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

Location: Wheat, Sorghum and Forage Research

2020 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
Wheat streak mosaic virus (WSMV) is an economically important wheat virus causing $100 million annual yield losses in the Great Plains. Since all viruses are obligate parasites, several viral proteins must interact with host proteins for viral replication, movement, and disease development. The coat protein (CP) of WSMV was identified as a multifunctional protein required for virion assembly, viral movement, and disease development. Interactions between WSMV CP and wheat proteins were examined and found that WSMV CP interacted with at least ten wheat proteins. Selective wheat proteins interacting with CP will be used for the development of WSMV-resistant wheat through genome-editing technology. In the field, WSMV is transmitted by the wheat curl mite. WSMV-encoded CP and helper component-proteinase (HC-Pro) proteins were identified as viral determinants required for WSMV transmission by wheat curl mites. To facilitate wheat curl mite transmission of WSMV, viral determinants must interact with wheat curl mite proteins. Wheat curl mite cDNA library screen was performed against WSMV CP and HC-Pro and found that 10 and 16 wheat curl mite proteins were interacted with WSMV CP and HC-Pro, respectively. Selective wheat curl mite proteins interacting with WSMV CP or HC-Pro will be utilized to downregulate through virus-induced gene silencing or transgenic RNAi constructs to mitigate the wheat curl mite transmission of WSMV. Leaf streak or black chaff is a bacterial disease of wheat common in irrigated fields or in areas with abundant rainfall with yield losses of up to 40%. Antimicrobial peptides (AMPs) elicit innate defense against bacterial and fungal infections. Thirteen AMPs have been screened for their activity against wheat bacterial streak disease by expressing AMPs in wheat using WSMV as an expression vector. Wheat plants infected with WSMV expressing four AMPs (one from onion and three from wheat) substantially reduced the size of lesions developed by the causal agent of bacterial streak disease. Triticum mosaic virus (TriMV) is a recently reported economically important wheat-infecting virus in the Great Plains and is transmitted by the wheat curl mite. Previously, the CP of TriMV has been reported to be involved in cross protection. In this study, TriMV CP amino acids 41 to 250 were identified as the minimal region required for cross protection in wheat. These findings revealed that CP amino acids 41-250 can be used to develop a protein-based disease management strategy against TriMV. The CP and NIa-Pro proteins of WSMV were identified as determinants of WSMV cross protection. The CP and NIa-Pro genes of WSMV were cloned in a binary vector and developed transgenic wheat at the Plant Transformation Core Facility, University of Nebraska-Lincoln. Several independent events of T0 transgenic plants with WSMV CP, NIa-Pro, or CP+NIa-Pro genes were obtained. These transgenic plants are being screened for resistance against WSMV. RNA interference-based transgenic wheat provided dual resistance against WSMV and TriMV at 25C or higher but not at 22C or below. In contrast, wheat cultivars with Wsm1 (Mace) or Wsm2 (KS06HW79) genes are resistant to WSMV and TriMV at or below 22C only. To obtain wheat cultivars that are resistant to WSMV and TriMV at a wide range of temperatures, the T4 transgenic wheat lines were crossed with Wsm1- or Wsm2-containing wheat cultivars. The first (F1) generation of wheat with both transgene and Wsm1 or Wsm2 genes were selfed through the single seed descent method up to the seventh (F7) generation. Several near homogenous populations of F7 generation wheat of different genetic backgrounds were obtained for resistance screening at a wide range of temperatures. Gene stacking of hairpin transgene together with Wsm1 and Wsm2 was performed to obtain durable dual resistance against WSMV and TriMV at a wide range of temperatures. The hairpin transgene, Wsm1, and Wsm2 genes were stacked by crossing a wheat line with a hairpin transgene and Wsm1 or Wsm2 genes with that of wheat cultivars containing Wsm2 or Wsm2 genes, respectively. The F1 generation of wheat with the transgene, Wsm1, and Wsm2 were selfed through the single seed descent method up to the second (F2) generation.

Accomplishments
1. Dual resistant transgenic wheat against synergistically interacting Wheat streak mosaic virus and Triticum mosaic virus. Wheat streak mosaic disease complex, caused by Wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), and High Plains wheat mosaic virus, is the most economically important viral disease of wheat in the Great Plains with about $100 million annual yield loss. Since all three viruses are transmitted by the wheat curl mite, mixed infections in a combination of any two or all three viruses have been reported in growers' fields with reduced yield. WSMV and TriMV synergistically interact in co-infected wheat with increased accumulation of both viruses and with enhanced disease severity. Hence, wheat cultivars with resistance to at least two of the three mite-transmitted viruses would minimize yield loss. ARS researchers at Lincoln, Nebraska, in collaboration with the University of Nebraska-Lincoln scientists developed RNA-interference-based transgenic wheat lines that provided dual resistance to both WSMV and TriMV at 25C or above. However, the transgenic wheat lines were susceptible to WSMV and TriMV at 20C, but both viruses accumulated at significantly lower levels compared to those in nontransgenic wheat. The availability of transgenic wheat lines in this study will facilitate stacking the low temperature-sensitive transgene with high temperature-sensitive Wsm1 or Wsm2 genes to obtain wheat cultivars with dual resistance at a wide range of temperatures, which will benefit wheat growers for the management of wheat streak mosaic disease.

Review Publications
Gupta, A.K., Tatineni, S. 2019. P1 binds to dsRNAs without size and sequence specificity and a GW motif is crucial for suppression of RNA silencing. Viruses. 11(5):472. https://doi.org/10.3390/v11050472.
Gupta, A.K., Tatineni, S. 2019. RNA silencing suppression mechanisms of Triticum mosaic virus P1: dsRNA binding property and mapping functional motifs. Virus Research. 269:197640. https://doi.org/10.1016/j.virusres.2019.197640.
Palmer, N.A., Rekalakunta Venka, C., Muhle, A., Tatineni, S., Yuen, G., Edme, S.J., Mitchell, R., Sarath, G. 2019. Transcriptome divergence during leaf development in two contrasting switchgrass (Panicum virgatum L.) cultivars. PLoS One. 14(9):e0222080. https://doi.org/10.1371/journal.pone.0222080.
Tatineni, S., Stewart, L.R., Sanfacon, H., Wang, X., Navas-Castillo, J., Hajimorad, R.M. 2020. Fundamental aspects of plant viruses-An overview on focus issue articles. Phytopathology. 110:6-9. https://doi.org/10.1094/PHYTO-10-19-0404-FI.
Tatineni, S., Sato, S., Nersesian, N., Alexander, J.A., Quach, T., Graybosch, R.A., Clemente, T.E. 2020. Transgenic wheat harboring an RNAi element confers dual resistance against synergistically interacting wheat streak mosaic virus and triticum mosaic virus. Molecular Plant-Microbe Interactions. 33(1):108-122. https://doi.org/10.1094/MPMI-10-19-0275-R.