De novo synthesis of potato spindle tuber viroid as measured by incorporation of 32P

Abstract

Potato spindle tuber viroid (PSTV) was radioactively labeled during its synthesis in PSTV-infected tomato plants whose roots had been placed in nutrient solution containing [ 32P]orthophosphate. The 32P was incorporated into PSTV when the radioactive label was introduced into infected plants 2 weeks after inoculation, when systemic symptoms of PSTV started to appear. Synthesis of 32P-labeled PSTV was not observed when 32P was introduced into infected plants, either 1 or 3 weeks after inoculation. Uninoculated control plants subjected to incorporation of 32P did not synthesize 32P-labeled PSTV. De novo synthesis of 32P-labeled PSTV, measured in a constant amount of the labeled low-molecular-weight RNA fraction, was higher in plants that incorporated the isotope for 2 days rather than 1 day and was similar in plants which incorporated 32P for 2–5 days. The total amount of 32P-labeled PSTV that was synthesized, however, increased with increasing time of incorporation. De novo synthesis of PSTV, compared with de novo synthesis of other RNAs, constituted 0.4% of the low-molecular-weight RNA and 0.08% of the total RNA extracted from tomato leaves. Infection with PSTV appreciably increased the synthesis of a low-molecular-weight RNA species that was larger than PSTV or 9 S RNA. Pure 32P-labeled PSTV was obtained by electrophoresis of the low-molecular-weight RNA fraction on slab gels followed by reelectrophoresis of the extracted labeled PSTV on cylindrical gels. The G + C content of the homogeneous 32P-labeled PSTV was 54.7 mol%.