Development of Virus Induced Gene Silencing Tools for Functional Genomics in Grapevine

Grapevine is perhaps the most widely grown fruit crop. To understand the genetic make-up so as to improve the yield and quality of grapes and grape products, researchers in Europe have recently sequenced the genomes of Pinot noir and its inbred. As expected, function of many grape genes is unknown. Functional genomics studies have become the major focus of grape researchers and breeders. Current genetic approaches for gene function studies include mutagenesis, crossing and genetic transformation. However, these approaches are difficult to apply to grapes and takes long periods of time to accomplish. It is thus imperative to seek new ways for grape functional genomics studies. Virus-induced gene silencing (VIGS) offers an attractive alternative for this purpose and has proven highly effective in several herbaceous plant species including tomato, tobacco and barley. VIGS offers several advantages over existing functional genomics approaches. First, it does not require transformation to silence a plant gene target. Instead, it induces silencing of a plant gene through infection with a virus that contains the target gene sequence, which can be accomplished within a few weeks. Second, different plant genes can be readily inserted into the viral genome via molecular cloning and functions of a large number of genes can be identified within a short period of time. Our long-term goal of this research is to develop VIGS-based tools for grapevine functional genomics, made of the genomes of Grapevine virus A (GVA) from Israel and Grapevine rupestris stem pitting-associated virus (GRSPaV) from Canada. GVA and GRSPaV are members of the Flexiviridae. Both viruses have single-stranded, positive sense RNA genomes, which makes them easy to manipulate genetically and excellent candidates as VIGS vectors. In our three years research, several major breakthroughs have been made by the research groups involved in this project. We have engineered a cDNA clone of GVA into a binary vector that is infectious upon delivery into plantlets of micropropagated Vitis viniferacv. Prime. We further developed the GVA into an expression vector that successfully capable to silence endogenous genes. We also were able to assemble an infectious full-length cDNA clones of GRSPaV. In the following sections Achievements and Detailed description of the research activities, we are presenting the outcome and results of this research in details.