Location: Grain, Forage, and Bioenergy Research2009 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
This research involves the interaction of streak mosaic virus (WSMV) with both plant hosts as well as with its mite vector. Experiments were designed identify and characterize WSMV determinant(s) of pathogenicity enhancement and suppression of the host defense RNA silencing pathway; identify and characterize WSMV determinant(s) responsible for semipersistent transmission by the wheat curl mite; and develop and evaluate transgenic wheat expressing WSMV non-structural proteins. An infectious clone of the type strain of WSMV was constructed for experiments designed to determine WSMV host range determinants. By exchanging genes between the sidney isolate (pathogenic on SDp2 corn) and the type isolate (nonpathogenic on SDp2 corn) we determined that the viral coat protein (CP) was the major determinant of host range. WSMV HCPro, NIa, and CP have been cloned in yeast new yeast two hybrid vectors for investigating protein-protein interactions. The first four amino acids of HCPro were found to be dispensable for mite transmission but deletion of eight amino acids abolished vector transmission. The reporter gene green fluorescent protein (GFP) was cloned downstream of the HCPro and NIa genes of WSMV and transferred into binary vectors for wheat transformation. Ten independent transgenic wheat lines expressing WSMV coat protein were developed. Antisera were made to all WSMV nonstructural proteins (P1, HCPro, P3, CI, NIa, and NIb) which will aid in screening transgenic wheat for gene and protein expression levels. cDNA of a new wheat virus, Triticum mosaic virus, was cloned and the complete genome sequence was determined. Phylogenetic analysis revealed that Triticum mosaic virus should be placed in a new genus in the plant virus family Potyviridae and this taxonomic proposal was approved by the Potyviridae Study Group of the International Committee on Taxonomy of Viruses. We found that wheat infected both Triticum mosaic virus and WSMV exhibited more severe symptoms than single infections of wheat with either virus. Single mite transmission assays revealed that Triticum mosaic virus is effieciently transmitted by the Nebraska biotype of the wheat curl mite.
1. Characterization of a new emerging virus of wheat. The complete genome sequence of Triticum mosaic virus, which recently has been found infecting wheat in the Great Plains region, has been determined by ARS virologists with the Grain, Forage and Bioenergy Research Unit. This sequence information is useful in several ways. It allowed the development of sensitive diagnostic assays using the polymerase chain reaction. The sequence allowed the identification of Triticum mosaic virus as a member of the Potyviridae family of plant viruses but distinct enough from other viruses in the family to be considered the type species of a new genus. The Potyviridae Study Group of the International Committee on Taxonomy of Viruses has accepted the proposed genus name "Poacevirus". Triticum mosaic virus is unlike any other virus found in the US and, in fact, its closest relative is Sugarcane streak mosaic virus found in South Asia.
2. Determination of the effects of mixed infections of Wheat streak mosaic virus and Triticum mosaic virus on wheat. Diseased wheat samples submitted for diagnosis have been found to be infected with both Wheat streak mosaic virus and Triticum mosaic virus over the past two years. This is not surprising because both viruses are carried by the same vector, the wheat curl mite. When plants are infected with two viruses at the same time disease symptoms are often much more severe than plants only infected by only one virus. Indeed this is the case in greenhouse tests for several wheat varieties, with doubly infected plants showing pronounced yellowing and stunting. Fortunately, the Wheat streak mosaic virus-resistant wheat variety Mace, released by an ARS plant geneticist with Grain, Forage and Bioenergy Research Unit, was shown to be tolerant to Triticum mosaic virus alone, and exhibit little to no increased yellowing and stunting when infected by both viruses.
3. Development of Wheat streak mosaic virus expressing a fluorescent protein as a genetic tool. The complete genome of Wheat streak mosaic virus has been cloned in bacteria from which infectious virus can be regenerated. This has allowed ARS virologists with the Grain, Forage and Bioenergy Research Unit to make mutations in particular genes to determine gene function, such as repression of the plant's innate anti-viral defences, and genes required for mite transmission. Importantly, extra genes can be added to Wheat streak mosaic virus' genome as well. Expression of a fluorescent protein allows the rate and extent of virus replication and movement to be monitored over multiple time points using a non-destructive assay. This can provide an easy way of screening germplasm for virus resistance. Work has been started to develop a similar assay for Triticum mosaic virus.
Tatineni, S., Afunian, M.R., Gowda, S., Hilf, M.E., Bar-Joseph, M., Dawson, W.O. 2009. Characterization of the 5’- and 3’-terminal subgenomic RNAs produced by a capillovirus: evidence for a CP subgenomic RNA. Virology 385 (2009) 521–528.