Location: Wheat, Sorghum and Forage Research
2019 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.
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.
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) and triticum mosaic virus (TriMV), two economically important viruses infecting wheat in the Great Plains region, are transmitted by the wheat curl mite. Co-infection with these two viruses exacerbates disease on wheat with increased yield losses. As part of Objective 1, identifying viral proteins that cause disease will facilitate the identification of targets to develop novel disease management strategies. TriMV genetic determinants involved in disease development were mapped by inserting select TriMV genes into the WSMV genome, followed by examining the symptom phenotype on wheat. Wheat plants infected by WSMV expressing TriMV encoded P1, HC-Pro, NIa, or coat protein (CP) elicited more severe symptoms compared with wild-type WSMV. These data revealed that TriMV encoded P1, HC-Pro, NIa and CP are pathogenicity determinants that are responsible for TriMV disease phenotype in wheat. Additionally, these data suggest that TriMV disease management strategies in wheat should target the P1, HC-Pro, NIa, and CP genes.
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, Nebraska, identified CP and NIa-Pro of WSMV as elicitors of superinfection exclusion. The complete CP, CP amino acids 100-300 and NIa-Pro coding sequences of WSMV were cloned in a binary vector between the ubiquitin promoter and NOS-terminator. The binary vectors with WSMV CP or NIa-Pro are being used for wheat transformation at the University of Nebraska-Lincoln.
The wheat curl mite transmits WSMV in a persistent propagative manner. Viral persistence and virus-vector coevolution may potentially influence vector gene expression to prolong viral association and increase the vector’s transmission efficiency. In order to understand the transcriptomic responses of wheat curl mite to WSMV, RNA sequencing was performed to generate differential transcriptomes of WSMV-viruliferous and aviruliferous mites. Among 7,785 de novo assembled unigenes, 1,513 were differentially expressed in viruliferous mites. The majority of these unigenes were downregulated in viruliferous mites in comparison to only a few unigenes that were upregulated. Transcriptomic data analyses revealed that transcriptional changes may inhibit the immune response of what curl mite, which prolongs viral association and alters wheat curl mite development to expedite population expansion, both of which could enhance viral transmission.
Interactions between wheat curl mite proteins and viral proteins facilitate the transmission of WSMV to wheat. Identification of wheat curl mite proteins that interact with viral factors facilitates disruption of interactions between viral and vector proteins. Total RNA was isolated from aviruliferous wheat curl mites collected from healthy wheat, and this RNA is being used for identification of wheat curl mite proteins that interact with WSMV proteins.
As part of Objective 2, transgenic wheat with a hairpin sequence from WSMV and TriMV are resistant to both WSMV and TriMV at 25°C or higher but not at 22°C or below. In contrast, wheat cultivars with Wsm1 (Mace) or Wsm2 (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 Wsm1- or Wsm2-containing wheat cultivars or lines. The F1 generation of wheat with both transgene and Wsm1 or Wsm2 gene were selfed through the single seed descent method up to the F6 generation to obtain homogenous populations of different genetic backgrounds. A large number of F6 generation wheat was obtained for resistance screening at a wide range of temperatures.
Understanding the key strategies viruses use to overcome natural defenses in plants could lead to the development of novel strategies for the management of viral diseases. Virus-encoded suppressors of RNA silencing play a pivotal role in combating host defense systems. Studying suppressors of RNA silencing and unraveling their molecular mechanisms are important not only to understand their diversity, regulation and evolution but also to develop and fine-tune approaches for developing new disease management strategies. WSMV and TriMV utilize their P1 proteins to suppress host-induced RNA silencing. The P1 proteins of these two viruses suppress host RNA silencing at multiple levels by protecting virus-specific RNAs from degradation. This research revealed that the P1 genes of WSMV and TriMV are primary targets for developing future molecular-based methods for managing these viruses. Additionally, the P1 proteins of WSMV and TriMV can be used to suppress host RNA silencing for efficient expression of transgenes in wheat.
HPWMoV is an economically important virus on wheat and maize in the Great Plains regions. This virus contains an octapartite negative-sense RNA genome that encodes eight proteins. The P7 and P8 proteins encoded by RNA 7 and 8, respectively, were identified as suppressors of RNA silencing. The P7 and P8 proteins of HPWMoV utilize two distinct pathways to suppress host defense mechanisms. This research also revealed that the P7 and P8 genes are primary targets for developing future molecular-based methods for the management of High Plains disease.
Accomplishments
1. Wheat streak mosaic virus (WSMV) manipulates wheat curl mite gene expression for efficient transmission. WSMV is one of the most damaging pathogens of wheat in the Great Plains as it can cause yield losses of up to 100%. WSMV is transmitted exclusively by the wheat curl mite, which can quickly reach high population densities in the field and can transmit many other viruses, such as High Plains wheat mosaic virus, brome streak mosaic virus, and triticum mosaic virus. ARS scientists at Lincoln, Nebraska, found that WSMV alters the biology and behavior of mites in order to facilitate and expedite viral transmission by increasing reproduction and population levels, expediting maturation to adulthood, and increasing feeding rates. In order to understand how WSMV alters the physiology of wheat curl mites, gene expression levels in infected and uninfected mites were compared. Overall, lower expression levels of genes linked to development and immunity were observed in infected wheat curl mites. This study facilitates the understanding of how WSMV manipulates wheat curl mites to increase viral transmission rates by enhancing the number and growth and development of wheat curl mites. This research will ultimately lead to better prevention and control tactics to reduce wheat infection rates and yield losses in the field.
Review Publications
Gupta, A.K., Hein, G.L., Graybosch, R.A., Tatineni, S. 2018. Octapartite negative-sense RNA genome of high plains wheat mosaic virus encodes dual suppressors of RNA silencing. Virology. 518: 152-162. https://doi.org/10.1016/j.virol.2018.02.013.
Tatineni, S., Hein, G.L. 2018. Genetics and mechanisms underlying transmission of Wheat streak mosaic virus by the wheat curl mite. Current Opinion in Virology. 33:47-54. https://doi.org/10.1016/j.coviro.2018.07.012.
Guptar, A.K., Scully, E.D., Palmer, N.A., Geib, S.M., Sarath, G., Hein, G.L., Tatineni, S. 2019. Wheat streak mosaic virus alters the transcriptome of its vector, wheat curl mite (Aceria tosichella Keifer), to enhance mite development and population expansion. Journal of General Virology. 100(5):889-910. https://doi.org/10.1099/jgv.0.001256.
Rekalakunta Venka, C., Palmer, N.A., Edme, S.J., Sarath, G., Kovacs, F., Yuen, G., Mitchell, R., Tatineni, S. 2019. A two amino acid difference in the coat protein of satellite panicum mosaic virus isolates is responsible for differential synergistic interaction with panicum mosaic virus. Molecular Plant-Microbe Interactions. 32:479-490. https://doi.org/10.1094/MPMI-09-18-0247-R.
Tatineni, S., Alexander, J.A., Gupta, A.K., French, R.C. 2018. Asymmetry in synergistic interaction between wheat streak mosaic virus and triticum mosaic virus in wheat. Molecular Plant-Microbe Interactions. [Online Journal] Available:https://apsjournals.apsnet.org/doi/10.1094/MPMI-07-18-0189-R
