Location: Cereal Crops Research
2018 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 3060-22000-050-00D, entitled “Host-Pathogen Interactions in Barley and Wheat,” which started at the end of March, 2017.
Net blotch on barley and Septoria nodorum blotch (formerly Stagonospora nodorum blotch) on wheat are two of the most destructive leaf diseases of cereals, both in the U.S. and worldwide. Our research has 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.
We have sequenced, assembled, and annotated the genomes of three P. nodorum isolates, one collected from spring wheat, one collected from durum wheat and the other collected from a wild grass. These P. nodorum isolates were sequenced using PAC-BIO SMRT long read sequencing technology. Isolates were annotated with the support of extensive RNA sequencing data.
Additionally, 200 P. nodorum isolates collected from different wheat production regions of the United States were re-sequenced to approximately 28X coverage using Illumina short read sequencing technology. This collection includes isolates from the Pacific Northwest, Midwest, Northern Great Plains, Northeast, and Southeast wheat growing regions. Single nucleotide polymorphism (SNP) and presence/absence variation (PAV) markers were identified using a marker identification pipeline. The 200 P. nodorum isolates were disease phenotyped on 24 wheat lines including spring, winter, and durum wheat. By combining the SNP and PAV markers with the phenotypic data, genome wide association study analysis (GWAS) was conducted to identify marker trait associations (MTAs) that defined regions significantly associated with virulence. Several regions harboring MTAs were localized to chromosomes, with strong candidate genes being identified which underlie each of these associated markers.
Candidate genes specific to each region were prioritized and are being validated using heterologous expression in both yeast and bacteria, gain of function transformation into avirulent isolates, and loss of function mutations using site directed gene disruption.
197 of the 200 genome sequences generated for P. nodorum were used in a population genomics study to show that the US population structure consists of two distinct populations made up primarily of isolates in the south and eastern U.S. in one population and isolates from the Northern Great Plains in a second population with isolates from Oklahoma being an admixture of the two populations. This work directly relates to Objective 1.
Pyrenophora teres f. teres candidate genes conferring virulence on Rika and Kombar barley have been identified using a population generated from parental isolates differing for their reaction to Rika and Kombar. Two loci conferring virulence on Rika (VR1, VR2) and two conferring virulence on Kombar (VK1, VK2) were previously identified. Candidate genes were evaluated and confirmed for VR2 using site directed gene disruption. Additional validation is underway. Candidate genes for other loci have been prioritized and are currently being confirmed for virulence activity.
Six P. teres f. teres reference quality genome sequences were generated resulting in telomere to telomere assemblies for most chromosomes. Sequenced isolates were originally collected in Ontario, Canada, North Dakota, California (2), Denmark, and Morocco. Additionally, 146 isolates collected in the US, Europe, and Australia, were sequenced using Illumina short read technology for use in GWAS analysis. All 146 isolates were phenotyped on 22 barley lines historically used in pathotype diversity studies worldwide. Several strong marker trait associations were identified and candidate genes have been identified and prioritized for confirmation.
Candidate genes for two P. teres f. maculata loci (PtmPin1 and PtmWel1) associated with virulence contributing to spot form net blotch on two barley varieties, have been identified using bi-parental mapping populations. Populations were previously generated from crosses of highly virulent P. teres f. maculata isolates from North Dakota and Montana with less virulent isolates from California and Australia. Site directed gene disruptions have been completed and are currently being evaluated to confirm the role of these genes in virulence. This work directly relates to Objective 2.
Accomplishments
1. Genome comparison of three fungal isolates that cause disease in wheat. Septoria nodorum blotch (SNB) is a major problem for wheat growers in the U.S. causing substantial yield and quality losses due to infection by the pathogen Parastagonospora nodorum. ARS researchers in Fargo, North Dakota obtained and compared genome sequences for three Parastagonospora nodorum strains, including a strain collected from wild grass, a strain collected from durum wheat, and a strain collected from bread wheat. These strains were sequenced and evaluation of the three genomes showed that one of the chromosomes was completely absent in the strain collected from grasses. This suggests the potential for dispensable chromosomes involved in disease and provides the necessary foundation for investigation of how wheat pathogens are causing disease. This knowledge is critical to intelligent disease control and wheat breeding.
2. Identification and validation of a gene important in causing Septoria nodorum blotch on wheat. Necrotrophic diseases of wheat, including Septoria nodorum blotch (SNB), are a major problem for wheat growers in the U.S. ARS researchers in Fargo, North Dakota obtained genome sequences of 197 isolates of Parastagonospora nodorum and evaluated all isolates for disease on popular spring, winter, and durum wheat lines. A P. nodorum gene was identified and confirmed to be involved in SNB disease induction on wheat. This information gives us insight into how this pathogen is manipulating host defenses to parasitize the plant and will contribute to the development of intelligent breeding strategies for producing disease resistant wheat.
Review Publications
Richards, J.K., Wyatt, N.A., Liu, Z., Faris, J.D., Friesen, T.L. 2018. Reference quality genome assemblies of three Parastagonospora nodorum isolates differing in virulence on wheat. G3, Genes/Genomes/Genetics. 8:393-399. https://doi.org/10.1534/g3.117.300462.
Wyatt, N.A., Brueggeman, R.S., Friesen, T.L. 2018. Reference assembly and annotation of the Pyrenophora teres f. teres isolate 0-1. G3, Genes/Genomes/Genetics. 8:1-8.
Ameen, G., Kariyawasam, G., Shi, G., Friesen, T.L., Faris, J.D., Ali, S., Rasmussen, J.B., Liu, Z. 2017. Molecular manipulation of the mating-type system and development of a new approach for characterizing pathogen virulence in Pyrenophora tritici-repentis. Fungal Genetics and Biology. 109:16-25. https://doi.org/10.1016/j.fgb.2017.10.004.
Lu, S., Edwards, M.C. 2017. Molecular characterization and functional analysis of PR-1-like proteins identified from the wheat head blight fungus Fusarium graminearum. Phytopathology. 108:510-520. https://doi.org/10.1094/PHYTO-08-17-0268-R.
Koladia, V.M., Richards, J.K., Wyatt, N.A., Faris, J.D., Brueggeman, R.S., Friesen, T.L. 2017. Genetic analysis of virulence in the Pyrenophora teres f. teres population BB25 x FGOH04Ptt021. Fungal Genetics and Biology. 107:12-19. https://doi.org/10.1016/j.fgb.2017.07.003.
Saini, J., Faris, J.D., Zhang, Q., Rouse, M.N., Jin, Y., Long, Y., Klindworth, D.L., Elias, E.M., McClean, P.E., Edwards, M.C., Xu, S.S. 2018. Identification, mapping, and marker development of stem rust resistance genes in durum wheat 'Lebsock'. Molecular Breeding. 38:77. https://doi.org/10.1007/s11032-018-0833-y.
Lu, S., Edwards, M.C. 2018. A simple culture method inducing sexual reproduction by Fusarium graminearum, the primary causal agent of Fusarium head blight. Plant Health Progress. 19:129-130. https://doi.org/10.1094/PHP-12-17-0079-BR.
Lu, S., Faris, J.D., Edwards, M.C. 2018. Molecular cloning and comparative analysis of a PR-1-RK hybrid gene from Triticum urartu, the A-genome progenitor of hexaploid wheat. Plant Molecular Biology. https://doi.org/10.1007/s11105-018-1098-7.
Syme, R.A., Martin, A., Wyatt, N.A., Lawrence, J.A., Muria-Gonzalez, M.J., Friesen, T.L., Ellwood, S.R. 2018. Transposable element genomic fissuring in Pyrenophora teres is associated with genome expansion and dynamics of host-pathogen genetic interactions. Frontiers in Genetics. 9:130. https://doi.org/10.3389/fgene.2018.00130.
