2011 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 abilty 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).
Wheat streak mosaic virus (WSMV) is transmitted by the wheat curl mite. One viral gene, Helper component-proteinase, is required for mite transmission. Six mutations were individually introduced into the carboxyl-terminus of the coat protein of WSMV to replace acidic amino acid residues with alanine. This region of the coat protein was chosen because it is thought to be close to the surface of virus particles. Mutated virus were inoculated to wheat and then tested for mite transmission. Transmission rates of an aspartic acid replacement at position 289 and a similar substitution at position 326 were reduced to 15% compared to 55% for wild type virus. The ability of WSMV and triticum mosaic virus (TriMV) to alter disease symptoms in mixed infections with two other cereal viruses was tested. Wheat infected with brome mosaic virus in combination with either WSMV or TriMV had much more severe symptoms than singly infected plants. Similar results were obtained with barley stripe mosaic virus. This suggests that TriMV is able to suppress RNA silencing in wheat to a similar degree as WSMV. Three populations of wheat curl mite were tested for their abilities to transmit TriMV. These mite populations, designated as Nebraska, Montana and South Dakota biotypes can be distinguished by mitochondrial DNA cytochrome oxidase subunit 1 gene sequences. All three biotypes are efficient vectors of WSMV as was shown by single mite transfers from infected source plants to test plants. However, only the Nebraska biotype was able to transmit TriMV using single mites. Transmission of TriMV by Montana and South Dakota biotypes required the transfer of hundreds of mites from infected source plants to test plants.
Wheat streak mosaic virus coat protein is a mite transmission determinant. Wheat streak mosaic is the most economically important virus in the Great Plains region of the US and is transmitted by wheat curl mites. Previously, the helper component-proteinase of Wheat streak mosaic virus was identified as a determinant of wheat curl mite transmission. ARS scientist in Lincoln, NE, in collaboration with scientists at the University of Nebraska-Lincoln identified the coat protein of Wheat streak mosaic virus as an additional viral determinant involved in efficient mite transmission. Mutations in the coat protein affected the mite transmission efficiencies by 2-3-fold compared to that of wild-type virus. Identification of helper component-proteinase and coat protein as mite transmission determinants will provide scientists with information on 'candidate genes' to produce virus resistant transgenic wheat by disrupting the virus life cycle.
Only one strain of wheat curl mite is an efficient vector of triticum mosaic virus. Triticum mosaic virus is an emergent virus infecting wheat in the US. It, like wheat streak mosaic virus, is transmitted by the wheat curl mite. Previous studies determined that there are genetically distinct strains of the wheat curl mite present in the region. Researchers in Lincoln, NE, in cooperation with the University of Nebraska, investigated whether all strains of the wheat curl mite are equally competent to transmit triticum mosaic virus. They demonstrated that one mite strain was much more efficient in transmitting the virus than other strains, while all mite strains are equally capable of transmitting wheat streak mosaic virus. Consequently the prevalence of triticum mosaic virus is expected to vary from place to place and from year to year depending on the strain composition of wheat curl mite populations. This information will be useful to extension plant pathologists for understanding why the incidence of triticum mosaic virus may differ from that of wheat streak mosaic virus. Ultimately, the information should assist wheat growers better manage the crop.
Tatineni, S., Mcmechan, A., Hein, G., French, R.C. 2011. Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: Formation of dense fluorescent aggregates for sensitive virus tracking. Virology. 410:268-281, doi:10.1016/j.virol.2010.10.043.
Tatineni, S., Van Winkle, D.H., French, R.C. 2011. The N-terminal region of Wheat streak mosaic virus coat protein is a host- and strain-specific long-distance transport factor. Journal of Virology. 85: 1718-1731.