INVOLVEMENT OF NUCLEAR DNA-DEPENDENT RNA-POLYMERASES IN POTATO SPINDLE TUBER VIROID REPLICATION - A REEVALUATION

Inhibitor studies on Potato Spindle Tuber Viroid (PSTVd) synthesis were performed with highly purified nuclei from suspension cultures of PSTVd-infected Solanum demissum cells in order to investigate which nuclear RNA polymerases are involved in PSTVd replication. The in vitro synthesized RNA was analyzed by dot blot hybridization with cDNA probes specific for the viroid RNA and for three cellular RNA species transcribed by the nuclear DNA-dependent RNA polymerases I, II and III, respectively. Application of different concentrations of alpha-amanitin revealed that PSTVd(+) and (-)RNA synthesis was inhibited at exactly the same toxin concentrations as the synthesis of the wun-2 mRNA, which is transcribed by DNA-dependent RNA polymerase II. RNA transcribed by DNA-dependent RNA polymerase I or III was not inhibited at that concentration of alpha-amanitin. On the other hand, PSTVd synthesis was not inhibited by actinomycin D even at high concentrations, indicating that PSTVd replication does not depend on the active transcription of chromosomal DNA. Our results demonstrate that the DNA dependent RNA polymerase II is directly involved in PSTVd(+) and (-)RNA synthesis.