Complete Genome Sequence of Grapevine Leafroll-Associated Virus 1 Isolates from Washington Vineyards
Bhanu Priya Donda, Sridhar Jarugula, and Rayapati Naidu*
*Department of Plant Pathology, Washington State University, Irrigated Agriculture Research and Extension Center, 24106 N. Bunn Rd, Prosser, WA 99350 (firstname.lastname@example.org)
Five distinct species of closteroviruses, designated as Grapevine leafroll-associated virus 1, 2, 3, 4, and 7 (GLRaV-1, -2, -3, -4, and -7) have been reported in grape-vines (Vitis vinifera) symptomatic or suspected of leafroll disease infection. The complete genome sequence has been determined for other GLRaVs and their strains, except for GLRaV-1. We determined the complete genome sequence of GLRaV-1 isolates from Washington vineyards. The genome of GLRaV-1 from Chardonnay (WA-CH) and Pinot noir (WA-PN) was amplified into four overlapping cDNA fragments using sequence-specific primers. The derived cDNA clones specific to each fragment were sequenced in both orientations by ‘primer walking’ using progressive sequence-specific primers. The 5’- and 3’-terminal sequences were determined using the RACE system. The sequences were annotated and assembled into a complete genome of 18,731 and 18,946 nucleotides, respectively, for WA-CH and WA-PN isolates. Both isolates showed similar genome organization, encoding nine putative open reading frames and with unusually long non-translated regions (NTRs). The most striking feature of GLRaV-1 isolates is a large 5’ NTR with a variable number of ~65 nt tandem repeat sequences. Differences in the 5’NTR sequences of GLRaV-1 isolates was exploited to develop a RT-PCR-based RFLP assay for discriminating virus isolates into three distinct variant groups. Northern blot hybridization of total RNA from virus-infected grape-vines with gene-specific riboprobes revealed the presence of sub-genomic RNAs (sgRNAs) corresponding to the coat protein (CP) and ORFs p21 and p24, with p24 sgRNA present at relatively higher levels than other sgRNAs. The 5’ termini of sgRNAs corresponding to the CP, CPd1, CPd2, p21, and p24 were mapped to the virus genome and the leader sequence for these sgRNAs determined. The results provide a foundation for further elucidation of the comparative molecular biology of GLRaVs.
Funding Support: WSU Agricultural Research Center, Wine Research Advisory Committee, Washington Wine Commission, Washington State Grape and Wine Research Program, Northwest Center for Small Fruits Research, and Altria – Chateau Ste. Michelle Wine Estates