GENETIC DIVERSITY AND MOLECULAR BIOLOGY OF GRAPEVINE LEAFROLL-ASSOCIATED VIRUSES

Abstract

Grapevine leafroll disease (GLRD) is an economically important virus disease of wine grapes (Vitis vinifera L.). Ten grapevine leafroll-associated viruses (GLRaVs, family Closteroviridae), designated as GLRaV-1 to -10 in the order of their discovery, have been reported in GLRD-infected grapevines. This research was focused on GLRaV-2 and GLRaV-3 that are frequently detected in Washington State vineyards. Genetic diversity among GLRaV-2 isolates collected largely from Washington vineyards was analyzed by sequencing the entire coat protein (CP) and a portion of the heat-shock protein-70 homolog (HSP70h). Phylogenetic analysis of CP and HSP70h sequences with corresponding Genbank sequences revealed segregation of global isolates of GLRaV-2 into six lineages (`PN', `93/955', `H4', `PV20', `BD' and `RG'), with virus isolates from Washington distributed in `PN', `H4' and `RG' lineages. The ratio of genetic diversity at nonsynonymous and synonymous sites computed separately for CP and HSP70h indicated diversifying selection pressures on the two viral genes. A molecular diagnostic assay, based on reverse transcription-polymerase chain reaction and restriction fragment length polymorphism, was developed to differentiate GLRaV-2 isolates in `PN', `H4' and `RG' lineages currently documented in Washington vineyards.The genome of the Washington isolate of GLRaV-3 was determined to be 18,498 nucleotides (nt) long with an unusually long 5'-nontranslated region of 737 nt and encoding eleven open reading frames. In Northern blots probed with gene-specific riboprobes, four 3'-coterminal subgenomic RNAs (sgRNAs) specific to CP, p21, p20A, and p20B genes were detected in total RNA extracted from a wine grape cultivar naturally infected with GLRaV-3. An analysis of their leader sequences indicated differences in transcriptional regulation of sgRNAs between GLRaV-3 and other closteroviruses. A multi-step cloning strategy was used to assemble a full-length cDNA clone of GLRaV-3 and demonstrated that in vitro RNA transcripts generated from the cDNA clone can replicate in Nicotiana benthamiana protoplasts. Similarly, the full-length cDNA clone of GLRaV-3 cloned into a binary vector showed replication when introduced into N. benthamiana leaves via agroinfiltration and formed authentic virion particles. The infectious cDNA clone of GLRaV-3 will help applying reverse genetics to study the molecular biology of grapevine-infecting closteroviruses and the etiology of GLRD