Precisely Monomeric Linear RNAs of Viroids Belonging to Pospiviroid and Hostuviroid Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure

Abstract

Infectious dimeric RNA transcripts are a powerful tool for reverse genetic analyses in viroid studies. However, the construction of dimeric cDNA clones is laborious and time consuming, especially in mutational analyses by in vitro mutagenesis. In this study, we developed a system to synthesize a precisely monomeric linear RNA that could be transcribed in vitro directly from the cDNA clones of four viroid species. The cDNA clones were constructed such that RNA transcription was initiated at the guanine nucleotide of a predicted processing and ligation site in the viroid replication process. Although the transcribed RNAs were considered to possess 5′-triphosphate and 3′-hydroxyl termini, the RNA transcripts were infectious even without in vitro modifications. Additionally, infectivity was detected in the monomeric RNA transcripts, in which transcription was initiated at guanine nucleotides distinct from the predicted processing/ligation site. Moreover, monomeric viroid RNAs bearing 5′-monophosphate, 5′-hydroxyl, or 5′-capped termini were found to be infectious. Northern blot analysis of the pooled total RNA of the plants inoculated with the 5′-terminal modified RNA of potato spindle tuber viroid (PSTVd) indicated that maximum PSTVd accumulation occurred in plants with 5′-monophosphate RNA inoculation, followed by the plants with 5′-triphosphate RNA inoculation. Our system for synthesizing an infectious monomeric linear viroid RNA from a cDNA clone will facilitate mutational analyses by in vitro mutagenesis in viroid research.