CLONING AND MOLECULAR ANALYSIS OF DOUBLE-STRANDED-RNA ASSOCIATED WITHGRAPEVINE LEAFROLL DISEASE

Eight major dsRNA species ranging from 1.0 to 19.5 kbp were detected i n a low-yielding clone of Sultana (Thompson seedless) grape (Vitis vin ifera L., cv. Sultana, clone B4L) affected by leafroll disease. Using total dsRNA from this Sultana line as template, a number of cDNA clone s were produced. The clones were used as probes for northern blot anal ysis of dsRNA extracted from Sultana B4L, and from six other grapevine leafroll-infected Sultana sources differing in yield performance. Bas ed on the hybridisation of each probe with dsRNA bands from various Su ltanas, the cDNA clones could be divided into three groups. One group of cDNA clones hybridised to high molecular weight dsRNA (19.5 kbp) fr om two low-yielding Sultanas, another group hybridised to high M(r) ds RNA 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 a t the 5'-end of a 1.15 kbp dsRNA (dsRNA 6) of B4L Sultana was determin ed. This RNA contains an open reading frame encoding a putative protei n of M(r) = 33 441 with no homology to known protein sequences. The se quence 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 i ndicate that more than one virus was present in Sultana B4L and that d sRNA 6 may be a subgenomic species of viral origin.