The replication of many viral and subviral pathogens as well as the 
amplification of certain cellular genes proceeds via a rolling circle mechanism. 
For potato spindle tuber (PSTVd) and related viroids, the possible role of a 
circular (-)strand RNA as a template for synthesis of (+)strand progeny is 
unclear. Infected plants appear to contain only multimeric linear (-)strand 
RNAs, and attempts to initiate infection with multimeric (-)PSTVd RNAs generally 
have failed. To examine critically the infectivity of monomeric (-)strand viroid 
RNAs, we have developed a ribozyme-based expression system for the production of 
precisely full length (-)strand RNAs whose termini are capable of undergoing 
facile circularization in vitro. Mechanical inoculation of tomato seedlings with 
electrophoretically purified (-)PSTVd RNA led to a small fraction of plants 
becoming infected whereas parallel assays with an analogous tomato planta macho 
viroid (-)RNA resulted in a much larger fraction of infected plants. 
Ribozyme-mediated production of (-)PSTVd RNA in transgenic plants led to the 
appearance of monomeric circular (-)PSTVd RNA and large amounts of (+)PSTVd 
progeny. No monomeric circular (-)PSTVd RNA could be detected in naturally 
infected plants by using either ribonuclease protection or electrophoresis under 
partially denaturing conditions. Although not a component of the normal 
replicative pathway, precisely full length (-)PSTVd RNA appears to contain all 
of the structural and regulatory elements necessary for initiation of viroid 
replication.