427 pages ; Plants utilize RNA silencing as a conserved and effective defense mechanism against viruses. To counteract this antiviral defense pathway, plant viruses have evolved to encode one or multiple proteins known as viral suppressors of RNA silencing (VSRs) that interfere with specific steps of the RNA silencing pathways. Although VSRs have been identified for numerous plant viruses, none is known for grapevine fanleaf virus (GFLV; genus Nepovirus, family Secoviridae). GFLV causes fanleaf degeneration, the most destructive viral disease of grapevine (Vitis spp.) in most vineyards worldwide. This disease causes devastating yield loss up to 80% and substantially reduces the lifespan of vineyards. My dissertation aims to enhance our understanding of how this economically concerning virus counteracts host antiviral RNA silencing defense by (i) identifying GFLV VSRs and characterizing their suppression activities, (ii) pinpointing GFLV VSRs amino acids essential for suppression of RNA silencing and host gene expression modulation abilities, and (iii) determining the subcellular localization and temporal translocation of GFLV VSRs in planta. I agroinfiltrated transgenic Nicotiana benthamiana expressing enhanced green fluorescent protein (EGFP) with a hairpin construct to trigger systemic silencing of EGFP. Then, I evaluated GFLV proteins for their ability to induce systemic suppression of EGPF silencing in apical leaves using fluorescence imaging, quantitative assessment of EGFP fluorescence intensity, measurements of EGFP transcript levels and changes in host gene expression by RT-qPCR. My results revealed that GFLV encodes three VSRs, 1A, 1B, and 1AB, that either individually or in combination, suppress systemic RNA silencing and differentially modify the expression of host genes involved in RNA silencing. Next, I identified the conservation of a tryptophan-glycine and glycine-tryptophan (WG/GW) motif in VSRs across different GFLV isolates. Using a reverse genetics approach, I determined that mutating W to A ...