VR2 act synergistically to colonize the host. See Accomplishments 1 for a more thorough description of this work. **Accomplishments**
                                

1. The net form net blotch barley pathogen uses effector proteins to induce disease. Net form net blotch, caused by the fungal pathogen Pyrenophora teres f. teres, is a major leaf disease of barley in the U.S. and globally. Successful pathogens adapt to locally planted cultivars through a change and fine tuning of virulence. Unfortunately, researchers lack a firm understanding of how P. teres f. teres uses virulence (effector) genes to manipulate barley, resulting in net form net blotch disease. ARS researchers in Fargo, North Dakota, identified, validated, and functionally characterized two effector genes that target barley to cause net form net blotch disease.  Understanding how this necrotrophic pathogen is manipulating its host is critical to disease control as well as helping us make advances on the host side where geneticists and breeders can work to remove the susceptibility genes targeted by these effectors. 2. Discovery of Xanthomonas translucens isolates that lack protease activity. Bacterial leaf streak of wheat and barley is a global disease of growing economic concern caused by the bacterium Xanthomonas translucens. ARS researchers in Fargo, North Dakota, found that inoculation of plants with bacterial isolates that lack protease activity resulted in reduced severity of disease symptoms compared to isolates that have protease activity. The complete genome sequencing of representative isolates showed that this protease appears to be differentially regulated in different pathogen genetic backgrounds. Understanding the contribution of extracellular protease to virulence and the mechanism of its expression is important to the development of control strategies and development of disease resistant varieties. 3. Improved visualization of plant fungal interactions. Laser scanning confocal microscopy’s ability to generate high-contrast 3D images has become essential to studying plant-microbe interactions. ARS researchers in Fargo, North Dakota, used wheat, barley, and sugar beet pathogens to show the utility of a staining and imaging technique that uses readily available stains that target the fungal cell wall and DNA/RNA to visualize the plant nucleus and cytoplasm. This technique is simple and robust and can be applied to any plant-fungal interaction. Therefore, this technique can be used by researchers to characterize model and non-model systems to better understand how fungal pathogens interact with their plant host. **Review Publications**
                            *Aboukhaddour, R., Hafez, M., Mcdonald, M., Moffat, C., Navathe, S., Friesen, T.L., Strelkov, S., Oliver, R., Tan, K., Liu, Z., Moolhuijzen, P., Phan, H., See, P. 2023. A revised nomenclature for ToxA haplotypes across multiple fungal species. Phytopathology. https://doi.org/10.1094/PHYTO-01-23-0017-SC.*
                                *Yuzon, J.D., Wyatt, N.A., Vasighzadeh, A., Clare, S., Navratil, E.M., Friesen, T.L., Stukenbrock, E.H. 2023. Hybrid inferiority and genetic incompatibilities drive divergence of fungal pathogens infecting the same host. Genetics. https://doi.org/10.1093/genetics/iyad037.*
                                *Richards, J., Li, J., Koladia, V., Wyatt, N.A., Reham, S., Brueggemann, R., Friesen, T.L. 2023. A Moroccan Pyrenophora teres f. teres population defeats the Rpt5 broad-spectrum resistance on barley chromosome 6H. Phytopathology.  https://doi.org/10.1094/PHYTO-04-23-0117-R.*
                                *Poddar, S., Tanaka, J., Running, K.L., Kariyawasam, G., Faris, J.D., Friesen, T.L., Cho, M., Kate, J.H., Staskawicz, B. 2023. Optimization of highly efficient exogenous-DNA-free Cas9-ribonucleoprotein mediated gene editing in disease susceptibility loci in wheat (Triticum aestivum L.). Frontiers in Plant Science. 13. Article 1084700. https://doi.org/10.3389/fpls.2022.1084700.*
                                *Alhashel, A., Fiedler, J.D., Nandety, R., Skiba, R., Bruggeman, R., Baldwin, T., Friesen, T.L., Yang, S. 2023. Genetic and physical localization of a major susceptibility gene to Pyrenophora teres f. maculata in barley. Theoretical and Applied Genetics. 136. Article e118. https://doi.org/10.1007/s00122-023-04367-1.*
                                *Peters Haugrud, A., Shi, G., Seneviratne, S., Running, K., Zhang, Z., Singh, G., Szabo-Hever, A., Acharya, K., Friesen, T.L., Liu, Z., Faris, J.D. 2023. Genome-wide association mapping of resistance to the foliar diseases septoria nodorum blotch and tan spot in a global winter wheat collection. Molecular Breeding. 43. Article 54. https://doi.org/10.1007/s11032-023-01400-5.*


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