Studies on viroid inducing RNA silencing from the view of plant pathology and agricultural impact

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When tomato plants were infected with Potato spindle tuber viroid (PSTVd) at the cotyledon stage, primary leaf curling appeared on the 3rd leaf 14 days after inoculation. PSTVd reached the detectable all the leaves but for the meristem. PSTVd-specific siRNA also reached the detectable level in the 2nd-9th leaves. Accumulation of PSTVd and the siRNA increased to the highest level in the leaves showing severe leaf curling, however, PSTVd concentration decreased in the 9th and the upper leaves recovered from severe leaf curling. The results indicated that PSTVd-specific RNA silencing was triggered soon after PSTVd reached its detectable level, and PSTVd-specific siRNA spread quickly throughout the plant except for the meristem. The leaves being differentiated after induction of RNA silencing are to be recovered, since they are placed under the RNA silencing from the initial stage of the differentiation which resulted in the suppression of PSTVd accumulation.Viroid-specific siRNAs of ca.21 and 23 nucleotides were detected in Hop stunt viroid-infected cucumber and hop, Hop latent viroid-infected hop and Apple fruit crinkle viroid-infected hop. On the other hand, only short siRNA of ca.21 nucleotides was detectable, in tomato infected with Cucumber mosaic virus.Using in silica analysis by BLAST search in DNA data base, several tomato EST sequences of 18-20 nucleotides were identified. They were PCR amplified, cloned and sequenced. Using the cloned cDNAs as probes, the expression of the genes were analyzed, however, any positive signal was not identified.Tomato seedlings obtained from healthy and viroid-infected fruits were equally infected with PSTVd challenge inoculation, which means that vertical transmission of viroid-induced RNA silencing was not apparent in tomato plants. On the other hand, the recovery was maintained in cuttings vegetatively propagated from PSTVd-infected tomato plants of the recovered stages.