RNA structural features responsible for potato spindle tuber viroid pathogenicity.

The native structure of potato spindle tuber viroid (PSTVd) contains a series of short double helices and small internal loops that are organized into five structural domains. Nucleotides within the pathogenicity domain are known to play a critical role in modulating PSTVd symptom expression, and it has been suggested that disruption of a comparatively unstable "premelting region" within the pathogenicity domain may be required for disease induction. We have used a combination of quantitative bioassays, temperature gradient gel electrophoresis of circularized RNA transcripts, and thermodynamic calculations to compare the biological and structural properties of 12 representative PSTVd sequence variants. Certain mutations appeared to act indirectly, downregulating pathogenicity by suppressing the rate of PSTVd replication/accumulation. The effects of other mutations appeared to be more direct, but there was no consistent correlation between symptom severity and melting temperature. Taking into account the three-dimensional shape of RNA helices, comparison of the optimal secondary structures for these variants point to major differences in the geometry of their pathogenicity domains; i.e., variants producing intermediate symptoms possess a linear arrangement of three consecutive helices, whereas for variants producing mild or severe symptoms this domain is bent in opposing directions. Such alterations in RNA structure together with concomitant alterations in RNA-protein interaction(s) may be the primary cause of viroid pathogenicity.