A chloroplastic RNA polymerase resistant to tagetitoxin is involved in replication of Avocado sunblotch viroid

Avocado sunblotch viroid (ASBVd), the type species of the family Avsunviroi dae, replicates and accumulates in the chloroplast. Two main chloroplastic RNA polymerases have been described: the plastid-encoded polymerase (PEP) w ith a multisubunit structure similar to the Escherichia coil enzyme and a s ingle-unit nuclear-encoded polymerase (NEP) resembling phage RNA polymerase s. On a different basis, sensitivity to tagetitoxin, two major RNA polymera se activities, tagetitoxin sensitive (TS) and resistant (TR), have been fou nd in plastids. The most plausible candidates for the TS and TR RNA polymer ases are PEP and NEP, respectively. To gain an insight into the enzymology of the polymerization of ASBVd strands, purified chloroplast preparations f rom ASBVd-infected leaves were assayed for their in vitro ability to transc ribe ASBVd RNAs together with some representative genes (psbA, 16SrDNA, acc D, and rpoB) of the three classes of chloroplastic genes according to their promoter structure. High concentrations of or-amanitin had no effect on ge ne or on viroid transcription, but tagetitoxin (5-10 mu M) prevented transc ription of all these genes without affecting synthesis of ASBVd strands; on ly at higher tagetitoxin concentrations (50-100 mu M) was a 255b 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, (C) 2000 Academic Press.