Subcellular localization of viroids in highly purified nuclei from tomato leaf tissue

Approximately 95% of the viroid RNA which is present in potato spindle tuber viroid (PSTV)-infected tomato plant leaf issue, is associated with the nucleolar fraction obtained from purified nuclei. Viroids were released from the nucleolar fraction by increasing the ionic strength of the medium to 0.66 suggesting that viroid RNA is present in these subnuclear components in a protein-nucleic acid complex. A purification procedure for nuclei from leaf tissue had to be newly developed; it involves two Percoll density centrifugations as final steps. The nuclei were sonicated and the sonicate fractionated into fractions either highly enriched in nucleoli or in broken chromatin and ribonucleoprotein particles. The viroid content in the different samples was determined by gel electrophoresis. Depending upon the progress of the disease, viroid copy numbers between 200 and 10,000 per cell were observed in homogenized tissue, purified nuclei and in the nucleolar fraction. In chloroplasts, practically no viroids were detected. The results are discussed in the light of current hypotheses about the replication, pathogenicity and origin of viroids.