Cymbidium mosaic virus RNA synthesis in isolated orchid protoplasts

An in vitro protoplast host system has been developed to facilitate the study of cymbidium mosaic virus (CyMV) replication mechanisms. CyMV, a member of the potexvirus group of plant viruses, is among the most significant pathogens of cultivated orchid plants, but a convenient system for studying its molecular genetics has not been available previously. A cDNA probe representing a segment of the CyMV genome was used to detect viral RNA following inoculation of Vanda floral protoplasts with virus particles. Detection was carried out by RNA-DNA hybridization methods (RNA slot blots). Newly synthesized viral RNA was detectable by 6-9 h postinfection and appeared to reach a maximum around 12 h. These results are consistent with the time course of viral RNA synthesis in protoplasts infected with other RNA viruses. Immunoblot detection methods revealed the presence of newly synthesized viral protein indicating that viral gene expression occurs in the protoplasts in addition to genome replication.