Infectivity andin vitromutagenesis of monomeric cDNA clones of citrus exocortis viroid indicates the site of processing of viroid precursors

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Abstract
Monomeric cDNA clones of citrus exocortis viroid (CEV) were constructed in the plasmid vector pSP6-4 and the infectivity of the clones plus in vitro-synthesized RNA transcripts determined by Inoculation onto tomato seedlings. Infectivity was dependent on the site of the viroid molecule used for cloning and the orientation of the cDNA insert. Only the plus BamHI cDNA clone was Infectious and produced progeny viroid with wild-type sequence at the region corresponding to the BamHI cloning site. Infectivity correlated with the terminal repetition of 11 nucleotides of viroid sequence, 5′GGATCCCCGGG 3′, in the vector adjacent to the insert. The 11-nucleotide sequence lies within the highly conserved central region of viroids. Site-directed mutagenesis of a single nucleotide in the repeat at the 5′-end of the CEV Insert to 5′ GGATCCCC(T, A)GG 3′ gave two point mutants. The two mutant CEV inserts, when excised from the vector, were not infectious. However, plasmid DNA and RNA transcripts from non-excised mutant CEV inserts were infectious. The progeny of one of these clones was examined and contained wild-type sequence. It was concluded that in vivo processing of longer-than-unit-length CEV occurs at one of three adjacent sites in the 11 nucleotide sequence and that the G nucleotide at position 97 is important for viroid replication.