Analyzing Virus Genomic Variability To Design And Test Genetic Constructs For Resistance

Abstract

An understanding of the genomic diversity of plant pathogenic viruses is essential for devising control strategies. Over the last two decades, improved sequencing technologies and the discovery of RNA silencing have profoundly impacted our ability to understand the diversity of virus populations and develop resistant plants. In Chapter 1 of this dissertation, I critically evaluate the literature regarding materials providing resistance against viruses and vectors in Vitis species and discuss their availability for disease management. This review indicates little or no useful resistance toward most virus diseases, and the critical need to develop resistant materials. In Chapter 2, in order to gain insights into the evolutionary mechanisms of Grapevine fanleaf virus (GFLV), sequence information was obtained from fourteen isolates collected in naturally infected vineyards in California. My results indicated that some isolates result from interspecies recombination between GFLV and Arabis mosaic virus, and suggest that recombination and purifying selection are important evolutionary mechanisms in the genetic diversification of GFLV. In Chapter 3, I designed various resistance constructs derived from GFLV based upon an analysis of sequence variability. These constructs were tested for resistance to GFLV using a transient expression system. Results indicated that some of these constructs are capable of reducing virus titers in GFLV-infected plants. In Chapter 4, I reviewed the literature regarding environmental and human safety issues related to virus-resistant transgenic horticultural crops. My analysis suggests that the use of virus-resistant transgenic plants is a safe and effective way to control viral diseases. In Chapter 5, I present the results of a survey for Prunus necrotic ringspot virus in an orchard of sour and sweet cherry trees. Sequence analysis of the viral coat protein gene from various isolates indicated one predominant and several minor molecular variants. Results revealed a higher rate of infection among sour cherry vs. sweet cherry trees, and also suggested that this virus may have been transferred from a single infected sour cherry tree into the orchard by pollen transfer. In Chapter 6, I present conclusions regarding the implications of my research and suggest future directions of the work presented in the preceding five chapters.