Cloning and molecular analysis of double-stranded RNA associated with grapevine leafroll disease*

Eight major dsRNA species ranging from 1.0 to 19.5 kbp were detected in a low-yielding clone of Sultana (Thompson seedless) grape (Vitis vinifera L., cv. Sultana, clone B4L) affected leafroll disease. Using total dsRNA from this Sultana line as template, a number of cDNA clones were produced. The clones were used as probes for northern blot analysis of dsRNA extracted from Sultana B4L, and from six other grapevine leafroll-infected Sultana sources differing in yield performance. Based on the hybridisation of each probe with dsRNA bands from various Sultanas, the cDNA clones could be divided into three groups. One group of cDNA clones hybridised to high molccular weight dsRNA (19.5 kbp) from two low-yielding Sultanas, another group hybridised to high M_r dsRNA from three low-yielding Sultanas and the third group hybridised to a number of smaller dsRNA species ranging in size between 1.15 and 6.5 kbp. Using the latter cDNA clones, the sequence of 965 nucleotides at the 5′-end of a 1.15 kbp dsRNA (dsRNA 6) of B4L Sultana was determined. This RNA contains an open reading frame encoding a putative protein of M, = 33 441 with no homology to known protein sequences. The sequence of dsRNA 6 was found to overlap larger dsRNAs of sizes between 2.2 to 6.5 kbp. This allowed us to determine the sequence upstream of the 5′-end of the positive strand of dsRNA 6. The nucleotide sequence neighbouring the 5′-end of the positive strand of dsRNA 6 conforms to a consensus sequence proposed as a subgenomic promoter element for the coat protein gene of positive strand RNA plant viruses. The results indicate that more than one virus was present in Sultana B4L and that dsRNA 6 may be a subgenomic species of viral origin.