Location: Grain, Forage, and Bioenergy Research2012 Annual Report
1a. Objectives (from AD-416):
Objective 1. Identify and characterize WSMV determinant(s) of pathogenicity enhancement (disease synergism) and suppression of the host defense RNA silencing pathway. Objective 2. Identify and characterize WSMV determinant(s) responsible for semipersistent transmission by the wheat curl mite. Objective 3. Develop and evaluate transgenic wheat expressing WSMV non-structural proteins (P1, HC-Pro, P3, NIa) for gene complementation and pathogen-derived resistance to WSMV.
1b. Approach (from AD-416):
Experiments will be conducted using a cloned cDNA copy of the wheat streak mosaic virus (WSMV) genome from which infectious viral RNA is generated in vitro and tested for biological activity in wheat and other cereal species. We will use a Agrobacterium tumefaciens/Nicotiana benthamiana system based on induced silencing of a green fluorescent protein (GFP) transgene. Individual protein coding regions of wheat streak mosaic virus (WSMV) will be cloned into a binary shuttle vector in A. tumefaciens. Each WSMV protein will be tested for the ability to restore GFP expression in infiltrated leaves. Protein domains involved in the suppression phenotype will be identified by in vitro mutagenesis. Effects of mutants on virus pathogenicity will be tested by introducing identified mutations into an infectious WSMV cDNA clone and tested for disease synergism in mixed infections with maize chlorotic mottle virus. Experiments will be done. Yeast two hybrid methodology will be used to determine if potential interactions between WSMV structural proteins (coat protein and NIa) with a known mite transmission determinant, HC-Pro, will be evaluated using immunoprecipitation, yeast two hybrid and in vitro binding assays. Relevant protein domains will be identified by in vitro mutagenesis and evaluated for effects on mite transmission. Four WSMV proteins (P1, HC-Pro, P3, NIa) will be expressed in transgenic wheat and evaluated for trans-complementation with deletion mutants of WSMV. A lethal HC-Pro mutant will be expressed in wheat and evaluated for potential dominant-negative interference effects on WSMV infection. This project has been approved by the University of Nebraska IBC on March 23, 2007 at Biosafety level 2 (BL-2).
3. Progress Report:
This is the final report for 5440-22000-023-00D, which expired in FY 2012 and was replaced by 5440-22000-024-00D. Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are the most important viral pathogens of wheat in the U.S. Great Plains region, and the latter virus was found to be prevalent in the 2008 growing season. Both these viruses are spread from plant to plant by the wheat curl mite. Research was undertaken to understand the etiology of TriMV, and interactions of WSMV and TriMV with wheat and their mite vector. The sequence of TriMV genome was determined and identified as a distinct virus from WSMV. The viral coat protein of TriMV was expressed in bacteria and used to produce a high quality antiserum, providing a much-needed method for high-throughput detection method for the management of TriMV and for germplasm screening in wheat breeding programs. This antiserum also can be used to develop TriMV diagnostic kits by biotech companies. A new wheat variety, Mace, was developed in collaboration with an ARS plant geneticist researcher at Lincoln, NE which provides significant genetic resistance to WSMV and TriMV. Since both viruses are transmitted by the same vector, mixed infections by WSMV and TriMV are expected to be commonplace. In co-infections, WSMV and TriMV synergistically interact with each other resulting in increased virus concentrations in wheat plants which causes severe effects on wheat growth and yield in susceptible cultivars compared to the resistant cultivar Mace. Therefore, cultivar selection will be an important management strategy for the control of the WSMV/TriMV disease complex in wheat. The double virus resistance of Mace should encourage other wheat breeders to incorporate its virus resistance locus in their breeding programs as well. The gene for green fluorescent protein (GFP) was inserted into WSMV genome to form bright fluorescent aggregates in infected cells. The availability of GFP-tagged virus has facilitated rapid monitoring of virus presence/spread within a wheat plant during virus infection. GFP-tagged virus can be used as a non-destructive screening tool in the development of new WSMV-resistant wheat varieties. The GFP-tagged virus will also facilitate examining virus-host and virus-vector interactions to better understand the virus life cycle for possible clues to develop new disease management strategies by breaking the disease cycle. In addition to genetic resistance, plants are known to have innate defenses against invading viruses. In turn, viruses have evolved with proteins to overcome host defense mechanisms. The P1 protein of WSMV was identified as a suppressor of RNA silencing. The coat protein and HC-Pro genes of WSMV were shown to play critical roles in transmission of WSMV by the wheat curl mite. The outcome of this research provides a new WSMV/TriMV disease management strategy to wheat growers, a new tool for breeders to screen for WSMV resistance, a new diagnostic method for TriMV for extension Plant Pathologists, and advances the understanding of WSMV biology.
Young, B.A., Stenger, D.C., Ou, F., Morris, T., Tatineni, S., French, R.C. 2012. Tritimovirus P1 functions as a suppressor of RNA silencing and an enhancer of disease symptoms. Virus Research. 163: 672-677.