Effect of actinomycin D on replication of satellite tobacco ringspot virus RNA in plant protoplasts.

We have developed a three-component system of host, tobacco ringspot virus (TobRV), and satellite tobacco ringspot virus RNA (sTobRV RNA) for investigating the specific contributions of host components or TobRV gene products to the propagative cycle of satellite RNA. Cowpea (Vigna unguiculata) protoplasts were inoculated with sTobRV and TobRV genomic RNAs by electroporation. An increase in sTobRV RNA was detected both by blot hybridization and by incorporation of [14C]uridine into material with the electrophoretic mobility of sTobRV RNA. DNA-dependent RNA synthesis in uninoculated protoplasts was effectively inhibited by 50 micrograms/ml actinomycin D (Act D) in the medium. Addition of Act D to protoplasts 24 or 48 hr after coinoculation with sTobRV RNA and TobRV genomic RNAs had little effect on accumulation of sTobRV RNA, whereas addition at 24 hr prior to coinoculation prevented any detected accumulation of sTobRV RNA of either polarity. Our results and previous findings of RNA complementary to encapsidated satellite RNA in extracts of infected tissue suggest that an RNA-dependent RNA polymerase is responsible for the synthesis of sTobRV RNA. The strongly inhibitory effect of Act D when added early implies a role for a host factor in the early phase of sTobRV RNA replication.