Project : USDA ARS

ARS Home » Plains Area » Fargo, North Dakota » Edward T. Schafer Agricultural Research Center » Cereal Crops Research » Research » Research Project #429074

Research Project: Host-Pathogen Interactions in Fungal Diseases of Wheat and Barley

Location: Cereal Crops Research

2021 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
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 U.S. 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: ARS researchers have used genome wide analysis (Subobjective 1.A. and Subobjective 1.B.) to identify SnTox5, a pathogen gene harbored by Parastagonospora nodorum that produces the necrotrophic effector protein SnTox5. SnTox5 was shown to target the wheat susceptibility gene Snn5 to induce cell death, resulting in a significant benefit of the pathogen by providing nutrients. We continued to characterize SnTox5 and showed that this gene not only targeted Snn5, but also facilitated the complete colonization of the wheat leaf, even in the absence of Snn5, indicating that SnTox5 has a second function in facilitating more efficient colonization of the wheat leaf. Analysis of the SnTox5 gene and protein sequences across a US collection of P. nodorum isolates identified a specific region that was under selection pressure. Changes in this region of the protein increased the level of disease induced by P. nodorum. Protein modeling of SnTox5 showed a high level of structural homology to SnTox3, another cell-death-inducing protein produced by P. nodorum, suggesting that these two secreted proteins exploit similar wheat vulnerabilities resulting in disease. This work is an extension of Subobjective 1.C. The same genome wide analysis used to identify SnTox5, was also used to validate a second necrotrophic effector gene, SnTox267. SnTox267 was named because it targets three independent wheat genes including Snn2, Snn6, and Snn7, all of which were classified as susceptibility genes that were targeted to cause cell death. We showed that Snn2 and Snn6 are both required to cause wheat cell death induced by SnTox267 and are therefore at different points in the same cell death pathway. Additionally, Snn7 was shown to be involved in a different cell death pathway. This work is also an extension of Subobjective 1.C. Objective 2: Pyrenophora teres f. teres is a fungal pathogen that causes net form net blotch 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 particular barley lines that carry the corresponding barley susceptibility gene(s). Gene disruption in virulent isolates and gain of function gene addition (complementation) in avirulent isolates was completed to validate candidate virulence genes at the VR1 and VR2 loci. Both VR1 and VR2 were shown to independently target the same barley susceptibility locus to facilitate virulence on barley. The VR1 protein had homology to secreted serine proteases and VR2 was predicted to be a hypothetical small, secreted protein with an unknown function like other pathogen produced effectors involved in disease. This work provides tools for the ongoing work of functional characterization of both pathogen virulence and barley resistance. Work on Sub-objective 2.B involved the genome sequencing of more than 150 P. teres f. teres isolates to be used to identify pathogen effector genes contributing to virulence and net form net blotch disease. Disease and marker data were collected on all 150 isolates to identify marker-trait (disease) associations that identified the genomic location of genes associated with virulence. Gene identification has been initiated at these genomic loci and will continue into the next fiscal year to characterize the genes contributing to virulence (disease). Additionally, a new pathogen population obtained from a cross of a North American and a North African P. teres f. teres isolate was generated and the genomes of 120 progeny were sequenced with the intent of identifying and characterizing virulence genes present in the North American P. teres f. teres population. This work is an extension of sub-objective 2.B. As noted above, P. teres f. teres causes net form net blotch on barley. A second form of P. teres, P. teres f. maculata, causes spot form net blotch of barley, an emerging disease of barley in many parts of the world, including the Northern Great Plains of the US. We previously developed a hybrid progeny population from a cross between isolates of P. teres f. teres and P. teres f. maculata. The 120 progenies of this cross were sequenced for marker identification and were phenotyped on Kombar barley. A single virulence locus was identified that corresponded to the VR2 locus mentioned above. Since the two forms rarely cross sexually in the field but readily cross in the lab, the sequencing data of the 120 progeny were also used in a collaborative effort to examine genetic barriers to outcrossing between P. teres f. teres and P. teres f. maculata in nature. This work is an extension of Sub-objective 2.C. Recently there have been reports in the literature that both forms of P. teres have made host jumps from barley to wheat. As an extension of Sub-objective 2.C., we screened diverse bread wheat and durum (pasta) wheat lines for susceptibility to a small global collection of the barley spot form net blotch pathogen P. teres f. maculata. We found susceptibility in four of the top seven durum cultivars planted in North Dakota and Montana, showing that in 2020 more than 750,000 acres were planted to susceptible cultivars in this region alone. The popular North Dakota durum cultivars Divide and Ben were two of the more susceptible cultivars identified and therefore disease evaluations were initiated on the durum wheat mapping populations Divide × PI272527 and Ben × PI41025. Genetic characterization of these populations is ongoing but initial characterization has shown two major quantitative trait loci (QTL) for susceptibility to P. teres f. maculata indicating it has likely made a host jump from barley to durum wheat in North Dakota and Montana.

Accomplishments
1. Validation of two pathogen genes that facilitate disease on barley. Net form net blotch of barley caused by the fungus Pyrenophora teres f. teres is a major problem in almost all barley growing regions of the world. It is a particularly important problem in barley growing regions of the US, regularly causing 5-10% yield losses and requiring fungicide applications to control the disease. ARS researchers in Fargo, North Dakota, identified two fungal genes (VR1 and VR2) that contributed to virulence (disease) on the experimental barley line Rika. Mutated versions of these genes caused less disease, on Rika barley. VR1 and VR2 are currently being used by barley geneticists to clone the corresponding barley resistance/susceptibility gene(s). This information will be useful to other scientists working to understand necrotrophic (tissue killing) pathogens of all crop plants as well as being useful to barley breeders and geneticists trying to breed resistance to this pathogen.

2. A fungal protein targets two distinct cell death pathways in wheat. Septoria nodorum blotch (SNB) of wheat caused by the fungal pathogen Parastagonospora nodorum, is a major leaf disease that causes significant yield reductions for wheat growers and is a model system for understanding necrotrophic (tissue killing) pathogen-host interactions. Previously, it was believed that there were three Parastagonospora nodorum proteins that interacted with the wheat susceptibility genes Snn2, Snn6, and Snn7 to induce cell death. ARS researchers in Fargo, North Dakota, showed that a single protein (SnTox267) targeted all three wheat susceptibility genes (Snn2, Snn6, and Snn7) to induce cell death, allowing the pathogen to colonize, gain nutrients, and reproduce. Further studies indicated that SnTox267 is exploiting at least two cell death pathways to cause disease. This information is being used by wheat breeders to develop improved varieties that are non-responsive to this fungal protein and thus more resistant to SNB disease.

U.S. DEPARTMENT OF AGRICULTURE

Cereal Crops Research: Fargo, ND