Project Number: 2072-21000-055-013-G
Project Type: Grant
Start Date: Aug 1, 2020
End Date: Oct 31, 2022
1. Construct infectious cDNA clones of GRBaV, and their insertion into embryogenic cultures using particle bombardment and Agrobacterium-mediated transformation. 2. Evaluate red and white wine grape cultivars for responses to GRBaV infection. 3. Examine different tissues (root, stem, leaves) of infected and healthy vines (greenhouse potted, field-grown, and transgenic healthy and infected vines) for specific symptomatology at the whole vine and ultrastructure levels.
For Objectives 1 and 2, infectious clones will be produced to characterize a virus at the molecular and biological levels. Cloning of the genome of Geminiviridae, including Grapevine red blotch-associated virus (GRBaV), should be relatively easy because of its small genome and the presence of the virus double-stranded (replicative) DNA form in infected plants. Plant samples used in this study will be collected from infected grapevines in CA, WA, and OR. To fully sequence and assemble the virus genome, total DNA will be isolated from infected tissues using the CTAB method. The viral genome will be cloned. The quality and quantity of DNA samples will be evaluated on 1% agarose gels and using a NanoDrop-1000 spectrophotometer (Thermo Fisher Scientific). Primers used for sequencing will be designed based on the predicated genomic sequence. Seven grape cultivars, Cabernet Franc, Cabernet Sauvignon, Shiraz, Chardonnay, White Riesling, Pinot noir, and Sevyal Blanc, will be used for optimization of biolistic bombardment for virus infection. For Objective 3, healthy (25) and infected (25) potted vines (Pinot noir) will be generated from rooted-cuttings of virus-free vines, and vines solely infected with the Red Blotch virus from vineyards tested positive for GRBaV. These vines will be used to compare the growth and development and cellular details of the infected and healthy potted vines with the transgenic and non-transgenic vines. Leaf, stem, and root samples from healthy and infected vines (at least 6 vines each) will be collected at different developmental stages for analysis.