Location: Wheat, Sorghum and Forage Research
2021 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
Objective 1: Wheat streak mosaic virus (WSMV) is an economically important wheat curl mite-transmitted virus of wheat in the Great Plains region of the U.S. Virions play a crucial role in the successful completion of the virus life cycle as virions were exposed to vector and plant factors. Hence, the content and composition of WSMV virions were examined through mass spectrometry, followed by Western blot using virions purified through 10-40% sucrose density gradient centrifugation. Mass spectrometric analysis of protein composition of purified WSMV virion preparations indicated that coat protein (CP) comprises 55-80% of total proteins in virion preparations. Additionally, virion preparations also contained four virus-encoded proteins as components of virions: helper component proteinase (HC-Pro; 3-8%), cylindrical inclusion protein (CI; 0.8%), protein 1 (P1; 0.8%), and nuclear inclusion protein a-viral genome-linked protein (NIa-VPg; 0.2-0.4%). These data suggest that components of virion proteins should be targeted to disrupt the virus life cycle for the management of wheat streak mosaic disease.
Objective 1: The CP of WSMV tolerates extensive deletions for systemic infection of wheat. WSMV lacking CP amino acids (aa) 36-57 elicited symptoms similar to those of wild-type (WT) virus, while deletions comprising aa 58-84 elicited severe chlorotic streaks and acute chlorosis with accelerated virus movement and replication. Interaction of wheat proteins with WSMV CP deletion mutants was examined by performing co-immunoprecipitation of proteins from WT- or CP deletion mutants-infected wheat with CP antibodies, followed by mass spectrometry. Analyses of mass spectrometry data revealed that severe-symptom inducing deletion mutants (SSID; D36-84aa and D58-84aa) efficiently interacted with the CI protein of WSMV compared to WT virus or mutant with no severe symptom-inducing deletion mutant (NSSID; D36-57aa). Additionally, the SSID mutants, but not WT or NSSID mutant, interacted with NIa-Pro of WSMV. The SSID mutants caused 17 to 24-fold enhanced interaction with dynamin-like wheat proteins compared to WT or NSSID mutant. The SSID mutants facilitated interaction with nine other wheat proteins but failed to interact at detectable levels with 16 wheat proteins compared to WT or NSSID mutant. These data suggest that deletion of CP aa 58-84 caused differential interaction with wheat proteins, possibly contributing to enhanced symptom severity in wheat.
Objective 2: Superinfection exclusion is defined as the phenomenon whereby initial infection by one virus prevents subsequent infection of pre-infected cells by closely related viruses. Plants expressing elicitors of superinfection exclusion most likely provide resistance against analogous viruses. Previously, scientists in Lincoln, NE, identified CP and NIa-Pro of WSMV as elicitors of superinfection exclusion. Several independent events of T0 transgenic plants harboring WSMV CP or NIa-Pro genes were obtained. These transgenic plants were screened for resistance against WSMV and found that two wheat plants with CP as transgene provided complete resistance against WSMV. Several transgenic wheat plants harboring the NIa-Pro gene from four transgenic events provided moderate to complete resistance against WSMV. Wheat seed collected from the T1 generation will be used to obtain a homogenous population through the single-seed descent method until T4 generation.
Objective 2: Interactions between wheat curl mite proteins and viral proteins facilitate most likely the transmission of WSMV to wheat. Identifying wheat curl mite proteins that interact with viral factors enables the disruption of interactions between viral and vector proteins. Yeast-two hybrid assay of total RNA from healthy wheat curl mites revealed that six wheat curl mite proteins interacted both with CP and HC-Pro proteins of WSMV. Sequences encoding these wheat curl mite proteins were inserted into the genome of WSMV to examine the effect of wheat curl mite proteins on the efficiency of vector transmission.
Objective 3: Twelve perennial wheat genotypes were screened for resistance against WSMV and Triticum mosaic virus (TriMV) under greenhouse conditions. Of these perennial wheat genotypes, PI611891 and PI611899 were rated as resistant to both WSMV and TriMV. Characterization of resistant genes putatively found in these perennial wheat lines and their relationship to Wsm1 and Wsm2, will facilitate the development of germplasm for the nation’s wheat breeding programs using traditional breeding approaches. F4-derived bulk populations (n=8) from two of those resistant perennial wheat genotypes are ready for harvest in the field: PI 611899 and PI 611891 (resistant perennial wheats) × Tomahawk (susceptible), × Mace (resistant, Wsm1), × N13MD2589W (resistant, Wsm1 and Wsm2) and × RonL (resistant, Wsm2). Recombinant inbred lines (n=200) will be selected from each population prior to harvest and advanced via single seed descent for future genetic mapping studies to determine if the perennial wheat resistance mechanisms are unique or allelic to genotypes carrying Wsm1, Wsm2, and Wsm1+Wsm2.
Objective 3: Bacterial leaf streak (BLS) disease is an emerging threat to wheat in the Great Plains region of the U.S. BLS disease can cause up to 40% yield loss and there is no effective chemical control to manage this disease. Sequences encoding several plant-based antimicrobial peptides (AMP) were engineered into the WSMV genome and wheat plants infected with WSMV harboring AMP sequences were examined for antimicrobial activity against BLS. Out of nine AMPs screened for antimicrobial activity against BLS disease, Ace AMP1 was found in onion, and Defensin TK AMP D1, -D3, and Ta PDF 13 showed a statistically significant reduction in lesion size caused by the bacteria in wheat. Wheat plants expressing Ace AMP1, Defensin TK AMP D1, -D3, and Ta PDF 13 elicited lesion sizes of 1.3 cm, 1.5 cm, 1.4 cm, and 2.2 cm, respectively, compared to WSMV-infected (8.3 cm) and mock-inoculated (9.1 cm) wheat. Additionally, the expression of two AMPs in tandem that worked effectively against the BLS disease elicited lesions similar to those inoculated with buffer. This study revealed that WSMV could be used as an efficient and high-throughput transient expression vector to screen a large number of AMPs for their activity in wheat.
Objective 3: Wheat cv. Mace containing a long chromosomal translocation with Wsm 1 gene provides resistance to WSMV and TriMV at 20°C or below. In the absence of viral infection, the Wsm1 gene has a 15-30% drag on yield relative to most current wheat cultivars due to the size of the chromosomal translocation. The development of wheat genotypes carrying shortened versions of the chromosomal translocation with the Wsm1 gene in combination with the Wsm2 gene is in progress to determine if the shortened translocation with Wsm1 will have any linkage drag on grain yield. Wheat breeding line KS08WGRC50 (carrying shortened Wsm1) was used to develop two new F6 bulk populations by crossing to wheat cultivars ‘Overland’ and ‘OK Rising’, which are two current high-yielding Great Plains cultivars. Seed from each F6 bulk population was planted in head rows in a field-based WSMV screen to evaluate their resistance against WSMV. The field-based WSMV screen indicated that each F6 planted headrow was resistant to WSMV. Single plants from headrows in the WSMV field nursery will be harvested and used in future yield trials to develop new wheat cultivars carrying the shortened version of the translocation carrying the Wsm1 gene.
Objective 5: 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. Fusarium head blight (FHB), also called scab, is a devastating fungal disease of wheat and barley in the U.S. and worldwide. Host resistance has been considered the most effective tactic to manage FHB disease. The limited genetic variability of the wheat genome has increasingly become a bottleneck for wheat improvement. However, wheat has a huge secondary and tertiary gene pool (i.e., wheat-related wild species) with tremendous genetic variability, representing a valuable gene source for wheat breeding. Wild grass-derived FHB resistance gene Fhb7 was incorporated into wheat by chromosome engineering (non-GMO) and new crosses were conducted with elite cultivars. The wheat germplasm containing Fhb7 will be released and made available to the wheat breeding community for variety development.
Accomplishments
1. Resistance against fusarium head blight in wheat. Wheat production has been continuously threatened by various biotic and abiotic stresses, such as disease pathogens, pests, and climate change. The genetic potential of wheat needs to be constantly improved by incorporating disease resistance genes for sustained wheat production under emerging disease threats. Fusarium head blight (FHB), also called scab, is a devastating fungal disease of wheat and barley in the U.S. and worldwide. Researchers in Lincoln, Nebraska, in collaboration with North Dakota State University scientists, incorporated a wild grass-derived FHB resistance gene Fhb7 into wheat by chromosome engineering (non-GMO). The Fhb7-containing wheat germplasm will be released and made available to the wheat breeding community for variety development.
2. Antimicrobial peptide resistance against the wheat bacterial leaf streak disease. 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. Researchers in Lincoln, Nebraska, found sequences encoding several plant-based antimicrobial peptides (AMP) provided resistance against the BLS disease. Wheat plants expressing AMPs through wheat streak mosaic virus (WSMV) as an expression vector were examined for antimicrobial activity against the bacterium causing the BLS. Ace AMP1 found in onion, and Defensin TK AMP D1, -D3, and Ta PDF 13 showed a statistically significant reduction in lesion size compared to WSMV-infected and mock-inoculated wheat. This study revealed that WSMV could be used as an efficient and high-throughput transient expression vector to screen a large number of AMPs for their activity in wheat.
3. Wheat germplasm with broad and specific disease resistance traits. Wheat germplasm with broad and specific disease resistance traits. Wheat production needs to be improved by identifying disease resistance traits from wild grasses and perennial wheat, followed by incorporating these genes into commercial wheat cultivars. Researchers in Lincoln, Nebraska, screened twelve perennial wheat genotypes for resistance against wheat streak mosaic virus and Triticum mosaic virus. Of these genotypes, PI 611891 and PI 611899 were rated as resistant to both viruses. Characterization of resistant genes found in these perennial wheat lines and their relationship to Wsm1 and Wsm2 genes will facilitate the development of germplasm for the nation’s wheat breeding programs by using traditional breeding approaches.
Review Publications
Tatineni, S., Hein, G.L. 2021. Tritimovirus and rymovirus (potyviridae). Encyclopedia of Virology. 4(3):797-804. https://doi.org/10.1016/B978-0-12-809633-8.21342-3.
Motta-Romero, H., Ferdinand, N., Boehm Jr, J.D., Rose, D.J. 2021. Effects of foliar fungicide on yield, micronutrients, and cadmium in grains from historical and modern Hard winter wheat genotypes. PLoS ONE. 16(3):e0247809. https://doi.org/10.1371/journal.pone.0247809.
