Location: Wheat, Sorghum and Forage Research2018 Annual Report
1a. Objectives (from AD-416):
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. Subobjective 1A: Examine virus-host interactions of Wheat streak mosaic virus (WSMV) to identify means of disruption and effect control of viral diseases. Subobjective 1B: Examine virus-virus interactions of WSMV and Triticum mosaic virus (TriMV) to identify proteins responsible for superinfection exclusion. Subobjective 1C: Examine interactions between WSMV and wheat curl mites to identify means of interrupting vector transmission. Objective 2: Develop and characterize transgenic wheat for resistance to WSMV and TriMV, and pyramid transgenes with natural resistance genes. Subobjective 2A: Develop transgenic wheat with WSMV and/or TriMV genes involved in superinfection exclusion (cross protection). Subobjective 2B: Pyramid transgenes of WSMV and TriMV with natural resistance genes, Wsm1 and/or Wsm2. Subobjective 2C: Knockout wheat curl mite transmission of WSMV by silencing WSMV-interacting mite gene(s) through RNAi approach in transgenic wheat. 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. Subobjective 3A: Characterize genes from perennial wheat conferring resistance to WSMV and TriMV, determine if they are unique or allelic to Wsm1 and Wsm2, and define mechanism of virus resistance. Subobjective 3B: Determine whether a truncated version of an intermediate wheatgrass chromosomal introgression would serve as an improved vehicle for deployment of Wsm1 in wheat cultivars. Subobjective 3C: Express anti-microbial peptides using TriMV-based expression vectors, and test for efficacy at control of bacterial streak in wheat. Objective 4: Develop and characterize adapted winter wheat germplasm with broad and specific disease resistance, and with improved grain nutritional quality. Subobjective 4A: Develop wheat with low levels of grain phytic acid, and effective field resistance to predominant Great Plains fungal and bacterial pathogens.. Subobjective 4B: Identify Great Plains adapted hard winter wheat germplasm with resistance to multiple forms of Ug99 stem rust. Subobjective 4C: Coordinate the Hard Winter Wheat Regional Nursery Program and use the nurseries to: 1) determine the yield potential and stability of newly developed low phytate and stem rust resistant germplasm, and 2) distribute germplasm to Great Plains breeding programs.
1b. Approach (from AD-416):
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.
3. Progress Report:
Wheat streak mosaic virus (WSMV) is a severe symptom-inducing virus on wheat compared to mild mosaic and mottling symptoms elicited by Triticum mosaic virus (TriMV). As part of Objective 1, WSMV genetic determinants involved in disease development were mapped by inserting each WSMV gene into the TriMV genome, followed by examining the symptom phenotype on wheat. Wheat infected by TriMV expressing WSMV encoded NIa-VPg, NIa-Pro, or TriMV coat protein (CP) elicited more severe symptoms than the wild-type TriMV. These data revealed that WSMV encoded NIa-VPg, NIa-Pro and CP most likely contribute for wheat streak mosaic disease phenotype in wheat. Hence, these data suggest that the NIa-VPg, NIa-Pro and CP genes are the most likely targets for the management of wheat streak mosaic disease in wheat. CP was reported as an effector protein of superinfection exclusion in wheat. Also relevant to Objective 1, the minimal region of TriMV CP involved in SIE was examined by introducing a series of genetic mutations, followed by insertion back into WSMV. Wheat plants infected by WSMV expressing TriMV CP harboring certain mutations failed to superinfect by a GFP-tagged TriMV. In contrast, wheat plants infected by WSMV a mutation resulting deletion of amino acids 41 to 250 from the TriMV coat protein gene were efficiently infected by a GFP-tagged TriMV. These data indicate that the middle portion of TriMV CP cistron (amino acids 41 to 250) is required for an effector function of superinfection exclusion activity. These studies indicate possible means by which transgenic wheat plants might ultimately defeat their viral invaders. As part of Objective 1, wheat curl mites propagated on WSMV-infected and buffer-inoculated wheat plants were collected at five-week postinfestation, followed by total RNA extraction. Wheat curl mite total RNA was used for high-throughput RNA sequencing. Analyses of wheat curl mite transcriptome indicated that several mite genes were differentially expressed in mites infested on WSMV-infected wheat compared to those on buffer-inoculated plants. WSMV and TriMV interact synergistically in co-infected wheat with dramatically increased disease severity and yield loss with elevated accumulation of both viruses. Since both these viruses are transmitted by wheat curl mites, double infections in field-grown wheat are common, resulting in disease synergism. Understanding the underlying mechanisms of synergistic interaction between WSMV and TriMV, a key component of Objective 1, would facilitate developing strategies to minimize losses from disease synergism. The effects of prior infection of wheat by either of ‘synergistically interacting partner’ (SIP) viruses (WSMV and TriMV) on the establishment of local and systemic infection by the other SIP virus are not known. This study found that prior infection of wheat by WSMV or TriMV negatively affected the onset and size of local foci elicited by subsequent SIP virus infection compared to those in buffer-inoculated wheat. These data revealed that prior infection of wheat by a SIP virus has no measurable advantage for another SIP virus on the initiation of infection and cell-to-cell movement. In TriMV-infected wheat, WSMV exhibited accelerated long-distance movement and increased accumulation of genomic RNAs compared to those in buffer-inoculated wheat, indicating that TriMV-encoded proteins complemented WSMV for efficient systemic infection. In contrast, TriMV displayed delayed systemic infection in WSMV-infected wheat with fewer genomic RNA copies in early stages of infection compared to those in buffer-inoculated wheat. However, during late stages of infection, TriMV accumulation in WSMV-infected wheat increased rapidly with accelerated long-distance movement compared to those in buffer-inoculated wheat. This study suggests that interactions between WSMV- and TriMV-encoded proteins result in disease synergism and disruption of these interactions most likely prevent synergistic interactions between viruses. Objective 2 includes screening T4 generation transgenic wheat with a hairpin sequence from WSMV and TriMV Transgenic wheat plants were resistant to both WSMV and TriMV at 25°C or higher but not at 22°C or below. In contrast, wheat cultivars with Wsm-1 (Mace) or Wsm-2 (KS06HW79) genes are resistant to WSMV and TriMV at or below 22°C. To obtain wheat cultivars that are resistant to WSMV and TriMV at a wide-range of temperatures, the T4 transgenic wheats were crossed with Wsm-1 or Wsm-2 containing wheat cultivars or lines. The F1 generation of wheat with both transgene and Wsm-1 or Wsm-2 gene were selfed to obtain the F2 progeny. 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 wheats is increasing, but these wheats must have low or nil levels of grain PPO. As part of Objective 4, mutations leading to a lack of PPO were found in 1930’s era wheats from Australia housed in the USDA collection. Through successive generations of mating and phenotypic recurrent selection, the nil PPO trait has been moved into Great Plains adapted wheats. PPO was measured in wheat breeding lines grown in Yuma, Arizona in 2017 and the nil PPO trait was found to be stable in 100+ breeding lines. These lines were seeded in Nebraska environments in the fall of 2017 for agronomic and disease resistance evaluations. Grain yield data from the 2018 harvest will be used to select and release to wheat breeding programs for use in developing more adapted hard white winter wheat cultivars.
1. Asymmetry in synergistic interaction between Wheat streak mosaic virus and Triticum mosaic virus in wheat. Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are economically important viruses infecting wheat in the Great Plains region of the USA. WSMV and TriMV interact synergistically in co-infected wheat with dramatically increased disease severity and yield loss with elevated accumulation of both viruses. Since both of these viruses are transmitted by wheat curl mites, 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. ARS scientists in Lincoln, Nebraska found that WSMV benefited from prior infection of wheat by TriMV during early stages of systemic infection while prior infection of wheat by WSMV negatively affected TriMV systemic infection. However, both viruses benefited from each other during late stages of synergistic interaction with acute symptom phenotype and increased accumulation of both interacting viruses. This study suggests that interactions between WSMV- and TriMV-encoded proteins enhance disease symptoms and suppress grain yield disruption of these interactions may prevent synergistic interactions between these viruses and help stabilize wheat grain yield under pathogen pressure.
2. Development and release of low phytic acid (LPA) germplasm. Low phytic acid mutants of wheat (Triticum aestivum L.) can reduce concentrations of this kernel anti-nutrient by one-third. The reduction of phytate increases the bioavailability and subsequently, the gut absorption of minerals in monogastric animals, including humans. ARS scientists at Lincoln, Nebraska developed novel breeding stockby mating of lines carrying a mutation (lpa-1) to Nebraska winter wheats. Multi-location grain yield testing, and selection for the low phytate (LPA) trait, resulted in the identification of eight LPA breeding lines adapted to the Great Plains of North America. The highest yielding LPA lines were not significantly different in grain yield from the adapted controls ‘Anton’ and ‘Intrada’, significantly higher in grain yield than ‘Big Sky’ and ‘Siouxland’, but significantly lower than the two highest yielding controls, ‘Freeman’ and ‘Ruth’. Overall, there were no significant differences in grain yield, grain volume weight, and grain protein concentration between the LPA lines and the mean values of the adapted controls. On average, the LPA lines had 18% more zinc than the adapted controls. Grain yield data obtained in diverse environments in Nebraska indicates no grain yield reduction associated with the low phytate trait, and shows the potential for development of high-yield cultivars with high mineral and low antinutrient concentrations. 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.