Molecular and cellular biology of grapevine leafroll-associated virus 3 and its effects on grape gene expression through transcriptomic analyses

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The grape & wine industry is an economic pillar for Canada and many other countries. The grape & wine industry, together with the related tourism, generate an annual economic impact of 4.4 billion and provides 37,000 jobs. A major obstacle to sustainable production of premium grape & wine is disease caused by viruses. Being the most destructive viral disease with a global distribution, grapevine leafroll (GLR) threatens the yield, quality, profitability and sustainability of the Canadian grape & wine sector. Five distinct viruses of the family Closteroviridae are associated with GLR; grapevine leafroll-associated virus 3 (GLRaV-3) is the predominant causal agent. As the type member of the genus Ampelovirus, GLRaV-3 has one of the largest RNA genomes among all viruses, and exhibits numerous unique features such as restriction to the phloem, targeting mitochondria for VRC (viral replication complexes) formation, possessing several open reading frames unique to GLRaV-3, and containing excessively long non-coding genomic regions. Unfortunately, the molecular and cellular aspects of the replication and mechanisms of infection remain understudied for GLRaV-3 and other GLR-associated viruses. This knowledge gap is due to multiple factors, including technical challenges in making full-length cDNA clones for such large RNA genomes, lack of efficient systems to launch infection in grapes with such clones, the very long time period required for vines to grow and to build up viral titer, among other factors. Our lab has been focusing on major grape viruses in hope to advance the grape virology field to the molecular and cellular level. In the next 5 years, we will concentrate our effort on 4 objectives: 1) global elucidation of the impact of GLRaV-3 infection on grape gene expression through RNA-Seq and transcriptome analysis; 2) viral gene and sequence determinants for mito targeting and VRC formation; 3) function and subcellular localization of the five ORFs unique for GLRaV-3 that are located near the 3' end of its genome; 4) the development and validation of virus-induced gene silencing vectors to confer resistance against GLRaV-3. Prior research on GRSPaV (a member of family Betaflexiviridae involved in the rugose woody disease complex), together with our recent establishment of an infectious GLRaV-3 clone and the grapevine infectivity system, make the proposed research highly feasible. Outcome of this research will enable us to gain deeper understanding of the molecular and cellular aspects concerning GLRaV-3 replication and its infection process. This pioneering research will also allow us to establish GLRaV-3 as a model system for the genus Ampelovirus. In the long run, this information will help us devise new strategies to mitigate the economic impact of disease by GLRaV-3 and other GLR-related viruses. Moreover, the interdisciplinary and collaborative nature of the proposed research will continue to serve excellent platforms for HQP training.