Rugose Wood Disease of Grapevine, Etiology and Virus Resistance in Transgenic Vines

Abstract

Rugose wood is a complex disease of grapevines, which occurs in all growing areas. The disease is spread in the field by vector transmission (mealybugs). At least five elongated-phloem- limited viruses are implicated in the various rugose wood disorders. The most fully characterized of these are Grapevine virus A (GV A) and GVB, members of a newly established genus, the vitivirus. GVC, a putative vitivirus, is much less well characterized than GV A or GVB. The information regarding the role of GVC in the etiology and epidemiology of rugose wood is fragmentary and no sequence data for GVC are available. The proposed research is aimed to study the etiology and epidemiology of rugose wood disease, and to construct genetically engineered virus-resistant grapevines. The objectives of our proposed research were to construct transgenic plants with coat protein gene sequences designed to induce post-transcriptional gene silencing (pTGS); to study the epidemiology and etiology of rugose wood disease by cloning and sequencing of GVC; and surveying of rugose wood- associated viruses in Californian and Israeli vineyards. In an attempt to experimentally define the role of the various genes of GV A, we utilized the infectious clone, inserted mutations in every ORF, and studied the effect on viral replication, gene expression, symptoms and viral movement. We explored the production of viral RNAs in a GV A-infected Nicotiana benthamiana herbaceous host, and characterized one nested set of three 5'-terminal sgRNAs of 5.1, 5.5 and 6.0 kb, and another, of three 3'-terminal sgRNAs of 2.2, 1.8 and 1.0 kb that could serve for expression of ORFs 2-3, respectively. Several GV A constructs have been assembled into pCAMBIA 230 I, a binary vector which is used for Angrobacterium mediated transformation: GV A CP gene; two copies of the GV A CP gene arranged in the same antisense orientation; two copies of the GV A CP gene in which the downstream copy is in an antigens orientation; GV A replicase gene; GV A replicase gene plus the 3' UTR sequence; and the full genome of GV A. Experiments for transformation of N. benthamiana and grapevine cell suspension with these constructs have been initiated. Transgenic N. benthamiana plants that contained the CP gene, the replicase gene and the entire genome of GV A were obtained. For grapevine transformation, we have developed efficient protocols for transformation and successfully grapevine plantlets that contained the CP gene and the replicase genes of GV A were obtained. These plants are still under examination for expression of the trans genes. The construction of transgenic plants with GV A sequences will provide, in the long run, a means to control one of the most prevalent viruses associated with grapevines. Our many attempts to produce a cDNA library from the genome of GVC failed. For surveying of rugose wood associated viruses in California vineyards, samples were collected from different grape growing areas and tested by RT-PCR for GV A, GVB and GVD. The results indicated that some of the samples were infected with multiple viruses, but overall, we found higher incidence of GVB and GV A infection in California vineyards and new introduction varieties, respectively. In this research we also conducted studies to increase our understanding of virus - induced rootstock decline and its importance in vineyard productivity. Our results provided supporting evidence that the rootstock response to virus infection depends on the rootstock genotype and the virus type. In general, rootstocks are differ widely in virus susceptibility. Our data indicated that a virus type or its combination with other viruses was responsible in virus-induced rootstock decline. As the results showed, the growth of the rootstocks were severely affected when the combination of more than one virus was present.