Grapevine fanleaf virus: Biology, biotechnology and resistance

Grapevine fanleaf virus (GFLV) causes fanleaf degeneration of grapevines. GFLV is present in most grape growing regions and has a bipartite RNA genome. The three goals of this research were to (1) advance our understanding of GFLV biology through studies on its satellite RNA, (2) engineer GFLV into a viral vector for grapevine functional genomics, and (3) discover a source of resistance to GFLV. This author addressed GFLV biology by studying the least understood aspect of GFLV: its satellite RNA. This author sequenced a new GFLV satellite RNA variant and compared it with other satellite RNA sequences. Forensic tracking of the satellite RNA revealed that it originated from an ancestral nepovirus and was likely introduced from Europe into North America. Greenhouse experiments showed that the GFLV satellite RNA has commensal relationship with its helper virus on a herbaceous host. This author engineered GFLV into a biotechnology tool by cloning infectious GFLV genomic cDNAs into binary vectors, with or without further modifications, and using Agrobacterium tumefaciens delivery to infect Nicotiana benthamiana. Tagging GFLV with fluorescent proteins allowed tracking of the virus within N. benthamiana and Chenopodium quinoa tissues, and imbuing GFLV with partial plant gene sequences proved the concept that endogenous plant genes can be knocked down. Infectivity of the viral vector depended on the identity of the GFLV strains or reassortants, on co-application of heterologous silencing suppressors and on lower ambient temperatures. No natural sources of resistance to GFLV exist within Vitis spp., but certain herbaceous hosts such as N. tabacum (tobacco) are resistant. This author used tobacco, its wild relatives, and hybrids between tobacco and wild relatives to evaluate the genomic and physiological basis of resistance. Resistance to GFLV in tobacco is governed by systemic recovery from virus infection that is additively inherited and likely multi-allelic. This research has opened new avenues to understand virus and plant evolution, and furnishes geneticists with a new tool to functionally characterize host genes. This dissertation also includes a history of pathogen-derived resistance with specific reference to plant virus resistance.