PRECISELY FULL-LENGTH, CIRCULARIZABLE, COMPLEMENTARY RNA - AN INFECTIOUS FORM OF POTATO SPINDLE TUBER VIROID

The replication of many viral and subviral pathogens as well as the am plification of certain cellular genes proceeds via a rolling circle me chanism. For potato spindle tuber (PSTVd) and related viroids, the pos sible role of a circular (-)strand RNA as a template for synthesis of (+)strand progeny is unclear. Infected plants appear to contain only m ultimeric linear (-)strand RNAs, and attempts to initiate infection wi th multimeric (-)PSTVd RNAs generally have failed. To examine critical ly the infectivity of monomeric (-)strand viroid RNAs, we have develop ed a ribozyme-based expression system for the production of precisely full length (-)strand RNAs whose termini are capable of undergoing fac ile circularization in vitro., Mechanical inoculation of tomato seedli ngs with electrophoretically purified (-)PSTVd RNA led to a small frac tion of plants becoming infected whereas parallel assays with an analo gous tomato planta macho viroid (-)RNA resulted in a much larger fract ion of infected plants. Ribozyme-mediated production of (-)PSTVd RNA i n transgenic plants led to the appearance of monomeric circular (-)PST Vd RNA and large amounts of (+)PSTVd progeny. No monomeric circular (- )PSTVd RNA could be detected in naturally infected plants by using eit her ribonuclease protection or electrophoresis under partially denatur ing conditions, Although not a component of the normal replicative pat hway, precisely full length (-)PSTVd RNA appears to contain all of the structural and regulatory elements necessary for initiation of viroid replication.