Location: Cereal Crops Research2019 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, 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. In FY2019, we completed the sequencing and assembly of 197 Parastagonospora nodorum genomes and used these genomes to identify more than 320,000 markers to be used in genome wide association studies (GWAS) as outlined in Objective 1 and Subobjective 1.A. In addition to the genome sequences and marker set, we have disease phenotyped a total of 24 winter, spring, and durum wheat lines using these 197 isolates as outlined in Subobjective 1B. This data set has proven to be extremely valuable in identifying pathogen genes associated with virulence as outlined in Sub objective 1.C. The 197 P. nodorum genome sequences were used in GWAS to identify marker trait associations that would locate pathogen effector genes important in virulence (disease). We identified the SnTox5-Snn5 interaction in 2012 but did not identify the genes associated with this interaction at that time. In FY2019, we identified a strong marker trait association on P. nodorum chromosome 8 associated with virulence on the Snn5 (SnTox5 sensitive) differential line. Gene transformation and gene disruption validated the SnTox5 candidate gene. Further validation using wheat populations segregating for SnTox5 sensitivity confirmed our SnTox5 candidate gene (See accomplishment 1). We identified the SnTox2-Snn2 interaction in 2007 but like the SnTox5-Snn5 interaction did not validate the genes associated with this interaction at that time. Using the same GWAS model mentioned above, candidate genes were identified and hypothesized to encode for the SnTox2 protein. The SnTox6-Snn6 interaction was identified in 2015. The SnTox6 differential line was also used in this GWAS model and a strong marker trait association was identified at the same chromosomal position as the SnTox2 candidate. Disruption of the SnTox2/6 candidate eliminated disease on both the Snn2 (SnTox2 sensitive) and Snn6 (SnTox6 sensitive) differential lines. Conversely, transformation of SnTox2/6 into an avirulent isolate made this new strain virulent on lines containing either Snn2 or Snn6 (See accomplishment 2). Using the GWAS model we have also initiated other phenotyping that could potentially identify genes important in the necrotrophic life style. We have initiated the phenotyping the 197 isolates mentioned above for sensitivity to reactive oxygen. Preliminary data has identified several marker trait associations for sensitivity to hydrogen peroxide, a commonly produced compound of the plant for defense from invading pathogens. In order to increase the level of diversity in our fungal genomes, we sequenced a P. nodorum isolate from a winter wheat growing region of the southeastern U.S. This isolate was sequenced by long read sequencing and was assembled into a reference quality sequence resulting in full chromosome assemblies. Using this isolate has more than doubled our number of markers from roughly 320,000 markers to more than 700,000 markers. This number of markers provides a high level of saturation of the genome, averaging approximately one marker per 50 bp, allowing us to better identify marker trait associations throughout the genome to better accomplish all aspects of Objective 1. In FY2019, we have introduced the CRISPR-CAS9 gene editing tool into our lab for use in gene editing and gene disruption. This technique has been tailored to be used in both P. nodorum and Pyrenophora teres. Using this technique, we have been able to more efficiently do gene disruptions and more precise gene editing including allele swaps of genes within the native genomic positions. This technique not only allows us to do precise gene editing that we were not able to do before but also streamlines previously used gene editing techniques. 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 barley. Two pathogen genes conferring virulence on Rika (VR1, VR2) and two conferring virulence on Kombar (VK1, VK2) were previously identified. In FY2019, we focused on validating VR1 and VR2 candidate genes. A CRISPR-Cas9 protocol developed for P. teres in our lab was used for site directed gene disruption. The VR1 candidate was transformed into an isolate that was previously avirulent on Rika. This transformed strain became highly virulent on Rika barley, giving validation that this gene was VR1. CRISPR-Cas9 gene editing was used to disrupt the VR2 candidate in an isolate that was virulent on Rika barley. Disruption of this gene made this virulent isolate avirulent on Rika barley. Additional validation is still needed to confirm these genes and this work is in progress. This work addresses subobjective 2A. A diverse set of six P. teres f. teres reference quality genome sequences were previously generated along with the resequencing of 146 isolates collected from North America, Europe, and Australia. These P. teres isolates were sequenced for use in GWAS analysis associated with Sub-objective 2B. 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. In previous work, we identified a locus that was associated with virulence/avirulence on Harbin barley referred to as AvrHar. A biparental population was used to locate this gene but no candidate AvrHar gene was validated using this population due to lack of a contiguous genome assembly. In FY2019, using the reference quality genome sequences and the GWAS data, we were able to identify marker trait associations with markers in a strong candidate gene that mapped to the same AvrHar region previously identified in the biparental population. Using the reference quality P. teres f. teres genomes, we conducted a genome wide comparative analysis to identify regions of similarity and regions of variability between P. teres f. teres isolates. We found that there were definitive regions of variability, termed accessory regions, located on the ends of chromosomes and these accessory regions had an over representation of pathogenicity related genes. We further found that 14 of 15 disease associated genomic regions published previously in our lab are in these accessory genomic compartments. These highly variable accessory genomic compartments have thus been shown to be important drivers of pathogenicity that are also able to evolve rapidly (see accomplishment 3).
1. SnTox5 is a single virulence gene important in septoria nodorum blotch (SNB) of wheat. Septoria nodorum blotch (SNB) of wheat caused by Parastagonospora nodorum, is a major leaf disease that causes significant yield reductions for wheat growers. This pathogen uses necrotrophic effectors to induce disease on wheat. ARS scientists in Fargo, North Dakota, previously identified the necrotrophic effector SnTox5 and its wheat susceptibility target Snn5. In FY2019, scientists identified and validated a SnTox5 candidate gene and showed that it targets the wheat gene Snn5 to induce disease. These results provide both information and a targeted means for screening wheat lines for SNB resistance. Purified protein of this cloned necrotrophic effector has already been requested by breeding programs worldwide for use in resistance breeding. Breeding programs will use this and other characterized necrotrophic effectors to select for resistance.
2. SnTox2 and SnTox6 are the same protein that target two different susceptibility genes in wheat. Septoria nodorum blotch (SNB), caused by the fungal pathogen Parastagonospora nodorum, is a major leaf disease of wheat that causes significant yield reductions for growers in the U.S. and worldwide. P. nodorum uses necrotrophic effectors to target wheat susceptibility genes to induce SNB disease. ARS scientists in Fargo, North Dakota, previously identified two necrotrophic effectors, including SnTox2 and SnTox6, that targeted the wheat susceptibility genes Snn2 and Snn6, respectively, to induce disease. Scientists cloned and validated the SnTox2 gene and showed that SnTox2 was also SnTox6, now referred to as SnTox2/6. The scientists therefore showed that the SnTox2/6 protein targets the two independent wheat susceptibility genes Snn2 and Snn6 to cause disease. This is the first time it has been shown that a single necrotrophic effector can target two different susceptibility genes in the host. These results provide additional information on how P. nodorum targets wheat to gain nutrients. Understanding this system is critical in controlling this pathogen through the use of resistance breeding. Products from this project (SnTox2/6) will be used by geneticists and breeders to eliminate the wheat susceptibility targets (Snn2 and Snn6) from breeding programs, ultimately resulting in more resistant wheat cultivar releases.
3. Variable regions of the genome are evolutionary drivers of pathogen virulence. Net form net blotch of barley caused by Pyrenophora teres f. teres is a major problem in almost all barley growing regions of the world and is a particularly important problem in barley growing regions of the U.S. ARS scientists in Fargo, North Dakota, compared the genome sequences of several diverse P. teres f. teres isolates collected from around the world and found that there were definitive regions of variability not present in all isolates, termed accessory regions. Many of the accessory regions were located on the ends of chromosomes and contained the majority of the genes associated with pathogen virulence (e.g. the ability to cause disease). These highly variable accessory regions were shown to evolve rapidly and thus to be important drivers of pathogenicity. These results add significantly to our understanding of how pathogens in general are able to rapidly adapt to a plant host. This information is critical to plant pathologists, barley geneticists, and breeding programs working toward durable resistance to net form net blotch of barley as well as other scientists investigating pathogen evolution.
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