Location: Grain, Forage, and Bioenergy Research2010 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.
3. Progress Report
The ability of the wheat curl mite to simultaneously transmit both wheat streak mosaic virus and the newly emergent wheat virus, triticum mosaic virus was experimentally confirmed. We found that the virus content of both Wheat streak mosaic virus and Triticum mosaic virus was two to seven fold higher in mixed infections compared to single infections with each virus in susceptible wheat varieties. On the other hand, virus content of both viruses was much lower in infected plants of the Wheat streak mosaic virus–resistant variety Mace. Therefore, the risk of spread of both viruses by the wheat curl mite may be increased in susceptible wheat varieties. 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.
1. Mixed infections of wheat streak mosaic virus (WSMV) and the newly emergent triticum mosaic virus (TriMV) multiply infection in susceptible wheats. As WSMV and, TriMV, are transmitted by the same mite vector, mixed infections by the two viruses are expected to be commonplace. ARS scientists in Lincoln, NE examined the effects of mixed infections in two wheat varieties susceptible to WSMV, and one variety, Mace, that is resistant to WSMV. There, research indicated that there was increased virus content in susceptible varieties infected by both viruses, which could enhance their natural spread by mites. Thus, it is very important that growers use resistant varieties where available and follow current cultural disease control practices such as delayed planting of susceptible varieties in the fall.
2. Development of an improved wheat streak mosaic virus clone for tracking viral infection. ARS scientists in Lincoln, NE have constructed a clone of Wheat streak mosaic virus containing a gene for green fluorescent protein (GFP). GFP gives a green fluorescence to infected tissue when viewed with a microscope under blue light. The GFP gene was modified so that it produces bright green aggregates in infected cells. This has greatly improved the ability to trace spread of the virus in infected plant tissues and will lead to a better understanding of virus-host interactions. It additionally provides a valuable tool for screening plants for disease resistance.
3. Development of infectious clones of Triticum mosaic virus. Triticum mosaic virus is the type member of the recently established Poacevirus genus of the potyvirus family. Little is known about the biology of these viruses and no molecular studies have been done. ARS scientists in Lincoln, NE have developed the first infectious clones for genus of viruses and established their biological activity. This will greatly facilitate studies of viral gene structure and function and may lead to new ways for controlling the disease and reducing yield losses.
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.