Location: Cereal Crops Research
2022 Annual Report
Objectives
Objective 1: Characterize the Septoria nodorum blotch of wheat interaction by identifying and characterizing necrotrophic effectors produced by Parastagonospora nodorum.
Sub-objective 1.A.Generate a highly saturated genome wide single nucleotide polymorphism (SNP) and presence-absence variation (PAV) marker set using 1) predicted small secreted protein genes with presence-absence variation and 2) full genome resequencing of U.S. P. nodorum isolates collected from spring, winter, and durum wheat).
Sub-objective 1.B. Collect disease data on wheat lines selected from different wheat classes including spring wheat, winter wheat, and durum wheat, and use this data in conjunction with subobjective 1A to identify genomic regions harboring virulence genes using a genome-wide association study (GWAS) analysis.
Sub-objective 1.C. Identify and validate candidate virulence genes in the MTA regions identified in the data collected in sub objective 1B.
Objective 2: Genetically characterize the mechanism of virulence used by Pyrenophora teres f. teres and P. teres f. maculata in causing barley net form and spot form net blotch, respectively.
Sub-objective 2.A.Use a characterized bi-parental mapping population of P. teres f. teres to identify genes associated with virulence on barley lines Rika and Kombar.
Sub-objective 2.B. Assemble, phenotype, and obtain whole genome sequences of a set of 124 P. teres f. teres isolates from the U.S., N. Africa, and Europe to be used in GWAS analysis to identify and characterize genomic regions associated with virulence/avirulence.
Sub-objective 2.C. Use a P. teres f. maculata bi-parental mapping population to identify and characterize genomic regions and the underlying genes associated with virulence.
Approach
Fungal diseases of small grains pose an economic threat to production throughout the US and the world. This project focuses on two fungal pathogens in an effort to better understand pathogenicity, virulence, and host resistance. It is our goal to identify and characterize pathogenicity/virulence factors of Pyrenophora teres f. teres (net form net blotch of barley), P. teres f. maculata (spot form net blotch of barley), and Parastagonospora nodorum (Septoria nodorum blotch of wheat), and evaluate their importance in each disease interaction. Our approach will be to: a) identify necrotrophic effectors and other components of virulence important in the Parastagonospora nodorum – wheat interaction using a genome wide association study (GWAS) approach involving full genome sequencing of a worldwide collection of P. nodorum isolates, b) Identify both virulence and avirulence factors in the P. teres f. teres – barley interaction by GWAS using a P. teres f. teres collection obtained from barley regions of the United States (North Dakota, Montana), Northern Europe, and North Africa (Morocco), and c) use previously characterized biparental mapping populations of both P. teres f. teres and P. teres f. maculata to identify and validate candidate genes that are associated with major virulence/avirulence QTL. These approaches will allow us to genetically characterize these interactions and will provide an opportunity to identify the genes underlying the virulence of each pathogen. Identification of virulence genes will allow us to better understand how these pathogens parasitize the plant. Understanding both how the pathogen infects the host and how the host defends itself are critical to defending against this disease.
Progress Report
This report documents progress for Project Number 3060-22000-050-000D, entitled “Host-Pathogen Interactions in Barley and Wheat,” which started at the end of March, 2017 and was terminated in fiscal year 2022.
Net blotch on barley and septoria nodorum blotch (SNB) (formerly Stagonospora nodorum blotch) on wheat are two of the most destructive leaf diseases of cereals, both in the United States. and worldwide. We have focused on the characterization of pathogen virulence as it relates to the interaction of plants and their corresponding pathogens for these important diseases. We have worked closely with collaborators focused on the host plant's involvement in these interactions.
Objective 1 – Using results from Sub-objective 1.A. and 1B, we previously showed that SnTox5 had a dual function in that it not only targeted the wheat susceptibility gene Snn5, but also facilitated the colonization of the mesophyll layer of the wheat leaf, allowing complete colonization and increased sporulation. To understand the pathogen and host transcriptional response to SnTox5, we have initiated a project using ribonucleic acid sequencing (RNAseq) to evaluate transcriptional differences in the host response to SnTox5. This has been done by inoculating wheat lines with P. nodorum isolates that are identical except for the presence of the necrotrophic effector gene SnTox5. Two combinations have been used including the avirulent isolate Sn79-1087 vs Sn79-1087+Tox5 as well as the virulent isolate Sn2000 vs the Sn2000 SnTox5 disruption strain Sn2000(delta)Tox5. Preliminary data has shown that several host processes are targeted by SnTox5. Additional work will be done to verify these genes and pathways.
SnTox267 was previously shown to target multiple pathways in wheat to facilitate disease. We have initiated a confocal microscopy study that is comparing infection dynamics of isolates with and without SnTox267. To do this, we have developed a new counterstaining technique to avoid the need for green/red fluorescent protein (GFP/RFP) transformations. This newly developed technique is highly effective and allows us to accurately calculate differences in fungal biomass in planta. This technique is now being used to evaluate infection strategies relative to SnTox267.
Objective 2 – Pyrenophora teres f. teres is a fungal pathogen that causes net form net blotch (NFNB) of barley. Work on Sub-objective 2.A is being accomplished by the continued evaluation and validation of the P. teres f. teres virulence genes VR1, VR2, VK1, and VK2, all of which confer virulence on barley lines that carry the corresponding barley susceptibility gene(s). Gene disruptions in both VR1 and VR2 were previously accomplished to show that these genes were important for infection. We have now perfected a clustered regularly interspaced short palindromic repeat - CRISPR associated protein 9 (CRISPR-Cas9) based approach to perform allele swaps at each of these loci as well as other loci in the future. An avirulent isolate with an avirulent allele at both VR1 and VR2 was used to create strains with VR1 or VR2 virulent alleles alone as well as a strain harboring both VR1 and VR2 virulent alleles. These near isogenic strains are now being used in comparative confocal microscopy experiments to identify the specific roles of VR1 and VR2 in the infection process.
A second P. teres f. teres pathogen population was developed from a cross of a Moroccan isolate (Mor40-3) that was virulent on the popular NFNB resistant line CI5791 with the reference isolate 0-1 which is avirulent on CI5791. Both mapping and phenotyping of the progeny of CI5791 was performed resulting in the identification of two quantitative trait locus (QTL) associated with virulence. The one QTL mapped to P. teres f. teres chromosome (Chr) 1 where the virulent allele was contributed by the virulent parent MorSM40-3 and a second QTL mapped to Chr 8, however, this virulence allele was contributed by the avirulent parent 0-1. Additionally, the virulence allele at the Chr 1 locus was necessary for the virulence allele at the Chr 8 locus to be effective and the two loci accounted for 22.5 and 14.9 % of the disease variation, respectively. Additionally, we used whole genome sequencing of 178 P. teres f. teres isolates in a genome wide association study (GWAS) to identify the same Chr 1 and Chr 8 loci. Several genes that encoded secreted proteins were present at both loci, one or more of which may be responsible for overcoming or triggering the CI5791 resistance. Work is ongoing to identify the genes contributing to virulence at these two loci.
Toward Sub-objective 2.B, we generated short read genome sequencing of an additional 38 P. teres f. teres isolates collected in Iran, the center of diversity for barley. These isolates are currently being phenotyped on the NFNB differential set to be used in GWAS analysis to identify genomic regions of P. teres f. teres contributing to virulence.
For Subobjective 2.C. a biparental population was generated from a cross of a highly virulent Montana isolate (P-A14) and an avirulent isolate (CAWB). This population was mapped, and progeny were phenotyped on several barley lines. QTL analysis identified major virulence loci associated with P. teres f. maculata Chr 1 and 2. QTL analysis of a barley population segregating for susceptibility showed major QTL on Chr 2H and 7H. The virulence loci on Chr 1 and 2 were subsequently shown to correspond to the susceptibility loci on barley chromosomes 2H and 7H, respectively, showing an inverse gene for gene interaction where pathogen effectors are targeting vulnerable host susceptibility genes to colonize the plant (see accomplishments).
During this year we have also established a laboratory for investigation of host-pathogen interactions between cereal crops and bacterial pathogens. This work has involved the acquisition of lab equipment for handling and storage of microbial samples. With focus on bacterial leaf streak of wheat and barley, we have begun initial collections of microbial isolates, including pathogenic bacteria of the genus Xanthomonas, non-pathogenic bacteria associated with wheat and barley, and bacteriophage specific to pathogenic Xanthomonads. A research priority this year has been development of methods for genetic manipulation of bacteria in the genus Xanthomonas. To this end, we have assessed several bacterial culture conditions, including testing nutritional content and susceptibility to common antibiotics, to determine those best suited for efficient laboratory manipulations and bacterial transformation. Random mutagenesis of Xanthomonas strains has been conducted, generating transposon mutant populations for use in virulence screening tests. We have further constructed novel plasmids with broad host range for expression of recombinase genes. These will facilitate development of methods enabling targeted mutagenesis in the chromosome of Xanthomonas species. These molecular tools will allow us to assess the interactions between pathogenic Xanthomonas and host barley and wheat at the molecular level.
Research closeout summary:
During this five-year research project it was our aim to characterize pathogen virulence genes involved in the Parastagonospora nodorum-wheat and P. teres barley interactions. To do this, we generated the tools needed to efficiently evaluate these interactions from the pathogen side. The resources we developed included five reference quality sequences of P. nodorum, six for P. teres f. teres, and five for P. teres f. maculata. Additionally, we generated 192 resequencing-quality short read sequences of P. nodorum, 190 of P. teres f. teres, and 124 of P. teres f. maculata. Highly saturated marker sets were generated from these sequences. All these isolates, which were collected from around the world, were also phenotyped on both locally and globally important host lines. Together the phenotyping and marker sets were used in GWAS studies to identify genomic regions contributing to virulence. Additionally, several P. teres f. teres and P. teres f. maculata biparental populations were also generated and QTL analysis was used to identify genomic locations associated with virulence. The genomic regions identified in these studies were then used to identify candidate genes that were contributing to pathogen virulence. In total, we have identified 14 P. teres f. teres and eight P. teres f maculata loci that are contributing to virulence on barley and more than ten P. nodorum loci contributing to virulence.
Using these genomic tools, we have validated and functionally characterized five necrotrophic effector genes from P. nodorum including SnToxA, SnTox1, SnTox267, SnTox3, and SnTox5. We are continuing to work on these genes/proteins to identify mode of action of each protein. In P. teres f. teres we have validated three genes including VR1, VR2, and VK2 and are in the process of characterizing the mode of action of the proteins. Collectively, the work that we have done in this 5-year project has made significant advances in our understanding of how necrotrophic pathogens infect and cause disease. Additionally, this work has laid a solid foundation for our next research project.
Accomplishments
1. Discovery of susceptibility in durum wheat cultivars to the barley pathogen P. teres f. maculata. Spot form net blotch of barley, caused by the fungus Pyrenophora teres f. maculata, is an emerging disease in major barley growing regions of the world. It is a particularly important problem in barley growing regions of the United States, however ARS researchers in Fargo, North Dakota, identified that P. teres f. maculata is also highly virulent on popular local durum wheat cultivars, a new discovery for this pathogen. A durum mapping population was used to identify the genetic location of a major gene contributing disease susceptibility in durum wheat. Knowledge that this pathogen has emerged as a durum wheat pathogen is critical for extension pathologists making recommendations to growers and breeders who will need to select for resistance to this newly emerged disease.
2. Spot form net blotch disease of barley involves an inverse gene-for-gene interaction. Spot form net blotch of barley, caused by the fungus Pyrenophora teres f. teres, has emerged as a major problem in almost all barley growing regions of the world. It is a particularly important problem in barley growing regions of the United States, often requiring fungicide applications to control the disease. ARS researchers in Fargo, North Dakota, identified two genomic locations in the pathogen for genes that target two independent susceptibility genes in barley, thereby causing disease. When present together in the pathogen, these two genes act synergistically to increase the level of disease when the two corresponding host susceptibility genes are present. This is the first time a gene for gene model has been identified in the spot form net blotch of barley interaction. Understanding how this pathogen is infecting barley and identifying the genetic location of the barley susceptibility genes is critical to developing control strategies for this disease. Geneticists and breeders can now use this information to develop disease resistant breeding lines and cultivars more effectively for barley growers.
Review Publications
Ameen, G., Solanki, S., Sager-Bittara, L., Richards, J., Tamang, P., Friesen, T.L., Brueggeman, R.S. 2021. Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability. PLoS Genetics. 17(12). Article e1009473. https://doi.org/10.1371/journal.pgen.1009473.
Schachterle, J.K., Shi, G., Baldwin, T., Liu, Z. 2022. Two complete genomes of Xanthomonas translucens pv. translucens isolated from barley. Microbiology Resource Announcements. https://doi.org/10.1128/mra.00010-22.
Schachterle, J.K., Gdanetz, K., Pandya, I., Sundin, G.W. 2022. Identification of novel virulence factors in Erwinia amylovora through temporal transcriptomic analysis of infected apple flowers under field conditions. Molecular Plant Pathology. 1-15. https://doi.org/10.1111/mpp.13199.
Clare, S., Duellman, K., Richards, J., Poudel, R., Merrick, L., Friesen, T.L., Brueggeman, R.S. 2022. Association mapping reveals a reciprocal virulence/avirulence locus within diverse US Pyrenophora teres f. maculata isolates. BMC Genomics. 23. Article 285. https://doi.org/10.1186/s12864-022-08529-1.
Alhashel, A., Poudel, R., Fiedler, J.D., Carlson, C.H., Rasmussen, J., Baldwin, T., Friesen, T.L., Brueggeman, R., Yang, S. 2021. Genetic mapping of host resistance to the Pyrenophora teres f. maculata isolate 13IM8.3. Genes, Genomes, Genetics. https://doi.org/10.1093/g3journal/jkab341.
Shi, G., Kariyawasam, G., Liu, S., Leng, Y., Zhong, S., Ali, S., Moolhuijzen, P., Moffat, C., Rasmussen, J., Friesen, T.L., Faris, J.D., Liu, Z. 2022. A conserved hypothetical gene is required but not sufficient for Ptr ToxC production in Pyrenophora tritici-repentis. Molecular Plant-Microbe Interactions. 35(4). https://apsjournals.apsnet.org/doi/10.1094/MPMI-12-21-0299-R.
Green, A.J., Mergoum, M., Frohberg, R., Underdahl, J., Walz, A., Simsek, S., Otteson, B., Heilman-Morales, A., Friskop, A., Rickertsen, J., Friesen, T.L., Rouse, M.N., Jin, Y., Chao, S. 2022. Registration of ND VitPro hard red spring wheat. Journal of Plant Registrations. http://doi.org/10.1002/plr2.20239.
