Location: Cereal Crops Research
2020 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. ARS researchers at Fargo, North Dakota, 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: The molecular marker set generated to fulfill Subobjective 1A along with the disease reaction data collected to fulfill Subobjective 1B were used to identify two P. nodorum candidate genes conferring virulence including SnTox2 and SnTox5. These two virulence genes were validated using gene knockouts and gene insertion transformation. We continued to characterize these two virulence genes and their corresponding proteins and used the expressed proteins to further characterize the wheat gene targets. In previous studies we showed that SnTox2 targeted the wheat susceptibility genes Snn2 and Snn6. In fiscal year 2020, we identified an additional wheat gene targeted by SnTox2 named Snn7. This was the first time a single virulence factor was shown to target multiple host genes and we have renamed SnTox2 to SnTox267. The disruption of SnTox267 resulted in the upregulation of three other virulence genes, indicating that the pathogen can compensate for the loss of SnTox267 virulence.
A second virulence factor, SnTox5, is highly expressed early in the infection process, peaking at 24 hours. Microscopy showed that SnTox5-disruption mutants could penetrate the outer epidermal layer of the leaf but clearly lost the ability to colonize beyond the first cell layer into the mesophyll layer of the leaf. Additionally, when the SnTox5 gene was transformed into an avirulent isolate, this SnTox5-expressing strain acquired the ability to colonize the mesophyll layer of the leaf, showing that SnTox5 plays a major role in fungal colonization. This work is an extension of Subobjective 1.C.
Objective 2: In previous studies involving net form net blotch of barley, caused by Pyrenophora teres f. teres, we have used P. teres f. teres populations to identify genomic regions harboring virulence genes. Work on Sub-objective 2.A. has progressed with the continued validation of four virulence genes including VR1 (virulence on Rika barley 1), VR2 (virulence on Rika barley 2), VK1 (virulence on Kombar barley 1), and VK2 (virulence on Kombar barley 2). Gene knockouts and gene insertion transformations validated the VR1 and VR2 genes as virulence factors involved in disease on Rika barley. Additional validation of VK1 and VK2 is ongoing.
In fulfilment of Subobjective 2B, 50 additional P. teres f. teres isolates were re-sequenced, increasing the total number to 191 with added diversity using populations from Azerbaijan, France, and Iran. Like the previously sequenced isolates, the 50 new isolates were disease phenotyped for virulence on a barley differential set comprised of 22 barley lines. This dataset will be used in the coming year to identify additional genes associated with net form net blotch on barley.
P. teres f. teres parental isolates collected in the U.S. and Denmark were used to develop a fungal population. This population was mapped, and all progeny were disease phenotyped on several differential barley lines including Pinnacle, a susceptible North Dakota barley cultivar and Beecher and Tifang, two lines commonly used as differentials for characterizing virulence in P. teres f. teres global populations. Candidate genes have been prioritized and gene knockouts have been completed in several of these genes.
Spot form net blotch of barley caused by P. teres f. maculata is an emerging disease in many parts of the world including the Northern Great Plains of the U.S. To characterize the P. teres f. maculata genes associated with virulence according to Subobjective 2C, we sequenced, assembled, and annotated three new reference quality genomes using a diverse set of isolates collected from barley regions throughout the world. This brings our total number of P. teres f. maculata reference genome assemblies to four. This will allow for future comparative work and marker development.
On the host side of the spot form net blotch interaction, we developed and mapped the PI67381 x Hockett and PI67381 x PI392501 barley populations. PI67381 is resistant to local isolates of P. teres f. maculata, whereas Hocket is a susceptible Montana barley cultivar and PI392501 shows partial resistance to spot form net blotch. Phenotyping of local isolates has been initiated and we will continue to characterize the resistance/susceptibility to the pathogen.
Accomplishments
1. A fungal pathogen targets a wheat gene, resulting in improved colonization of the leaf. Septoria nodorum blotch (SNB) of wheat caused by Parastagonospora nodorum, is a major leaf disease that causes significant yield reductions in the 5-15% range for wheat growers. This pathogen uses necrotrophic effectors to induce disease on wheat. Scientists in Fargo, North Dakota, previously identified the necrotrophic effector (NE) SnTox5 and its wheat susceptibility target Snn5. Scientists labeled various strains of P. nodorum with red fluorescent protein and used laser confocal microscopy to show that the pathogen secretes SnTox5 which targets the wheat gene Snn5, allowing the pathogen to fully colonize the wheat leaf. This information is critical to wheat breeders and geneticists trying to breed resistance to this pathogen, as well as scientists working on other agriculturally important necrotrophic pathogens. This NE is currently being used by geneticist and breeders to eliminate the Snn5 gene.
2. A broad spectrum source of barley net blotch resistance is overcome by a North African pathogen population. 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. Resistance to the pathogen has been mapped to several genomic regions; however, resistance on chromosome 6H has been reported by several groups from around the world and 6H resistance is being used in breeding programs in the U.S. because it is broadly effective. Scientists in Fargo, North Dakota, identified a P. teres f. teres population from Morocco that overcame the broad resistance found in barley lines carrying the 6H resistance. A barley population segregating for 6H resistance was analyzed and showed that indeed, these P. teres f. teres isolates had overcome the broadly effective 6H resistance; however, resistance found on 3H that is not effective against most U.S. isolates was still effective against the Moroccan isolates. Because the 6H source of resistance is being used in barley breeding programs in the U.S., this is critical information for public and private barley breeders to more effectively breed for highly durable resistance against the net form net blotch disease.
Review Publications
Richards, J.K., Stukenbrock, E.H., Carpenter, J., Liu, Z., Cowger, C., Faris, J.D., Friesen, T.L. 2019. Local adaptation drives the diversification of effectors in the fungal wheat pathogen Parastagonospora nodorum in the United States. PLoS Genetics. 15(10):e1008223. https://doi.org/10.1371/journal.pgen.1008223.
Wyatt, N.A., Richards, J.K., Brueggeman, R.S., Friesen, T.L. 2020. A comparative genomic analysis of the barley pathogen Pyrenophora teres f. teres identifies sub-telomeric regions as drivers of virulence. Molecular Plant-Microbe Interactions. 33:173-188. https://doi.org/10.1094/MPMI-05-19-0128-R.
Clare, S.J., Wyatt, N.A., Brueggeman, R.S., Friesen, T.L. 2020. Research advances in the Pyrenophora teres-barley interaction. Molecular Plant Pathology. 21(2):272-288. https://doi.org/10.1111/mpp.12896.
Sharma, J.S., Zhang, Q., Rouse, M.N., Klindworth, D.L., Friesen, T.L., Long, Y., Olivera, P.D., Jin, Y., McClean, P.E., Xu, S.S., Faris, J.D. 2019. Mapping and characterization of two stem rust resistance genes derived from cultivated emmer wheat accession PI 193883. Theoretical and Applied Genetics. https://doi.org/10.1007/s00122-019-03417-x.
