Precisely full length, circularizable, complementary RNA: an infectious form of potato spindle tuber viroid

• Published in: Proceedings of the National Academy of Sciences - PNAS 1998-05, Vol.95 (11), p.6560-6565
• Main Authors: Feldstein, P.A. (Center for Engineering Plants for Resistance Against Pathogens, Davis, CA.), Hu, Y, Owens, R.A
• Format: Article
• Language: English
• Subjects: ARN; Base Sequence; Biological Sciences; Botany; Circles; CIRCULAR RNA; Electrophoresis; EXPERIMENTACION IN VIVO; EXPERIMENTAL INFECTION; EXPERIMENTATION IN VIVO; Gels; IN VIVO EXPERIMENTATION; INFECCION EXPERIMENTAL; INFECTION EXPERIMENTALE; INFECTIVITY; Inoculum; LYCOPERSICON ESCULENTUM; MOLECULAR CONFORMATION; Molecular Sequence Data; Molecules; NEPOVIRUS; NEPOVIRUSES; NICOTIANA; NICOTIANA BENTHAMIANA; Nucleic Acid Conformation; PATHOGENICITY; Pathology; PATOGENICIDAD; PLANT VIRUSES; PLANTAS TRANSGENICAS; PLANTE TRANSGENIQUE; Plasmids; Potatoes; POUVOIR PATHOGENE; REPLICACION; REPLICATION; Ribonucleic acid; RIBOZYMES; RNA; RNA probes; RNA, Viral - chemistry; RNA, Viral - genetics; Solanum tuberosum - virology; TOMATO PLANTA MACHO VIROID; TOMATO RINGSPOT NEPOVIRUS; TRANSGENIC PLANTS; VIROIDE; VIROIDES; VIROIDS; Viroids - physiology; VIRUS DE LAS PLANTAS; VIRUS DES VEGETAUX; Virus Replication
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.11.6560

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 temini 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