Location: Cereal Crops Research2017 Annual Report
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.
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.
This report documents progress for Project Number 3060-22000-050-00D, which started at the end of March 2017 and continues research from Project Number 3060-22000-048-00D, entitled “Host-Pathogen Interactions in Barley and Wheat.” 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 interactions of plants and pathogens for these important diseases. We have worked closely with collaborators focused on the host plant’s involvement in these interactions. Spot form net blotch (SFNB) of barley. Highly virulent isolates of the fungus Pyrenophora teres f. maculata (causal agent of SFNB) collected in Eastern Montana were crossed to either an avirulent P. teres isolate collected from wild barley in California or to a less virulent P. teres isolate collected in New Zealand. These two mapping populations were used to investigate a recent virulence shift specific to the Northern Great Plains. Genetic markers were identified to be used in locating genomic regions harboring genes that confer virulence on barley. Two major virulence regions were identified that were important in causing disease on popular local barley varieties. Candidate genes, including genes encoding small secreted proteins, were identified and are now being validated by expression, transformation, and gene disruption. This work directly relates to Objective 2. Net form net blotch (NFNB) of barley. In previous work, a population was generated by crossing a highly virulent P. teres f. teres isolate collected in the Northern Great Plains with a less virulent isolate collected in Denmark. Progeny isolates from this population were tested for their ability to induce disease on a geographically diverse set of 10 barley lines as part of a project to better understand how North American isolates are able to overcome several sources of NFNB resistance. Genetic markers were used in conjunction with disease phenotyping to identify genomic regions accounting for as much as 75% of the disease variation. Full genome sequences of the parental isolates were assembled, annotated for gene content, and used to identify candidate genes in the identified genomic regions. Several small secreted proteins were identified and are being validated by expression, transformation, and gene disruption. This work directly relates to Objective 2. Septoria nodorum blotch (SNB) of wheat. The fungus Parastagonospora nodorum, causal agent of SNB of wheat, produces several proteins known as necrotrophic effectors (NE) that are important virulence factors. We have now obtained full genome sequences of 178 isolates of this fungus and assembled a bioinformatics pipeline to identify both SNP and presence/absence variation (PAV) markers for use in genome wide association study (GWAS) analysis. More than 480,000 SNP and indel markers have been identified between these isolates and these markers have been used to identify marker trait associations (MTA) via disease phenotyping. Validation of these candidate genes is in progress. Additionally, a protocol has been worked out to show that SnTox1 is not only a necrotrophic effector that induces cell death, but is also a chitin binding protein that protects P. nodorum from wheat chitinases both in vitro and in planta. This work directly relates to Objective 1.
1. Validation of the virulence gene VR2. Characterization of the genetics of pathogen virulence is critical to understanding the interactions between plants and pathogens; this understanding is, in turn, foundational to effective resistance breeding. ARS researchers in Fargo, North Dakota studied the genetics of virulence in the fungal pathogen that causes net form net blotch disease in barley and identified several genomic regions harboring virulence. Candidate genes for VR2 were verified using site directed gene disruption. This information will be used to describe how this pathogen is manipulating host defenses and how the plant is able to defend itself from this important pathogen. This work will contribute to the development of intelligent breeding strategies for producing disease resistant barley, and will also facilitate progress in understanding the basis of this and other similar diseases.
2. Identification of SnTox2, 5, and 6 candidate genes in the Septoria nodorum blotch (SNB) pathogen using genome wide association. SNB, caused by the fungus Parastagonospora nodorum, results in 5-15% yield losses annually in wheat; however, little genetic resistance is available to growers partially due to the lack of understanding of this disease interaction. ARS researchers in Fargo, North Dakota have sequenced the genomes of 178 P. nodorum isolates collected from winter wheat, spring wheat, and durum wheat regions and identified more than 480,000 markers that are useful in identifying genes involved in pathogen virulence on wheat. Several strong marker-trait associations were identified, providing strong candidate genes for SnTox2, SnTox5 and SnTox6 as well as novel genomic regions associated with uncharacterized virulence. Understanding this interaction both locally and globally will provide pathologists and breeders valuable information needed to deliver solutions to growers through resistance breeding and other control recommendations.