Nucleotides within the pathogenicity domain of potato spindle tuber viroid 
(PSTVd) are known to play an important role in regulating symptom expression, 
but the underlying molecular mechanism is unknown. In order to determine more 
precisely how structural features within the pathogenicity domain regulate 
symptom expression, we have characterized a series of mutations that 
progressively stabilize premelting region 1 and the rest of the "virulence 
modulating" region. The structural effects of these mutations were monitored by 
temperature gradient gel electrophoresis of circularized RNA transcripts, and 
their biological effects were assessed by quantitative bioassays in tomato. 
Closure of a 4-nucleotide loop within the premelting region 1 virtually 
abolished PSTVd infectivity, especially when a nearby 2-nucleotide loop was also 
closed. Although RNA transcripts containing less stabilizing mutations were 
readily infectious, none of the four single and one double substitutions 
examined were stably maintained in vivo. The pattern of spontaneous, apparently 
compensatory sequence changes observed in the progeny suggests that PSTVd 
variants with less stable secondary structures enjoy a selective advantage. 
Mutations which stabilize the pathogenicity domain of PSTVd in vitro also 
suppressed symptom expression, but at least one other mutation having no obvious 
structural effects was associated with a similar phenotype. Conformational 
stability appears to be only one of several factors regulating PSTVd replication 
and pathogenicity.