Longer-than-unit-length viroid minus strands are present in RNA from infected plants

Nucleic acids isolated from uninfected and potato spindle tuber viroid-infected Rutgers tomato plants were fractionated on agarose gels under two different sets of denaturing conditions and hybridized to (125)I-labeled viroid in a series of blot hybridization experiments. Complementary strand nucleic acids detected in extracts of infected plants were heterogeneous in size, with four discrete bands containing molecules approximately 700, 1050, 1500, and 1800 nucleotides long. Enzymatic studies indicated that these viroid minus strands are composed exclusively of RNA and, as extracted, are present in complexes containing extensive double-stranded regions. After treatment with several RNases under conditions favoring digestion of single-stranded regions, the high molecular weight minus strands can no longer be detected and roughly unit-length minus strands appear. A model for the structure of the viroid replication intermediate is proposed.