INHIBITION OF POTATO SPINDLE TUBER VIROID (PSTVD) INFECTIVITY WITH ANTISENSE RNA TRANSCRIPTS

Antisense RNA was mixed with natural potato spindle tuber viroid (PSTVd) at 200-fold molar excess and hybridized in vitro before inoculation of tomato plants. Inhibition of viroid infectivity was then characterized by percent infected plants of inoculated plants (percent of infectivity) and amount of PSTVd in infected leaves. For short RNA complementary to nucleotides 42-78, the inhibitory effect was much lower than for the corresponding antisense DNA oligonucleotides under the same experimental conditions. An almost complete block of PSTVd infection was observed, however, using complete minus monomers of PSTVd, suggesting that double stranded PSTVd RNA is not infectious. For antisense RNA complementary to nucleotides 338-359/1-87, 264-359/1-94, and 88-337, percent of infectivity was 67%, 8% and 17% and levels of PSTVd relative to control (100%) were 48%, 20% and 35%, respectively. Double stranded complexes formed between PSTVd and antisense RNAs were detectable using antibody specific for double stranded RNA and the sizes of the double-stranded stretches were estimated using a nuclease S1-protection assay. It was found that double-stranded RNA was formed during co-transcription of antisense RNA and PSTVd monomers of plus polarity at 37-degrees-C and, to a lesser extent, during hybridization of antisense RNA and PSTVd monomers at lower temperature (after initially heating the samples to 40-degrees-C). In addition, antisense RNA inhibited infectivity of dimeric PSTVd transcripts of plus polarity with high efficiency if added during transcription. Co-transcription of PSTVd dimers with antisense RNAs yielded RNA species showing virtually no infectivity. Nuclease S1-protection assays revealed double-stranded RNAs having lengths predominantly in the range of the corresponding antisense RNAs.