Research Project: Improved Winter Wheat Disease Resistance and Quality through Molecular Biology, Genetics, and Breeding

Location: Wheat, Sorghum and Forage Research

2023 Annual Report

Objectives
Objective 1: Identify candidate viral and host genes, through use of mutational analysis, protein-protein interaction, and genomic studies for enhanced control and management of Wheat streak mosaic and Triticum mosaic viruses. Objective 2: Develop and characterize transgenic wheat for resistance to WSMV and TriMV, and pyramid transgenes with natural resistance genes. Objective 3: Identify, characterize, and deploy biologically active peptides and genes from the primary and secondary gene pool of wheat for resistance to viral, fungal, and bacterial diseases of wheat. Objective 4: Develop and characterize adapted winter wheat germplasm with broad and specific disease resistance, and with improved grain nutritional quality. Objective 5: Develop scab resistant winter barley and winter wheat germplasm.

Approach
The primary objectives of this project are to develop improved wheat germplasm by enhancing disease resistance and grain quality traits. The project will characterize genes of Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) responsible for pathogenicity and vector transmission. This information will be used to develop transgenic wheat with resistance to both viruses, and to the common vector, the wheat curl mite. The project also will use TriMV to express biologically active peptides in wheat, to effect control of bacterial and fungal diseases. Natural (non-transgenic) sources of virus resistance will be used to develop and select germplasm with such resistance, and distribute it to breeding programs world-wide. The project will complete the evaluation and distribution of wheat breeding materials with resistance to Ug99 forms of stem rust, and with low levels of grain phytic acid. The latter will lead to wheat with improved mineral nutrition and diminished anti-nutrient properties. Developed germplasm will be characterized and distributed via the USDA-ARS Lincoln coordinated Winter Wheat Performance Nursery Program. The project consists of three integrated components: germplasm development and evaluation, viral genetics, and plant pathology. Molecular and conventional methodologies will be utilized, and the project scale will range from DNA molecules to field-level. The project also has extensive formal and informal collaborations enhancing our ability to conduct this research. Anticipated products include improved wheat germplasm for the wheat seed industry with value-added traits and biotic stress tolerance, and new targets to continue the laudable goal of developing host-plant resistance.

Progress Report
Summary of five-year Project 3042-21000-033-00D. Wheat production has been continuously threatened by various biotic and abiotic stresses, such as disease pathogens, pests, and climate change. There is a constant need to enhance the genetic potential of wheat for sustained wheat production under emerging threats. Objective 1: Wheat streak mosaic virus (WSMV) is the main causal agent of wheat streak mosaic (WSM) disease of wheat, the most economically important viral disease. Wheat resistance against WSMV across a wide range of temperatures is not available; hence, novel management strategies are needed. Determination of viral proteins involved in disease development would facilitate the identification of host susceptibility genes through protein-protein interaction studies. Four WSMV-encoded proteins were identified as pathogenicity determinants involved in different stages of WSM disease in wheat. Since viruses are obligate parasites, some of the viral proteins must interact with host susceptibility genes for viral replication, movement, and disease development. Identifying host susceptibility genes would facilitate the development of novel management strategies by disrupting virus-host interactions. Ten wheat proteins interacting with WSMV coat protein (CP) were identified, and these susceptibility genes can be used to disrupt virus-host interactions to provide virus-resistant wheat plants. WSMV and Triticum mosaic virus (TriMV) interact synergistically in co-infected wheat, dramatically increasing disease severity and yield loss. Since both viruses are transmitted by wheat curl mites (WCM), double infections in field-grown wheat are common, resulting in disease synergism. Understanding the underlying mechanisms of synergistic interaction between WSMV and TriMV would facilitate developing strategies to minimize losses from disease synergism. Sequential inoculation of wheat by WSMV and TriMV revealed that interaction between WSMV- and TriMV-encoded proteins likely enhances disease symptoms, and disruption of these interactions could prevent synergistic interactions, which will lead to virus resistance. Objective 2: Wheat streak mosaic disease complex, caused by WSMV, TriMV, and High Plains wheat mosaic virus, is the most economically important viral disease of wheat in the Great Plains. Since all three viruses are transmitted by the WCM, mixed infections in combination with any two or all three viruses have been reported in growers’ fields with reduced yield. Hence, wheat cultivars with resistance to at least two of the three mite-transmitted viruses would minimize yield loss. Transgenic wheat lines were developed by inserting a hairpin RNA element with sequences of WSMV and TriMV. These transgenic wheat lines provided dual resistance to WSMV and TriMV at 25°C or above but not at 20°C. Wheat cultivars with the Wsm-1 gene are resistant to WSMV and TriMV at 20°C or below. Wheat lines resistant to WSMV and TriMV at a wide range of temperatures were obtained by stacking NIb hairpin transgenic wheat with Wsm-1 gene-containing wheat cultivars, suggesting gene staking provides broad-spectrum resistance. Wheat curl mites (WCM) transmit WSMV in a persistent propagative manner. WSMV-encoded CP and HC-Pro were identified as viral determinants required for WSMV transmission by WCM. Some of the WCM proteins are involved in interaction with WSMV CP and/or HC-Pro for WCM transmission. WSMV CP and HC-Pro interacted with 10 and 16 WCM proteins, respectively. These WCM proteins will be selected to downregulate WCM genes to mitigate the WCM transmission of WSMV. Additionally, transcriptome analyses from WSMV-viruliferous and aviruliferous WCM revealed that transcriptional changes may inhibit the immune response of WCM, which prolongs viral association and alters WCM development to expedite population expansion, both of which could enhance viral transmission. This research will ultimately lead to better prevention and control tactics to reduce wheat infection rates and yield losses in the field due to WCM. Objective 3: WSMV and TriMV are economically important wheat curl mite-transmitted viruses of wheat with 5-7% annual yield loss in the Great Plains region. Wheat cultivar Mace containing a long chromosomal translocation with Wsm 1 gene provides resistance to WSMV and TriMV at 20°C or below. The Wsm1 gene was originally transferred from intermediate wheatgrass. However, the presence of a large amount of the wheatgrass DNA in Mace resulted in a 15-30% yield reduction. New wheat germplasm was developed to minimize this yield drag, possessing a truncated chromosome from intermediate wheatgrass carrying the Wsm1 resistance gene. Two of the highest-yielding lines from single plot field trials in 2021, possessing the shortened chromosome segment from intermediate wheatgrass, NW13MD108-3 and NW13MD109-1, were resistant to WSMV in high disease pressure, confirming the presence of the Wsm1 gene. Yield trial results further revealed no significant differences in seed yield of wheat lines with truncated Wsm 1 gene compared to widely used cultivars, and these wheat lines will be targeted for cultivar development. Wheat breeding lines with stem rust (Sr) resistance to evolved Ug99 Sr races were developed to protect U.S. wheat from the worldwide threat of this virulent strain of rust. The Sr resistance gene is on the same chromosome segment as Wsm1 and provides resistance to all known evolved races of Ug99. Sixty-eight Sr-resistant breeding lines were entered into a preliminary yield trial during the 2022 field season. Ten Sr-resistant breeding lines outperformed the high-yielding check varieties. These lines will be used to develop new cultivars with dual WSMV and Sr resistance adapted to the Great Plains. Bacterial leaf streak (BLS) disease is an emerging threat to wheat in the U.S. BLS disease can cause up to 40% yield loss, and there is no effective chemical control to manage this disease. New BLS resistance genes have been identified in the rye genome of triticale and incorporated into rye chromosome 5R containing the wheat BLS resistance gene. These lines have been cross-pollinated to a wheat ph1b mutant line to shuffle the genes on rye chromosome 5R with wheat chromosomes and to eliminate rye linkage drag. Additionally, sequences encoding several plant-based antimicrobial peptides (AMP) provided resistance against the BLS disease. Wheat plants expressing four AMPs through WSMV as an expression vector significantly reduced BLS lesion size. This research will lead to the development of new BLS-resistance wheat lines that can be used to breed new BLS-resistant varieties. Objective 4: Low phytic acid (LPA) mutants of wheat can reduce concentrations of the anti-nutrient phytate by one-third in the wheat kernel. The reduction of phytate increases the bioavailability and, subsequently, the gut absorption of minerals in monogastric animals, including humans. Novel LPA wheat lines were developed by mating the lines carrying the LPA mutation to Nebraska winter wheat. Multi-location grain yield testing and selection for the LPA trait resulted in the identification of eight high-yielding LPA breeding lines adapted to the Great Plains region. The LPA lines had 18% more zinc with no significant differences in grain yield, grain volume weight, and grain protein concentration compared to adapted controls. These eight LPA germplasm lines were released and deposited in the USDA-ARS National Small Grains Collection for use by wheat breeding programs across the globe. Polyphenol oxidase (PPO) is found in nearly all plants and is responsible for product discoloration and loss of economic value. Market demand for hard white winter (HWW) wheats is increasing, but these wheats must have low or nil levels of grain PPO. Mutations leading to a lack of PPO were found in the 1930s era wheats from Australia and were housed in the USDA collection. Through successive generations of mating and phenotypic recurrent selection, the nil PPO trait was moved into Great Plains-adapted HWW wheats. PPO was measured in flours derived from these wheat breeding lines, and the nil PPO trait was found to be stable in more than 100 breeding lines. These lines will be further evaluated for agronomic and disease resistance and have been released to wheat breeding programs for the development of low PPO HWW wheat cultivars adapted to the Great Plains. Objective 5: Fusarium head blight (FHB), also called scab, is a devastating fungal disease of wheat and barley in the U.S. and worldwide. A novel FHB resistance gene, Fhb7, was found in tall wheatgrass and integrated into wheat by chromosome engineering (non-genetically modified organism approach), enabling Fhb7 to improve common and durum wheat. Fhb7-specific DNA markers were developed to enhance the utility of this novel resistance gene in wheat breeding efforts, which will aid the deployment of Fhb7 into all wheat classes, including hard red winter wheat and durum wheat in the Great Plains. FHB-resistant wheat germplasm, WGC002, was developed through a chromosome engineering-based breeding pipeline that removed deleterious traits from its wild grass donor; hence, it is ready for immediate use in cultivar development. In addition, Fhb7-specific DNA markers were developed to enhance the utility of this novel resistance gene in wheat breeding. Utilization of WGC002 in wheat breeding for FHB resistance will strengthen and diversify the host resistance of wheat to FHB disease. Additionally, Fhb7 was deployed in three adapted hard red spring wheat (HRSW) accessions through a marker-assisted backcrossing breeding pipeline. The presence of the Fhb7 gene was confirmed via FHB screening efforts in greenhouse and field experiments. These three wheat lines now exhibit significant resistance to FHB compared to their susceptible HRSW parents in both environments. These efforts will enhance and diversify the genetic resistance of HRSW to FHB.

Accomplishments
1. Development and release of fusarium head blight-resistant spring wheat germplasm ‘WGC002’. A wheat line containing a novel wild grass-derived Fusarium head blight (FHB) resistance gene (Fhb7) was released. ARS scientists in Lincoln, Nebraska, in collaboration with North Dakota State University scientists, developed FHB-resistant wheat germplasm through a chromosome engineering-based breeding pipeline. Development of this wheat makes this wild grass-originated FHB resistance gene usable in wheat breeding. WGC002 does not contain wild grass-derived deleterious traits and is ready for immediate use for variety development. In addition, the resistance gene-specific DNA markers were developed to enhance the utility of this novel resistance gene in wheat breeding. Utilization of WGC002 in wheat breeding for FHB resistance will strengthen and diversify the plant resistance of wheat to FHB and reduce the economic losses of wheat growers caused by this devastating fungal disease.

2. Deployment of the novel fusarium head blight resistance gene Fhb7 in the adapted hard red spring wheat. Novel wild grass-derived fusarium head blight (FHB) resistance gene Fhb7 was deployed in three hard red spring wheats (HRSW) accessions through the marker-assisted backcrossing breeding pipeline. ARS scientists in Lincoln, Nebraska, developed three HRSW germplasm lines containing Fhb7 and have been evaluated for FHB resistance in the greenhouse and field. These wheat lines exhibited significant resistance to FHB compared to their susceptible HRSW parents in both environments. This work enables HRSW to gain this new FHB resistance gene, which will enhance and diversify the genetic resistance of HRSW to FHB and reduce the economic losses caused by FHB to the HRSW growers in the upper Great Plain regions.

3. Disease determinants of wheat streak mosaic virus. Wheat streak mosaic virus (WSMV) is the causal agent of wheat streak mosaic (WSM) disease, the most economically important viral disease in wheat, accounting for 5 to 7% annual yield losses in the Great Plains region. Wheat resistance against WSMV across a wide range of temperatures is not available; hence, novel management strategies are needed. Determination of viral proteins required for disease development would facilitate identifying host susceptibility genes through protein-protein interaction studies. ARS scientists in Lincoln, Nebraska, identified four WSMV proteins that are collectively involved in different stages of the disease in wheat. Future WSM disease management strategies will focus on disrupting interactions between WSMV and wheat susceptibility proteins required for disease progression.

Review Publications
Tatineni, S., Hein, G.L. 2023. Plant viruses of agricultural importance: current and future perspectives of virus disease management strategies. Phytopathology. 113(2):117-141. https://doi.org/10.1094/PHYTO-05-22-0167-RVW.
Zhang, W., Danilova, T.V., Mingyi, Z., Ren, S., Zhu, X., Zhang, Q., Zhong, S., Dykes, L., Fiedler, J.D., Xu, S.S., Frels, K., Wegulo, S., Boehm Jr, J.D., Cai, X. 2022. Cytogenetic and genomic characterization of a novel tall wheatgrass-derived Fhb7 allele integrated into wheat B genome. Journal of Theoretical and Applied Genetics. https://doi.org/10.1007/s00122-022-04228-3.
Talukder, Z.I., Underwood, W., Misar, C.G., Seiler, G.J., Liu, Y., Li, X., Cai, X., Qi, L. 2021. Unraveling the Sclerotinia basal stalk rot resistance derived from wild Helianthus argophyllus using a high-density SNP linkage map. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2020.617920.
Tatineni, S., Hein, G.L. 2021. High Plains wheat mosaic virus: An enigmatic disease of wheat and corn causing the high plains disease. Molecular Plant Pathology. 22:1167-1179. https://doi.org/10.1111/mpp.13113.
Kiszonas, A., Ibba, M., Boehm Jr., J.D., Morris, C.F. 2021. Effects of Glu-D1 gene introgressions on soft white spring durum wheat (Triticum turgidum ssp. durum) quality. Cereal Chemistry. 98(5):1112-1122. https://doi.org/10.1002/cche.10459.
Kiszonas, A.M., Ibba, M., Boehm Jr., J.D., Morris, C.F. 2021. Effects of the functional Gpc-B1 allele on soft durum wheat grain, milling, flour, dough, and breadmaking quality. Cereal Chemistry. 98(6):1250-1258. https://doi.org/10.1002/cche.10477.
Alam, M., Kashif, M., Easterly, A.C., Wang, F., Boehm Jr, J.D., Baenziger, P.S. 2021. Coleoptile length comparison of three winter small grain cereals adapted to the Great Plains. Cereal Research Communications. 50:127-136. https://doi.org/10.1007/s42976-021-00151-3.
Gill, B., Klindworth, D.L., Rouse, M.N., Zhang, J., Zhang, Q., Sharma, J.S., Chu, C.N., Long, Y., Chao, S., Olivera, P.D., Friesen, T.L., Zhong, S., Jin, Y., Faris, J.D., Fiedler, J.D., Elias, E.M., Liu, S., Cai, X., Xu, S.S. 2021. Function and evolution of allelic variation of Sr13 conferring resistance to stem rust in tetraploid wheat (Triticum turgidum L.). Plant Journal. 106:1674-1691. https://doi.org/10.1111/tpj.15263.
Athiyannan, N., Long, Y., Kang, H., Chandramohan, S., Bhatt, D., Zhang, Q., Klindworth, D.L., Rouse, M.N., Friesen, T.L., MciIntosh, R., Zhang, P., Forrest, K., Hayden, M., Patpour, M., Hovmoller, M.S., Hickey, L.T., Ayliffe, M., Cai, X., Lagudah, E.S., Periyannan, S., Xu, S.S. 2022. Haplotype variants of Sr46 in Aegilops tauschii, the diploid D genome progenitor of wheat. Theoretical and Applied Genetics. 135: 2627-2639. https://doi.org/10.1007/s00122-022-04132-w.
Talukder, M.I., Underwood, W., Misar, C., Seiler, G.J., Cai, X., Li, X., Qi, L. 2022. A quantitative genetic study of Sclerotinia head rot resistance introgressed from the wild perennial Helianthus maximiliani into cultivated sunflower (Helianthus annuus L.). International Journal of Molecular Sciences. 23(14). https://doi.org/10.3390/ijms23147727.
Nunna, H., Qu, F., Tatineni, S. 2023. P3 and NIa-Pro of turnip mosaic virus are independent elicitors of superinfection exclusion. Viruses. https://doi.org/10.3390/v15071459.