Location: Grain, Forage & Bioenergy Research
2010 Annual Report
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.
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.
The gene for green fluorescent protein (GFP) was inserted into an infectious clone of Wheat streak mosaic virus (WSMV). Expression of GFP requires it to be cleaved from the WSMV polyprotein by viral proteinases. We examined a range proteinase cleavage sites for release of GFP from the viral polyprotein. Expression of GFP was stable for at least seven serial passages and for 120 days after inoculation. This provides a valuable tool to study transient gene expression in plants and for screening for virus resistance.
A stable, infectious cDNA clone of the genome of triticum mosaic virus was developed. RNA transcribed in vitro was highly infectious to wheat, barley, and triticale, demonstrating that this clone has biological properties identical to native virus. This will greatly facilitate studies of viral gene structure and function. Improved understanding of virus replication and transmission may lead to new ways for controlling the disease and reducing yield losses.
Resistance of the maize line SDp2 to the type strain of wheat streak mosaic virus was shown to be due to a block in systemic infection but not to a lack of replication in inoculated leaves. By making gene exchanges between the type strain and a resistance breaking strain, Sidney 81, and by site-directed mutagenesis, we have identified four specific amino acid residues in the viral coat protein that enable the virus to overcome resistance to infection exhibited by SDp2. These differences in coat protein sequences likely correspond to resistance/susceptibility genes in the host and may impact studies designed to identify resistance genes in corn.
Albiach-Marti, M.R., Robertson, C., Gowda, S., Tatineni, S., Belliure, B., Garnsey, S.M., Folimonova, S.Y., Moreno, P., Dawson, W.O. 2010. The Pathogenicity Determinant of Citrus Tristeza Virus Causing the Seedling Yellows Syndrome is Located at the 3’-Terminal Region of the Viral Genome. Molecular Plant Pathology, Volume 11 (1), pages 55-67.
Tatineni, S., Graybosch, R.A., Hein, G.L., Wegulo, S.N., French, R.C. 2010. Wheat Cultivar-Specific Disease Synergism and Alteration of Virus Accumulation During Co-Infection with Wheat Streak Mosaic Virus and Triticum Mosaic Virus. Phytopathology, volume 100, pages 230-238.
Tatineni, S., Ziems, A.D., Wegulo, S.N., French, R.C. 2009. Triticum Mosaic Virus: A Distinct Member of the Family Potyviridae with an Unusually Long Leader Sequence. Phytopathology. 99:943-950.
Tatineni, S., Gowda, S., Dawson, W.O. 2010. Heterologous Minor Coat Proteins of Citrus Tristeza Virus Strains Affect Encapsidation, but the Coexpression of HSP70h and p61 Restores Encapsidation to Wild-Type Levels. Virology. Volume 402:262-270.