DESTABILIZATION OF POTATO SPINDLE TUBER VIROID BY MUTATIONS IN THE LEFT TERMINAL LOOP

Infectivity studies with highly infectious RNA inocula generated by ri bozyme cleavage were used to compare the biological properties of thre e apparently nonviable mutants of potato spindle tuber viroid (PSTVd). One of these mutants (PSTVd-P) contains three nucleotide substitution s in the left terminal loop, and mechanical inoculation of tomato seed lings with RNA transcripts at levels equivalent to 10(3)-10(5) times t he ID50 for PSTVd-Intermediate failed to result in systemic infection. Viable progeny containing a spontaneous C --> G change at position 4 could, however, be recovered from transgenic Nicotiana benthamiana pla nts that constitutively expressed PSTVd-P RNA. The initial mutations i n PSTVd-P led to an overall weakening of its native structure in vitro , and the precisely-full-length molecule released by ribozyme cleavage in vivo was also unstable. Even RT-PCR analysis failed to reveal dete ctable amounts of circularized PSTVd-P among the RNAs isolated from un infected plants. Predicted stabilizing effects of a spontaneous mutati on at position 4 suggest that the appearance of viable progeny was dep endent on a combination of events: errors by host RNA polymerase II du ring transcription of the mutant transgene coupled with a strong selec tive pressure against alterations in the native structure of PSTVd.