Soluble, template-dependent extracts from Nicotiana benthamiana plants infected with potato virus X transcribe both plus- and minus-strand RNA templates

We have developed a method to convert membrane-bound replication complexes isolated from Nicotiana benthamiana plants infected with potato virus X (PV X) to a soluble, template-dependent system for analysis of RNA synthesis. A nalysis of RNA-dependent RNA polymerase activity in the membrane-bound, end ogenous template extracts indicated three major products, which corresponde d to double-stranded versions of PVX genomic RNA and the two predominant su bgenomic RNAs. The endogenous templates were removed from the membrane-boun d complex by treatment with BAL 31 nuclease in the presence of Nonidet P-40 (NP-40). Upon the addition of full-length plus- or minus- strand PVX trans cripts, the corresponding-size products were detected. Synthesis was not ob served when red clover necrotic mosaic dianthovirus (RCNMV) RNA 2 templates were added, indicating template specificity for PVX transcripts. Plus-stra nd PVX templates lacking the 3' terminal region were not copied, suggesting that elements in the 3' region were required for initiation of RNA synthes is. Extracts that supported RNA synthesis from endogenous templates could a lso be solublized using sodium taurodeoxycholate and then rendered template -dependent by BAL 31 nuclease/NP-40 treatment. The solubilized preparations copied both plus-and minus-strand PVX transcripts, but did not supper? syn thesis from RCNMV RNA 2. These membrane-bound and soluble template-dependen t systems will facilitate analyses of viral and host components required fo r PVX RNA synthesis. (C) 2000 Academic Press.