Replication of Brome Mosaic Virus RNA in Chloroplasts

Abstract

The replication site of brome mosaic virus (BMV) RNA was investigated in BMV-infected barley cells. Subcellular fractions were separated from protoplast lysates by differential centrifugations and Percoll gradient centrifugation. The activity of RNA synthesis was assayed in the each fraction in the presence of actinomycin D. The RNA dependent RNA polymerase activity was detected in a purified chloroplast fraction and the activity per chlorophyll increased with purification of chloroplasts. The existence of RNAs hybridized with cDNA of BMV RNA was detected in the purified chloroplasts in the dot blot assay. Replicative forms were synthesized in the in vitro RNA synthesis system adding chloroplasts isolated from BMV-infected cells instead of enzyme fraction. By the microautoradiographic observation, the silver grains were observed on the thylakoid when the purified chloroplasts isolated from BMV-infected cells were added to the reaction mixture and incubated in the presence of actinomycin D and ³H-UTP. These results strongly suggested that at least BMV RNA can replicate in chloroplasts of BMV-infected barley cells.