Infectious cucumber mosaic virus RNA transcribed in vitro from clones obtained from cDNA amplified using the polymerase chain reaction

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Abstract
Full-length cDNA to RNA 1, RNA 2 and RNA 3 of cucumber mosaic virus strain Q (CMV-Q) was amplified using the polymerase chain reaction (PCR). The first-strand primer contained BamHI site and sequences complementary to the 3'' terminus of the RNA. The second-strand primers contained a BamHI site, a T7 promoter and sequences corresponding to the 5'' terminus of each RNA. After cleavage with BamHI, the PCR products were cloned into the BamHI site of the vector pEMBL9(+). Five clones of each RNA were selected and RNA transcripts were synthesized in vitro from each clone using T7 RNA polymerase. The constructs were designed to allow transcription to initiate precisely at the 5'' terminus of each RNA. All the transcripts were found to be infectious when inoculated onto Nicotiana tabacum cv. Samsun plants in sets of three, corresponding to RNA 1, RNA 2 and RNA 3. Of the transcript sets, four induced symptoms indistinguishable from symptoms induced by CMV-Q RNAs. However a fifth transcript set induced much more severe symptoms. Plasmids were also constructed to allow synthesis of transcripts with one or two additional G residues at the 5'' terminus of each RNA. Although the yields of such transcripts synthesized in vitro with T7 RNA polymerase were higher, their infectivity was lower than of those with no additional residues at their 5'' termini.