The Interference of Cytoplasmic Membrane-bound Material from Plant Cells with the Detection of a Plant Rhabdovirus Transcriptase

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Abstract
Summary Nuclei and chloroplasts isolated from cell-free extracts of Nicotiana glutinosa L. leaves by sedimentation at 1000 g contain DNA-directed RNA polymerase activity. Whereas only traces of RNA polymerase activity were detected in fractions prepared from the 1000 g supernatant of healthy leaf extracts, RNA-directed RNA polymerase was readily detected in similar extracts from lettuce necrotic yellows virus (LNYV)-infected plants. This virus-associated enzyme activity was strongly inhibited by fractions containing membrane material from ruptured leaf cells of both healthy and virus-infected plants. Fractions with LNYV-associated RNA polymerase activity were prepared after treating the 1000 g supernatant with the non-ionic detergent Nonidet P-40. RNA polymerase activities both of cell-free leaf extract fractions and of purified LNYV preparations were rapidly lost during storage; loss of activity was more rapid in the presence of the detergent. Results presented in the paper are discussed in relation to the difficulties encountered in demonstrating RNA transcriptase activity associated with purified rhabdovirus preparations. We conclude that to detect such enzymes it is necessary to use rapid procedures to obtain virus preparations free of contaminating cellular membranes.

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