Effects of random mutagenesis upon potato spindle tuber viroid replication and symptom expression

A combination of random chemical mutagenesis plus temperature gradient gel electrophoresis was used to isolate a collection of 57 potato spindle tuber viroid (PSTV) cDNAs containing mutations distributed throughout the entire 359 nucleotide genome. Although the presence of multiple mutations was often associated with a loss of cDNA infectivity, infectious PSTV cDNAs containing as many as four unlinked alterations could be isolated. Several mutations in the pathogenicity domain and left terminal loop were stably maintained in the resulting progeny, but those which affect base pairing in secondary hairpins I and II were either lethal or rapidly reverted to wild-type. One stable C----U substitution which may promote significant structural rearrangement within the right side of the pathogenicity domain had no detectable effect upon symptom expression. The variable domains of several noninfectious mutants contained an A----G substitution which is likely to inhibit the in vitro formation of secondary hairpin II via stabilization of the native structure, and the lethal nature of this mutation was confirmed by oligonucleotide-directed mutagenesis. Several lines of evidence now point toward an essential role for secondary hairpin II in the replication of PSTV and related viroids.