|
|
Nucleic Acids Res. 2001 June 1; 29(11): 2395–2400. | PMCID: PMC55696 |
Replicating potato spindle tuber
viroid RNA is accompanied by short RNA fragments that are characteristic
of post-transcriptional gene silencing Ioannis Papaefthimiou,1,2 Andrew Hamilton,3 Michela Denti,1,2 David Baulcombe,3 Mina Tsagris,1,2 and Martin Tabler1a 1Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology, PO Box 1527, GR-71110 Heraklion, Crete, Greece , 2Department of Biology, University of Crete, GR-71110 Heraklion, Crete, Greece , and 3The Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK Received December 8, 2000; Revised March 9, 2001; Accepted April 1, 2001. The low molecular weight fraction of tomato plants inoculated
with potato spindle tuber viroid (PSTVd) contains a population of
short PSTVd-specific RNAs of either polarity. The main constituents
were RNAs of 22 and 23 nt representing different domains of the viroid
genome. The occurrence of such distinct RNA species indicated that
the nuclear replicating PSTVd RNA induces post-transcriptional gene
silencing. The short RNAs were slightly more abundant at 30 days
post-inoculation than at later stages and were present in plants
infected with a mild, severe or lethal isolate of PSTVd. There was
no apparent correlation between the quantity of small PSTVd-specific
RNAs and the degree of virulence of the viroid isolate. Post-transcriptional gene silencing (PTGS) results in the sequence-specific
degradation of single-stranded RNAs. The phenomenon was first described
in plants as ‘co-suppression’ when transgenes
with highly active promoters were analysed ( 1, 2). In many examples the affected RNA transcripts, including
those expressed from viruses and recombinant viruses (reviewed in 3, 4),
were transcribed at high levels. However, poorly transcribed RNAs
(reviewed in 5, 6)
or even ectopic promoterless DNA constructs have been shown to initiate PTGS
( 7). Once PTGS is induced it spreads
systemically and homologous RNAs are degraded regardless of whether
they had been expressed from nuclear genes, transgenes or cytoplasmic
RNA viruses. It is likely that PTGS represents a host defence system
against viruses ( 8), transposons
( 9) and potentially other forms
of invading RNA. Transgenes or double-stranded (ds) RNA, also called
interfering RNA (RNAi), can initiate similar processes in Neurospora
crassa ( 10), Caenorhabditis
elegans ( 11), Drosophila
melanogaster ( 12) and vertebrates
( 13, 14),
including mice ( 15). According to current models of PTGS, an endogenous RNA-dependent
RNA polymerase or a virus-encoded RNA polymerase ( 16)
synthesises a long dsRNA that is processed into 21–25 nt
RNA fragments of both polarities. Such RNAs were first described
in plants ( 17) and have recently
been identified in Drosophila cells treated with
RNAi ( 18, 19).
Current evidence suggests that the short RNAs are incorporated into
a ribonuclease and that they confer sequence-specificity upon this
enzyme ( 18, 20– 22). Hamilton and Baulcombe ( 17)
detected the characteristic RNAs in plants exhibiting PTGS that
had been initiated in each of three different ways, including transgene-induced
PTGS in the presence or absence of a homologous endogenous gene
and systemic PTGS induced by local infiltration with Agrobacterium tumefaciens carrying
a transformation vector. Since the size of the small RNA was identical
in these examples of PTGS it was concluded that the occurrence of
such RNA species can be used to diagnose PTGS and related processes
in situations where it had not previously been suspected or confirmed.
For example, the presence of potato virus X (PVX)-specific RNAs in
PVX-infected plants was used as supporting evidence for the proposed
role of PTGS as a natural anti-viral protection mechanism. Further
examples of the occurrence of PTGS-related short RNAs in plants
have been reported recently ( 23– 25). Viroids represent a distinct form of an RNA that can invade and
infect some higher plant hosts. Like viruses they are pathogenic,
autonomously replicating RNAs. However, their genomic RNA is unique.
It consists of a single-stranded, covalently closed and thus circular
RNA molecule ranging from 246 to 375 nt (reviewed in 26).
Unlike RNA viruses, the viroid RNA is devoid of any protecting coat
protein, does not use a pathogen-encoded replicase, and does not
encode any protein. Potato spindle tuber viroid (PSTVd), which is
the type-member of the largest groups of viroids, the pospiviroids, is
replicated in the nucleus by the DNA-dependent RNA polymerase II
( 27, 28)
which also synthesises RNA transcripts from endogenous genes or
transgenes. However, unlike a typical mRNA, PSTVd accumulates primarily
in the nucleolus ( 29). In this
report we show that PSTVd can induce the PTGS-typical response of
its host as monitored by the occurrence of small RNAs. Infections with PSTVd The tomato cultivar ‘Rentita’ was inoculated
with in vitro synthesised RNA transcripts as previously
described ( 30). Three different
PSTVd isolates were used: (i) the mild isolate KF5 (accession number
S54933; 31), (ii) the severe
isolate KF440-2 (accession number X58388; 32, 33) and (iii) the lethal isolate RG1 (accession
number U23058; 34). Photographs showing
the symptoms obtained with different viroid isolates have been published
previously ( 32). Plants were
kept under greenhouse conditions at ~20°C. Extraction of plant materials RNA extraction shown in Figure was
carried out according to the method of Hamilton and Baulcombe ( 17). For extractions shown in Figures –, ~2–3
g of leaf material was harvested, frozen in liquid nitrogen and
homogenised in a mortar. To the frozen powder, 7 ml of TEMS buffer
(100 mM Tris–HCl pH 7.5, 100 mM NaCl, 10 mM EDTA)
supplemented with 100 mM 2-mercaptoethanol prior to usage,
was added. This was followed immediately by the addition of 10 ml
extraction phenol (1 kg phenol, 300 ml TEMS buffer, 1 g
8-hydroxyquinoline and 250 ml chloroform). The mixture was vortexed and
centrifuged at 4°C, 4000 g for
30 min. The aqueous phase was extracted once more with phenol then
once with chloroform–isoamyl alcohol (24:1) (v/v),
in each case followed by a further centrifugation step. After increasing
the sodium concentration to 200 mM by the addition of sodium acetate pH 5.6,
the mixture was precipitated with 2.5 vol ethanol. After collecting
the precipitate by centrifugation the samples were washed with 70% ethanol
and dried. One millilitre of 8 M LiCl was added to the dried pellet of
the crude extract and shaken at 4°C
overnight. Following centrifugation at 4°C,
4000 g for 30 min, the supernatant was collected and
the extraction procedure repeated for 6 h. The combined supernatants
were precipitated with 2.5 vol ethanol. The samples were again collected by centrifugation, followed
by washing several times in 70% ethanol and drying. The
LiCl-soluble fraction was dissolved by adding 100 µl water. Hybridisation probes Hybridisation was performed using in vitro synthesised 32P-labelled
RNA transcripts generated as previously described [( 17) (Fig. ) or
( 35) (Figs –)]. Templates used were (i) pHa106
( 33) linearised with HindIII
which was transcribed with T7 RNA polymerase for synthesis of a
full-length PSTVd(–) RNA probe and (ii) plasmids containing
either of the two AvaI fragments of PSTVd cDNA
( 36) to synthesise sense or antisense
probes of the ‘left’ and ‘right’ halves
of PSTVd. Markers Markers shown in Figure were 32P-labelled
mGFP5 RNA, transcribed in vitro and digested with
RNAase T1. In Figures – the following DNA oligonucleotides were end-labelled
with [γ-32P]ATP:
23mer 5′-GGGGTAGGTGCGGGTTTCTTCAC; 25mer
5′-AGGTTTGGGTTTCGCCACTCTCTCG. For the transcription
marker, we used a mixture of plasmid pT3T7lac (Boehringer Mannheim)
linearised with either AvaI or SalI. Transcription
with T7 RNA polymerase resulted in RNA transcripts of ~27
and 39 nt. Northern analysis The analysis shown in Figure was carried
out according to Hamilton and Baulcombe ( 17).
In those shown in Figures –4, samples
equivalent to ~100 mg of plant tissue
were heat-treated in formamide buffer and loaded onto a 12% polyacrylamide
slab gel (12% acrylamide, 0.6% bisacrylamide)
containing 7 M urea and 50 mM TBE buffer (50 mM Tris, 41.5 mM boric
acid, 0.5 mM EDTA) and separated by electrophoresis. The samples were
electroblotted to Nytran ®N membrane (Schleicher & Schuell)
and fixed by UV cross-linking. Pre-hybridisation and hybridisation,
which included the RNA probe at ~10 6 c.p.m./ml,
was carried out in 5× SSC, 1× Denhardt
solution ( 37), 1% SDS,
0.25 mg/ml tRNA carrier at 58°C
for 2 and 16 h, respectively. After hybridisation, the
following washing steps were used: two washes with 2× SSC
for 5 min at room temperature followed by two washes with 2× SSC,
0.5% SDS for 30 min at 58°C.
Signals were visualised by autoradiography. In vitro synthesised RNA transcripts were used
to inoculate tomato plants at the four-leaf stage with the severe
PSTVd isolate KF440-2. After the plants developed PSTVd symptoms,
leaves were harvested at day 30 post-inoculation, which is an early
stage of viroid symptom development at the low temperature conditions
applied. Low molecular weight RNA was detected in PSTVd infected
tomato which was apparently identical in size to the PTGS-specific,
low molecular weight GFP RNA from GFP-silenced transgenic Nicotiana
benthamiana (Fig. ). This size class
of RNA has been found in all examples of PTGS tested in this way
but in none of the corresponding controls that did not exhibit PTGS
( 16– 25). As an alternative to the previously described procedure for extraction
of these RNAs ( 17), we tested
whether they could be enriched by LiCl solubilisation from total
RNA and DNA extracts of infected plants as used for the enrichment
of viroid RNA ( 38), except that
the LiCl concentration was increased to 8 M. Under these conditions
tRNAs, 5S rRNA, DNA and traces of rRNA were dissolved in the LiCl
extraction solution and the longer RNAs remained in the pellet.
Since the LiCl-soluble fraction includes most of the low molecular
weight RNAs (and chromosomal DNA) this extraction resulted in enrichment
for small RNAs. The LiCl extracted material was desalted by ethanol precipitation
and extensive washing and subjected to northern blot analysis by
hybridisation with a full length PSTVd antisense RNA probe (Fig. A). Two prominent low molecular weight RNA
fragments could be detected in extracts originating from infected
plants. Based on the migration of oligonucleotide markers (Fig. B), we concluded that these RNAs were ~22 and
23 nt long. However, neither of these RNAs migrated exactly in parallel
with the markers, and precise size determination was not possible.
Nevertheless, the size of small RNAs in viroid-infected plants appeared
to be identical to that in plants exhibiting PTGS of transgenes
(Fig. ). Next we assayed for representation of the sense and antisense
strands of viroid RNA in the population of the small RNAs in viroid-infected
plants. We also determined whether the small RNAs could hybridise
to different regions of the PSTVd genome and whether the production
of these RNAs varied at different stages of the infection or with
mild and aggressive strains. To carry out these tests we inoculated tomato plants with three
PSTVd strains of different virulence. Of these, KF5 was the least
aggressive, inducing only mild symptoms. Strain KF440-2 induced
severe symptoms but isolate RG1, which arose spontaneously during
an infection experiment ( 34),
was the most aggressive. Plant tissues were harvested at 30 days post-inoculation
and a second sample of the same plants was collected 62 days after
inoculation. LiCl-fractionated RNA extracts of these samples were
analysed by northern blotting using three different probes. Probe
A was an antisense RNA that covered the left half of the PSTVd,
ranging from nucleotides 284 to 359/1–97 (Fig. , left). Probe B was also of antisense polarity
and detected the right half of the PSTVd molecule, ranging from
nucleotides 92 to 289 (Fig. , middle).
Probe C was the sense equivalent to probe B (Fig. , right). The characteristic small RNAs were detected in all PSTVd-infected
samples. The occurrence of the small RNAs in the left and the right
domain of PSTVd indicated that different domains of the PSTVd RNA
sequence are represented in the population and that RNAs of both
polarities are produced. The antisense probes showed additional signals originating from
larger PSTVd(+) RNAs that were absent in the healthy control.
These larger PSTVd(+) RNAs are most probably unrelated
to PTGS. In accordance with the lower concentration of PSTVd replication
intermediates of (–) polarity ( 39)
there was a lower background hybridisation detected with the sense probe
C. However, there was an additional signal in the healthy control
that is due to cross hybridisation of the chloroplast large 5S rRNA
of tomato ( 40). This 121 nt RNA
is 100% identical to its tobacco homologue (accession number
M10360; 41) and can cross hybridise
due to a match of 15/16 nt between positions 215 and 230
of PSTVd(+) RNA. This cross hybridisation to 5S RNA was used as an internal standard
in our analysis for PSTVd-specific small RNAs in plants infected
with PSTVd strains of different virulence. The hybridisation signals
of 5S rRNA and small PTGS-induced PSTVd(–) RNAs were quantified
with a phosphorimager. Using the 5S rRNA as a reference, we found
that the samples taken at day 30 contained ~2–4-fold
more small RNAs than the samples taken at day 62. Re-probing of
the blot shown in Figure , right, with
a U1 antisense RNA confirmed this slight difference (data not shown).
Although we cannot exclude some variation in the concentration of
short RNAs in individual plants, these data indicate that PTGS is
induced at the early stage of PSTVd replication, when plants start
to develop symptoms. Comparing independent experiments we could
not, however, detect any significant difference in the PTGS response between
PSTVd isolates of different virulence (Fig. ). Small 21–25 nt RNAs have been associated with several
examples of PTGS. These include different examples of PTGS in plants
( 16, 17, 23– 25).
Similar RNA species have been detected in Drosophila in
response to treatment with interfering dsRNAs (RNAi) ( 18– 22). Recently, these RNAs have been shown
to be the specificity determinants of a sequence-specific ribonuclease
which carries out the degradation of mRNA that is characteristic
of PTGS ( 18– 22). Nevertheless, it is possible to use
the presence of these RNAs as an indicator of PTGS or PTGS-like
mechanisms in plants. Here we show the presence of small PSTVd homologous
RNAs of both polarities covering different domains of the PSTVd
genome in infected plants. This indicates for the first time that
plants respond to viroid infection by initiating PTGS directed against
the viroid RNA. Originally the short PTGS-associated RNAs were described as
25 nt in length. However, similar RNAs in extracts of Drosophila were
assigned lengths of 21 and 23 nt ( 19)
and here, using DNA markers, we determined a length of 22 and 23 nt.
Whatever the true length of these molecules, it does seem likely
that they exist in at least two major size classes. In addition,
two less abundant RNAs were observed in the PSTVd-infected plants.
One of these was 21 nt, and the other was ~26
nt. One way in which these classes of molecules could differ in
a manner that affects migration on gels is in the chemical nature
of the terminal residue. Recently, Hutvágner et
al. ( 25) showed that the
PTGS-typical short RNAs can be phosphorylated, indicating that they
have a 5′-hydroxyl group. However, that analysis
did not distinguish between the two classes of molecule and it remains
possible that they differ in the nature of the terminal residues.
The chemical nature of the terminal residue could explain the anomalous
migration of the short RNAs relative to the marker oligonucleotides
that had 5′-terminal phosphate groups. PTGS in plants can be initiated by cytoplasmically replicating
viruses and is usually targeted against mature rather than precursor
mRNAs. However, it is not yet clear whether the process is exclusively
cytoplasmic and the results reported here add to this ambiguity
because PSTVd replicates in the nucleus. PTGS can also be induced
by transgenes (reviewed in 42),
and in some instances causes an increase in the level of precursor
mRNAs corresponding to the target species ( 43).
In virus-infected cells there are several lines of evidence to indicate that
PTGS is part of a defence system that prevents unrestricted accumulation
of the viral RNA. Initially, as the virus enters the cell, viral
RNA accumulates rapidly. However, at later times, after PTGS has
been targeted against the viral RNA, its rate of accumulation slows
down and may even stop. It is tempting to speculate that similar
processes are active in PSTVd-infected cells. Thus, at 30 days post-inoculation
when viroid replication is most rapid, the small PSTVd-specific
RNAs are more abundant than at later stages when the viroid RNA
is present at a lower level. According to the idea that PTGS protects
against viroids, the reduced level of viroid RNA in the later stages
of infection would be due to PTGS induced by the rapidly replicating
viroid RNA in the early stages of the infection process. To test
this idea it would be necessary to assess viroid accumulation in
mutant plants that are defective for PTGS or in the presence of
virus-encoded suppressors of PTGS. If viroids are susceptible to
restriction by PTGS then this also provides a likely molecular basis
for the observed cross protection between related viroid strains. The finding that PTGS restricts viroid RNA accumulation implies
that the execution of PTGS could take place in the nucleus where
PSTVd replicates and accumulates. Attempts at detecting short RNAs
in nuclear RNA preparations have failed so far. Perhaps the PTGS
machinery has both cytoplasmic and nuclear phases. However, viroid
RNA would have to be present in the cytoplasm as it moves from cell
to cell and spreads systemically in the entire plant and it cannot
be ruled out that the results reported here reflect a response to
the cytoplasmic phase of the viroid RNA. On the other hand, it has already
been shown that agents known to induce PTGS ( 44– 47) also induce homology dependent transcriptional
gene silencing (TGS; a nuclear process by definition) and implies that
the signaling molecules conveying sequence specificity may be active
in both nucleus and cytoplasm. Notably, TGS was also accompanied
by the accumulation of the same sized short RNAs characteristic
of PTGS. Our works extends this interrelationship of TGS and PTGS
to viroids as PSTVd was previously shown to induce methylation of
homologous cDNA sequences ( 48– 50). It was notable that we could not correlate aggressiveness of the
viroid isolates with the PTGS response as determined by the concentration
of the PTGS-specific small RNAs. This is reminiscent of
the observation that virulence of different viroid isolates is not
correlated with the titer of accumulating viroid RNA ( 34).
It is unlikely, therefore, that the variation in aggressiveness
is related to the differential effects of PTGS on the overall accumulation
of the viroid RNA. A second untested possibility is that PTGS is
directly involved with symptom development in viroid-infected plants.
Since the specificity determinants of PTGS are short RNAs, similarly
short lengths of chance homology between parts of the viroid genome
and cellular mRNAs may be sufficient to render those host mRNAs targets
of the PTGS response induced by the viroid. In this hypothesis,
the differential virulence of viroids might be explained not by
their overall level of accumulation in the plant, but by subtle
sequence variation in the region sharing homology with the targeted
host genes. We thank Dr Gerhard Steger, University of Düsseldorf
for providing cDNA clones of PSTVd isolate RG1 and KF5, and A. Emilio
Martinez de Alba (IMBB) for subcloning the AvaI fragments
of PSTVd cDNA. This work was in part supported by the Greek Ministry
of Development, (PENED 99ED376) and the European Union (Biotech
BIO4 CT97-2300). A.J.H. and D.C.B. are grateful to the Gatsby Charitable
Foundation for financial support. 1. Jorgensen R. (1990)
Altered gene expression in plants due to trans interactions between
homologous genes. Trends Biotechnol., 8, 340–344. [PubMed] 2. van
der Krol A.R., Mur,L.A., Beld,M.,
Mol,J.N. and Stuitje,A.R. (1990) Flavonoid genes in
petunia: addition of a limited number of gene copies may lead to
a suppression of gene expression. Plant Cell, 2, 291–299. [PubMed] 3. Baulcombe D.C. (1999)
Viruses and gene silencing in plants. Arch. Virol., 15, S189–S201. 4. Baulcombe D.C. (1999)
Fast forward genetics based on virus-induced gene silencing. Curr.
Opin. Plant Biol., 2, 109–113. [PubMed] 5. Kooter J.M.,
Matzke,M.A. and Meyer,P. (1999) Listening to the silent genes:
transgene silencing, gene regulation and pathogen control. Trends Plant
Sci., 9, 340–347. [PubMed] 6. Meins F. (2000)
RNA degradation and models for post-transcriptional gene silencing. Plant
Mol. Biol. 43, 261–273. [PubMed] 7. Voinnet O.,
Vain,P., Angell,S. and Baulcombe,D.C. (1998) Systemic
spread of sequence-specific transgene RNA degradation in plants
is initiated by localized introduction of ectopic promoterless DNA. Cell, 95, 177–187. [PubMed] 8. Ratcliff F.G.,
MacFarlane,S.A. and Baulcombe,D.C. (1999) Gene silencing
without DNA. RNA-mediated cross-protection between viruses. Plant
Cell, 11, 1207–1216. [PubMed] 9. Ketting R.F.,
Haverkamp,T.H., van Luenen,H.G. and Plasterk,R.H. (1999)
Mut-7 of C. elegans, required for transposon silencing
and RNA interference, is a homolog of Werner syndrome helicase and
RNaseD. Cell, 99, 133–141. [PubMed] 10. Cogoni C. and
Macino,G. (1997) Isolation of quelling-defective (qde) mutants
impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa. Proc.
Natl Acad. Sci. USA, 94, 10233–10238. [PubMed] 11. Fire A.,
Xu,S., Montgomery,M.K., Kostas,S.A., Driver,S.E. and Mello,C.C.
(1998) Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806–811. [PubMed] 12. Kennerdell J.R. and
Carthew,R.W. (1998) Use of double-stranded RNA-mediated
genetic interference to demonstrate that frizzled and frizzled 2 act
in the wingless pathway. Cell, 95, 1017–1026. [PubMed] 13. Wargelius A.,
Ellingsen,S. and Fjose,A. (1999) Double-stranded RNA induces
specific developmental defects in zebrafish embryos. Biochem. Biophys.
Res. Commun., 263, 156–161. [PubMed] 14. Li Y.X.,
Farrell,M.J., Liu,R., Mohanty,N. and Kirby,M.L. (2000)
Double-stranded RNA injection produces null phenotypes in zebrafish. Dev.
Biol., 217, 394–405. [PubMed] 15. Wianny F. and
Zernicka-Goetz,M. (2000) Specific interference with
gene function by double-stranded RNA in early mouse development. Nat.
Cell Biol., 2, 70–75. [PubMed] 16. Dalmay T.,
Hamilton,A., Rudd,S., Angell,S. and Baulcombe,D.C. (2000) An
RNA-dependent RNA polymerase gene in Arabidopsis is
required for posttranscriptional gene silencing mediated by a transgene
but not by a virus. Cell, 101, 543–553. [PubMed] 17. Hamilton A.J. and
Baulcombe,D.C. (1999) A species of small antisense RNA
in posttranscriptional gene silencing in plants. Science, 286, 950–952. [PubMed] 18. Hammond S.M.,
Bernstein,E., Beach,D. and Hannon,G.J. (2000) An RNA-directed
nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 404, 293–296. [PubMed] 19. Zamore P.D.,
Tuschl,T., Sharp,P.A. and Bartel,D.P. (2000) RNAi: double-stranded
RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide
intervals. Cell, 101, 25–33. [PubMed] 20. Yang D.,
Lu,H. and Erickson,J.W. (2000) Evidence that processed
small dsRNAs may mediate sequence-specific mRNA degradation during
RNAi in Drosophila embryos. Curr. Biol., 10, 1191–1200. [PubMed] 21. Bernstein E.,
Caudy,A.A., Hammond,S.M. and Hannon,G.J. (2001) Role for
a bidentate ribonuclease in the initiation step of RNA interference. Nature, 409, 363–366. [PubMed] 22. Elbashir S.M.,
Lendeckel,W. and Tuschl,T. (2001) RNA interference
is mediated by 21-and 22-nucleotide RNAs. Genes Dev., 18, 188–200. [PubMed] 23. Dalmay T.,
Hamilton,A., Mueller,E. and Baulcombe,D.C. (2000) Potato virus
X amplicons in arabidopsis mediate genetic and epigenetic gene silencing. Plant
Cell, 12, 369–379. [PubMed] 24. Llave C.,
Kasschau,K.D. and Carrington,J.C. (2000) Virus-encoded suppressor
of posttranscriptional gene silencing targets a maintenance step
in the silencing pathway. Proc. Natl Acad. Sci. USA, 97, 13401–13406. [PubMed] 25. Hutvágner G.,
Mlynárová,L. and Nap,J.P. (2000)
Detailed characterization of the posttranscriptional gene-silencing-related
small RNA in a GUS gene-silenced tobacco. RNA, 6, 1445–1454. [PubMed] 26. Flores R.,
de la Peña,M., Navarro,J.A., Ambrós,S. and Navarro,B.
(1999) Molecular biology of viroids. In Mandahar,C.L.
(ed.) Molecular Biology of Plant Viruses. Kluwer
Academic Press, Dordrecht, The Netherlands, pp. 225–239. 27. Mühlbach H.-P. and
Sänger,H.L. (1979) Viroid replication is inhibited
by α-amanitin. Nature, 278, 185–188. [PubMed] 28. Schindler I.M. and
Mühlbach,H.P. (1992) Involvement of the nuclear DNA-dependent
RNA polymerases in potato spindle tuber viroid replication: a re-evaluation. Plant
Sci., 84, 221–229. 29. Harders J.,
Lukacs,N., Robert-Nicoud,M., Jovin,T.M. and Riesner,D. (1989) Imaging
of viroids in nuclei from tomato leaf tissue by in situ hybridisation and confocal laser scanning microscopy. EMBO
J., 8, 3941–3949. [PubMed] 30. Tabler M. and
Sänger,H.L. (1984) Cloned single- and double-stranded DNA
copies of potato spindle tuber viroid (PSTV) and co-inoculated subgenomic
DNA fragments are infectious. EMBO J., 3, 3055–3062. [PubMed] 31. Lakshman D.K. and
Tavantzis,S.M. (1993) Primary and secondary structure
of a 360-nucleotide isolate of potato spindle tuber viroid. Arch. Virol., 128, 319–331. [PubMed] 32. Schnölzer M.,
Haas,B., Ramm,K., Wang,Z.F. and Sänger,H.L. (1985) Correlation
between structure and pathogenicity of potato spindle tuber viroid
(PSTV). EMBO J., 4, 2181–2190. [PubMed] 33. Tsagris M.,
Tabler,M. and Sänger,H.L. (1991) Ribonuclease
T1 generates circular RNA molecules from viroid-specific RNA transcripts
by cleavage and intramolecular ligation. Nucleic Acids Res., 19, 1605–1612. [PubMed] 34. Gruner R.,
Fels,A., Qu,F., Zimmat,R., Steger,G. and Riesner,D. (1995) Interdependence
of pathogenicity and replicability with potato spindle tuber viroid. Virology, 209, 60–69. [PubMed] 35. Hammann C.,
Hormes,R., Sczakiel,G. and Tabler,M. (1997). A spermidine-induced
conformational change of long-armed hammerhead ribozymes: ionic
requirements for fast cleavage kinetics. Nucleic Acids Res., 25, 4715–4722. [PubMed] 36. Martinez
de Alba A.E. (2000)
Isolation and Characterisation of Viroid-Binding Proteins. PhD thesis,
Universidad del Pais Vasco, Bilbao, Spain. 37. Sambrook J.,
Fritsch,E.F. and Maniatis,T. (1989) Molecular
Cloning: A Laboratory Manual, 2nd Edn. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 38. Tabler M.,
Günther,I., Kern,R. and Sänger,H.L. (1989)
A microscale procedure for isolating and sequencing the viroid RNA
present in one gram of infected leaf tissue. J. Virol. Methods, 23, 111–126. [PubMed] 39. Spiesmacher E.,
Mühlbach,H.P., Tabler,M. and Sänger,H.L. (1985) Synthesis
of (+) and (–) RNA molecules of potato spindle
tuber viroid (PSTV) in isolated nuclei and its impairment by transcription
inhibitors. Biosci. Rep., 5, 251–265. [PubMed] 40. Herold T. (1990)
Zusammenhänge zwischen der Struktur und Pathogenität
der Viroide am Beispiel des Potato Spindle Tuber Viroids, sowie
die sequenzierung und molekulare klonierung einer zelleigenen RNA,
die mit PSTV (+) RNA hybridisiert. PhD thesis, University
of Giessen, Germany. 41. Takaiwa F. and
Sugiura,M. (1981) Heterogeneity of 5S RNA species in tobacco
chloroplasts. Mol. Gen. Genet., 182, 385–389. 42. Sijen T. and
Kooter,J.M. (2000) Post-transcriptional gene-silencing: RNAs
on the attack or on the defense? Bioessays, 6, 520–531. [PubMed] 43. Mishra K.K. and
Handa,A.K. (1998) Post-transcriptional silencing of pectin
methylesterase gene in transgenic tomato fruits results from impaired
pre-mRNA processing. Plant J., 14, 583–592. 44. Jones L.,
Hamilton,A.J., Voinnet,O., Thomas,C.L., Maule,A.J. and Baulcombe,D.C.
(1999) RNA–DNA interactions and DNA methylation
in post-transcriptional gene silencing. Plant Cell, 11, 2291–2302. [PubMed] 45. Mette M.F.,
Aufsatz,W., van der Winden,J., Matzke,M.A. and Matzke,A.J. (2000)
Transcriptional silencing and promoter methylation triggered by
double-stranded RNA. EMBO J., 19, 5194–5201. [PubMed] 46. Wang M.B.,
Wesley,S.V., Finnegan,E.J., Smith,N.A. and Waterhouse,P.M. (2001)
Replicating satellite RNA induces sequence-specific DNA methylation
and truncated transcripts in plants. RNA, 7, 16–28. [PubMed] 47. Thomas C.L.,
Jones,L., Baulcombe,D.C. and Maule,A.J. (2001) Size constraints
for targeting post-transcriptional gene silencing and for RNA-directed
methylation in Nicotiana benthamiana using Potato
virus X vector. Plant J., in press. 48. Wassenegger M.,
Heimes,S., Riedel,L. and Sänger,H.L. (1994)
RNA-directed de novo methylation of genomic sequences
in plants. Cell, 76, 567–576. [PubMed] 49. Pelissier T. and
Wassenegger,M. (2000) A DNA target of 30 bp is sufficient
for RNA-directed DNA methylation. RNA, 6, 55–65. [PubMed] 50. Wassenegger M. (2000)
RNA-directed DNA methylation. Plant Mol. Biol., 43, 203–220. [PubMed]
|
This article has been 
