Small-scale isolation of viral RNA-Dependent RNA polymerase from protoplasts inoculated with in vitro transcripts

Cowpea chlorotic mottle virus (CCMV) replicated in tobacco suspension cell protoplasts inoculated with in vitro transcripts of CCMV RNA1, 2, and 3. CC MV RNA-dependent RNA polymerase (RdRp) isolated from these protoplasts spec ifically recognized CCMV and Brome mosaic virus (BMV) subgenomic RNA promot ers and directed in vitro RNA synthesis in a manner indistinguishable from CCMV RdRp more laboriously isolated from systemically infected cowpea leave s. Omission of CCMV RNA3 from the protoplast inoculum or replacement with i n vitro transcripts of BMV RNA3 reduced CCMV (+)-strand RNA1 and 2 accumula tion to =1/40 and =1/10, respectively, of the level attained when CCMV RNA3 was present. The absence of CCMV RNA3 did not prevent assembly and isolati on of highly active, template-dependent and template-specific CCMV RdRp, wh ich directed synthesis of products identical in size to those of RdRp isola ted from protoplasts inoculated with all three CCMV genomic RNAs. These res ults demonstrate that CCMV RNA1 and 2 are sufficient for CCMV replication a nd RdRp assembly in tobacco protoplasts. This approach for isolation of fun ctional viral RdRp will be especially useful for viruses for which large qu antities of infected tissue are unavailable, such as those with specific ti ssue tropisms or mutants incapable of systemic movement.