Cell-free synthesis and processing of an infectious dimeric transcript of potato spindle tuber viroid RNA

Abstract

A full-length cloned cDNA insert containing the sequence of potato spindle tuber viroid (PSTV) was dimerized and placed in the plasmid vector pSP65 adjacent to a promoter for bacteriophage SP6 RNA polymerase. In vitro transcription of this region yielded a single linear RNA chain 760 bases in length containing two copies of PSTV RNA with about 20 bases of vector sequence at each end. Bioassay on tomato plants revealed that this transcript has infectivity comparable to that of PSTV itself, yielding circular progeny RNAs indistinguishable from PSTV grown in vivo Comparative RNA fingerprinting analysis revealed that the viroid sequence in the dimeric transcript breeds true (compared to control viroid strains of different sequence grown in parallel) but loses the vector-specific sequences during growth in plants. Incubation of 32P-labeled dimeric PSTV RNA at neutral pH and 39° gave a 1–5% yield of three RNA segments, one comigrating with unit-length linear PSTV and two smaller fragments. Reaction of the unit-length cleavage product with wheat germ RNA ligase gave a high yield of circular molecules indistinguishable from PSTV circles arising in vivo, suggesting that the cleavage reaction yields 2′,3′ cyclic phosphate termini and could represent a part of the in vivo PSTV replication cycle.