Location: Grain, Forage, and Bioenergy Research2013 Annual Report
1a. Objectives (from AD-416):
Develop transgenic wheat cultivars resistant to Wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), and Wheat mosaic virus (High Plains virus, HPV).
1b. Approach (from AD-416):
WSMV, TriMV, and HPV are economically important viruses in the Great Plains region of the USA, and all three viruses are vectored by wheat curl mites. Wheat cultivars with a broad-spectrum resistance to multiple viruses are ideally suitable for the effective management of viral diseases in wheat. It has been demonstrated that a transgene with separate short hairpins (200-300 nucleotides) derived from different viruses can provide resistance to multiple viruses. Hence, we proposed to develop hairpin constructs from the conserved regions of WSMV, TriMV and HPV genomic RNA sequences between the maize ubiquitin promoter and NOS-terminator sequences in a binary vector. Similarly, putative microRNA precursor sequences from the genomic RNAs of WSMV, TriMV and HPV will be identified and cloned in a binary vector between the maize ubiquitin promoter and NOS-terminator. The binary plasmids containing expression cassettes comprising the hairpin or microRNA precursor sequences from WSMV, TriMV and HPV will be used to transform wheat. The transformants containing hairpins or microRNA precursor sequences will be screened for resistance against WSMV, TriMV and HPV.
3. Progress Report:
Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are economically important wheat viruses in the Great Plains region. Because these two viruses are transmitted by wheat curl mites, mixed infections with increased yield losses are common in growers’ fields. Wheat cultivar Mace with Wsm-1 gene, developed by ARS scientists, provides temperature-sensitive resistance against WSMV and TriMV. The temperature-sensitive resistance of Mace needs to be improved by developing new wheat cultivars with additional sources of broad-spectrum resistance against multiple wheat viruses. Recently, RNA-interference (RNAi) based transgenic plants, using multiple hairpin sequences from different viruses, provided a robust and broad-spectrum resistance to multiple viruses. A hairpin construct of WSMV and TriMV, each with ~200 nucleotide RNA sequence, was engineered under the ubiquitin promoter in a plant transformation vector. This construct was used to transform wheat cv. CB 037, and 10 independent events of transgenic wheat lines were obtained. Transgenic wheat lines were screened for the presence of transgene by NPTII assay and by Northern blot hybridization for the presence of small interfering RNAs generated from the transgene as the result of host defense mechanisms. Transgenic wheat lines from the T1 generation were screened for resistance against WSMV and TriMV. Wheat lines from four independent transgenic events provided mild to moderate resistance to WSMV but not to TriMV. Seeds from moderately resistant T2 wheat lines were collected for further screening against WSMV and TriMV resistance. Additional RNAi constructs with a single hairpin sequence comprising WSMV and TriMV with an intron loop and another construct with two independent cassettes of WSMV and TriMV hairpins with intron loops were developed in a plant transformation vector. These two constructs will be used for wheat transformation to obtain additional transgenic wheat lines.