Achloroplastic RNA polymerase resistant to tagetitoxin is involved in replication of avocado sunblotch viroid

Avocado sunblotch viroid (ASBVd), the type species of the family Avsunviroidae, replicates and accumulates in the chloroplast. Two main chloroplastic RNA polymerases have been described: the plastid-encoded polymerase (PEP) with a multisubunit structure similar to the Escherichia coli enzyme and a single-unit nuclear-encoded polymerase (NEP) resembling phage RNA polymerases. On a different basis, sensitivity to tagetitoxin, two major RNA polymerase activities, tagetitoxin sensitive (TS) and resistant (TR), have been found in plastids. The most plausible candidates for the TS and TR RNA polymerases are PEP and NEP, respectively. To gain an insight into the enzymology of the polymerization of ASBVd strands, purified chloroplast preparations from ASBVd-infected leaves were assayed for their in vitro ability to transcribe ASBVd RNAs together with some representative genes (psbA, 16SrDNA, accD, and rpoB) of the three classes of chloroplastic genes according to their promoter structure. High concentrations of alpha-amanitin had no effect on gene or on viroid transcription, but tagetitoxin (5-10 microM) prevented transcription of all these genes without affecting synthesis of ASBVd strands; only at higher tagetitoxin concentrations (50-100 microM) was a 25% inhibition observed. These results suggest that NEP is the RNA polymerase required in ASBVd replication, although the participation of another TR RNA polymerase from the chloroplast cannot be excluded.