J. Matousek et al., INHIBITION OF POTATO SPINDLE TUBER VIROID (PSTVD) INFECTIVITY WITH ANTISENSE RNA TRANSCRIPTS, Journal of phytopathology, 141(1), 1994, pp. 10-24
Antisense RNA was mixed with natural potato spindle tuber viroid (PSTV
d) at 200-fold molar excess and hybridized in vitro before inoculation
of tomato plants. Inhibition of viroid infectivity was then character
ized by percent infected plants of inoculated plants (percent of infec
tivity) and amount of PSTVd in infected leaves. For short RNA compleme
ntary to nucleotides 42-78, the inhibitory effect was much lower than
for the corresponding antisense DNA oligonucleotides under the same ex
perimental 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 c
omplementary to nucleotides 338-359/1-87, 264-359/1-94, and 88-337, pe
rcent 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 usi
ng antibody specific for double stranded RNA and the sizes of the doub
le-stranded stretches were estimated using a nuclease S1-protection as
say. It was found that double-stranded RNA was formed during co-transc
ription of antisense RNA and PSTVd monomers of plus polarity at 37-deg
rees-C and, to a lesser extent, during hybridization of antisense RNA
and PSTVd monomers at lower temperature (after initially heating the s
amples to 40-degrees-C). In addition, antisense RNA inhibited infectiv
ity 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. N
uclease S1-protection assays revealed double-stranded RNAs having leng
ths predominantly in the range of the corresponding antisense RNAs.