The Plant Journal (2004) 37, 762±769
doi: 10.1111/j.1365-313X.2003.02001.x
Short interfering RNAs speci®c for potato spindle tuber
viroid are found in the cytoplasm but not in the nucleus
Michela Alessandra Denti1,2,y,z, Alexandra Boutla1,2,z, Mina Tsagris1,2 and Martin Tabler1,
1
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Hellas, PO Box 1527,
GR-71110 Heraklion/Crete, Greece, and
2
Department of Biology, University of Crete, GR-71110 Heraklion/Crete, Greece
Received 18 September 2003; revised 2 December 2003; accepted 4 December 2003.
For correspondence (fax ‡30 810 394408; e-mail [email protected]).
y
Present address: Department of Genetics and Molecular Biology, University of Rome `La Sapienza', Rome, Italy.
z
These two authors share ®rst authorship.
Summary
Short interfering (si) and micro (mi) RNAs in¯uence gene expression at post-transcriptional level. In plants,
different classes of DICER-LIKE (DCL) enzymes are responsible for the generation of these small regulatory
RNAs from different precursors. To characterize the cellular site of their generation and accumulation, we
puri®ed nuclei from tomato plants infected with potato spindle tuber viroid (PSTVd) RNA, which is known
to replicate in the nucleus via double-stranded (ds) RNA intermediates. We could detect PSTVd-speci®c
siRNAs in the cytoplasmic fraction, but not in the nuclear fraction. To correlate the localization of the
PSTVd-speci®c siRNAs with that of similarly sized small RNAs, we studied the compartmentalization of
a naturally occurring miRNA. We could detect the precursor of miR167 in the nucleus, but the mature
miRNA was found only in the cytoplasmic fraction. We discuss the consequences of this ®nding for the
model of viroid replication and heterochromatin formation.
Keywords: siRNA, miRNA, Dicer, RNA interference (RNAi), post-transcriptional gene silencing (PTGS),
regulatory non-coding RNA.
Introduction
Two classes of small RNAs downregulate gene expression.
Short interfering (si) RNAs are small double-stranded (ds)
RNA fragments that mediate speci®c cleavage of singlestranded target RNAs via the RNA-induced silencing complex (RISC) in a process called RNA interference (RNAi) in
animals and post-transcriptional gene silencing (PTGS) in
plants (Cerutti, 2003; Dykxhoorn et al., 2003; Matzke et al.,
2001; Mlotshwa et al., 2002; Zamore, 2002). Micro (mi)
RNAs are short single-stranded RNAs that may arrest or
impair mRNA translation (Ambros et al., 2003). The discrimination between the two classes of regulatory RNAs is not
absolutely strict, since siRNAs may function as well as
miRNAs (Doench et al., 2003) and miRNAs may also enter
the PTGS pathway in plants (Llave et al., 2002; Palatnik
et al., 2003; Tang et al., 2003). Besides the direct in¯uence
of small regulatory RNAs on mRNA stability or mRNA
translation, it was recently shown that the RNAi machinery,
including siRNAs, is also responsible for heterochromatin
formation and thus for transcriptional silencing (Hall et al.,
762
2002; Schramke and Allshire, 2003; Volpe et al., 2002;
reviewed by Matzke and Matzke, 2003).
siRNA and miRNA exert their inhibitory post-transcriptional effect in the cytoplasm, but the effect of siRNAs on
heterochromatin formation must occur in the nucleus. This
raises the question: in which cellular compartment are the
two classes of small RNAs generated; and to what extent do
they accumulate in different cellular compartments. It is
established that siRNAs are the processing product of
perfectly longer dsRNAs that are cleaved by the ribonuclease III-like enzyme Dicer (Bernstein et al., 2001). There is
no doubt that generation of siRNA can proceed well in the
cytoplasm, as the replicative ds intermediates of plant RNA
viruses, including those that are supposed to stay in the
cytoplasm during their entire replication cycle, are effectively converted into siRNAs, followed by speci®c cleavage
of single-stranded viral RNA. This process is believed to be
the major defense strategy of plants against RNA viruses
(Voinnet, 2001). The Dicer enzyme is also responsible for
ß 2004 Blackwell Publishing Ltd
Cellular localization of viroid-specific siRNA
releasing mature miRNAs from nuclear-encoded precursor
RNAs that assume a hairpin-type secondary structure
(Grishok et al., 2001; Hutvagner et al., 2001). However,
different classes of Dicer enzymes are found depending
on the organism. While mammals (human, mouse), Caenorhabditis elegans, and the ®ssion yeast Schizosaccharomyces pombe contain only one class of Dicer enzyme,
Drosophila melanogaster contains two and there are four in
Arabidopsis thaliana (Schauer et al., 2002). Two of those,
DICER-LIKE 1 (DCL1) and DCL4, contain nuclear localization
signals (NLS). Recently, it was shown that DCL1 is required
for the generation of miRNA, but not for the generation of
siRNA (Finnegan et al., 2003). In accordance with the NLS
signal in DCL1 and based on the expression of nuclear and
cytoplasmic variants of P19, which is a suppressor of PTGS
derived from tomato bushy stunt virus (TBSV) and known
to bind speci®cally siRNAs, it was recently concluded that
plant miRNAs are processed in the nucleus and then
exported (Papp et al., 2003). This processing pathway in
plants is at variance with the maturation of mammalian
miRNAs. In human cell lines, it was shown that the long
primary transcripts (pri-miRNA) are processed by the
nuclear RNaseIII Drosha into the monomeric stem-loop
precursors (pre-miRNA; Lee et al., 2003), which are then
exported into the cytoplasm and processed to mature
miRNAs (Lee et al., 2002).
To clarify whether in plants siRNAs accumulate in the
nucleus, we made use of a viroid of the family Pospiviroidae, which represents a unique case of nuclearly localized
dsRNA that is, however, not encoded by the chromosome.
Viroids are naked single-stranded, covalently closed circular RNAs that cause infectious diseases in higher plants
(Flores, 2001). Their RNA genome does not encode any
peptide so that their replication and proliferation requires
host factors. Like viruses, they replicate via ds RNA±RNA
intermediates; however, the site of replication of Pospiviroidae is con®ned to the nucleus, where RNA synthesis is
carried out by the DNA-dependent RNA polymerase II (Pol
II; Schindler and MuÈhlbach, 1992). Along with others, we
could recently show that tomato plants infected with potato
spindle tuber viroid (PSTVd) RNA generate viroid-speci®c
siRNAs (Itaya et al., 2001; Papaefthimiou et al., 2001), at
®rst view suggesting that the small RNAs are also formed in
the nucleus. According to current models of silencing,
nuclear PSTVd-speci®c siRNA would serve no function.
First, they could not direct chromatin changes because
so far no homologous DNA sequences have been identi®ed
in a host plant. However, if viroid DNA sequences are
arti®cially introduced, they get methylated depending on
the presence of replicating viroid RNA (Wassenegger et al.,
1994). Second, it is not clear whether a nuclear RISC complex exists (compare Cerutti, 2003) and if so, whether it can
direct speci®c RNA cleavage. In the absence of such a
nuclear RISC-driven RNA degradation, viroid-speci®c
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 762±769
763
siRNAs would not be able to participate in the defense
against nuclear viroids. If PSTVd-speci®c siRNAs would
be translocated to the cytoplasm and then incorporated
into RISC, they would, according to the model of nuclear
viroid replication, not ®nd a speci®c single-stranded target
RNA that they could cleave. Despite the lack of function of
PSTVd-speci®c siRNAs, it should be added that both size
classes typical for RNA virus-derived plant siRNA and consisting of about 21/22 and 24±26 nucleotides (Hamilton
et al., 2002) could be discriminated. Moreover, viroid-speci®c siRNAs could also be observed for the peach latent
mosaic viroid (PLMVd) and chrysanthemum chlorotic mottle viroid (CChMVd) of the family Avsunviroidae (Martinez
de Alba et al., 2002), whose replication and accumulation is
restricted to the chloroplast. As there is no evidence for
Dicer activity in chloroplasts, those siRNAs must be generated in the cytoplasm. It was our objective to ®nd out
where in the plant host cell PSTVd-speci®c siRNAs actually
accumulate. For this purpose, we prepared RNA from puri®ed nuclei originating from PSTVd-infected tomato. We
found no experimental indication for viroid-speci®c siRNA
in the nucleus, but they were abundant in the cytoplasmic
fraction.
Results
Detection of PSTVd-specific siRNA
We harvested leaves from PSTVd-infected tomato plants
and corresponding healthy controls. From each type of
plants, a single leaf was taken for the preparation of a total
RNA extract, and the residual leaves were used to purify
nuclei. Monitoring the preparation under the microscope
con®rmed: ®rst, the absence of major contamination by
chloroplasts; and second, it showed that almost all nuclei
were intact. Next, we extracted RNA from the puri®ed
nuclei. The four RNA preparations were then subjected
to Northern analysis, and initially, we separated the samples on an 8% polyacrylamide gel in order to allow simultaneous detection of all size classes from circular viroid
RNA down to siRNAs. Using an RNA probe speci®c for
PSTVd (‡) RNA, we could detect circular and linear PSTVd
RNAs in the nuclear and in the total RNA fractions
(Figure 1a, lanes 2 and 5). The nuclear fraction contained
some smaller-than-unit-size RNA fragments, but no
siRNAs, which were however clearly visible in the total
fraction. The same membrane was then stripped and reprobed for U1 small nuclear (sn) RNA known to occur in
both cellular compartments. As expected, U1 could be
detected in all RNA fractions (Figure 1b), but the nuclear
fractions were distinct as some smaller RNAs could be
seen. The membrane was mildly stripped in order to maintain some of the U1 signals and re-hybridized with an RNA
764 Michela Alessandra Denti et al.
Figure 1. Northern analysis of nuclear and total RNA fractions from healthy and PSTVd-infected tomato, separated on a denaturing 8% polyacrylamide gel.
(a) Detection of PSTVd (‡) RNAs; lanes 1 and 2 contain RNA from nuclei originating from infected (In) and healthy (H) tomato; lanes 3 and 4 contain total RNA
fractions from healthy and infected plants; lane 5, the in vitro synthesized PSTVd (‡) RNA transcript Ha106 consisting of 406 nucleotides (Con). The position of
the linear (lin) and circular (cir) PSTVd RNA is indicated, as well as the position of the siRNA, which was determined in a separate lane by an unlabeled marker
prior to blotting.
(b) The same membrane as that in (a), after stripping and probed with U1 snRNA antisense probe. The position of U1 snRNA is indicated; the nuclear fractions
contain two smaller RNAs that might represent nascent incompletely synthesized U1 RNA that is not found in the cytoplasm.
(c) The same membrane as in (b), after mild stripping probed with an RNA probe speci®c for PSTVd ( ) RNA. The position of the U1 snRNA, the chloroplast 5S
RNA (5S), which is known to cross-hybridize with PSTVd (‡) RNA is indicated, as well the position of the siRNAs.
(d) The same membrane as that in (c), after stripping probed with an U6 snRNA antisense probe.
probe speci®c for PSTVd ( ) RNA. As anticipated, we found
in the total RNA some cross-hybridization with the chloroplast 5S RNA (Figure 1c) as it shares with PSTVd ( ) RNA
some sequence similarity (Papaefthimiou et al., 2001). We
also could detect siRNAs of ( ) polarity, however, again
only in the total, but not in the nuclear fraction derived from
infected plants. Finally, the same membrane was again
stringently stripped and re-hybridized with a probe
speci®c for U6 snRNA known to be retained in the nucleus
(Boelens et al., 1995; Figure 1d). This experiment essentially repeated the situation as seen for U1 snRNA.
Our ®nding that PSTVd-speci®c siRNA was undetectable
in the nucleus prompted us to make a more detailed analysis to exclude that this was the result of RNA leakage
during the preparation of the nuclei. So far, we could
discriminate only between a nuclear RNA fraction and a
total RNA fraction, which should contain both nuclear and
cytoplasmic RNAs. Therefore, we repeated the preparation
of nuclei, but this time we included further cell fractions in
the RNA analysis. As described in Experimental procedures, we ®rst homogenized the pool of harvested leaves
and kept a part to prepare a total extract (Supplementary
Material, Figure S1b). We continued in our preparation of
nuclei (Supplementary Material, Figure S1d) and subsequent RNA extraction, but we extracted also RNAs from
the remaining fraction, which represents cytoplasmic
RNAs (Supplementary Material, Figure S1c) and potentially
should contain RNAs that leak out from broken nuclei.
To evaluate the quality of this cell fraction, we analyzed
the presence of U6 snRNA, but this time separating the
RNAs on a denaturing 12% polyacrylamide gel, better
suited to resolve small RNAs. Figure 2 shows that the
nuclear and total RNA fractions contained U6 snRNA as
expected, but that even under overexposure, the cytoplasmic fraction contained only traces of it, demonstrating the
ef®ciency of our fractionation and the quality of our
nuclei preparation. It is interesting to note that shorter
U6-speci®c RNA could be found in the nucleus, which could
be degradation products, or more likely, they could represent nascent, incompletely synthesized U6 snRNAs of as
little as about 40 nucleotides, as puri®ed nuclei are able to
continue RNA transcription till they run out on nucleoside
triphosphates. There is no indication for leakage of these
small U6-speci®c RNAs into the cytoplasmic fraction. The
membrane was then stripped and re-probed for PSTVd (‡)
and later for PSTVd ( ) siRNAs. Consistent with the ®rst
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 762±769
Cellular localization of viroid-specific siRNA
experiment, PSTVd-speci®c siRNA could be detected in the
total and also in the cytoplasmic fractions from infected
tissues, but not in the nuclear fraction (Figure 2). It is
unlikely that leakage of the nuclei is responsible for the
identi®cation of siRNAs in the cytoplasmic fraction. If that
was the case, also U6-speci®c smaller RNA should at least,
in part, leak out. Moreover, it is hard to envisage
that leakage of viroid-speci®c siRNA would be complete
so that even detectable traces would not remain in the
nucleus fraction.
Detection of miRNA167 and its precursor
Micro RNAs are generated from a precursor RNA that
assumes a hairpin-type secondary structure unlike the
Figure 2. Northern analysis of nuclear, cytoplasmic, and total RNA fractions
from healthy and PSTVd-infected tomato, separated on a denaturing 12%
polyacrylamide gel.
The top shows the entire membrane probed for U6 snRNA. Lane 1 (TL)
contains a total leaf extract from PSTVd-infected tomato; lanes 2±4 contain
RNAs from total (T), cytoplasmic (C), and nuclear fraction (N) of PSTVdinfected tomato; and lanes 5±7 contain the corresponding fractions from
healthy control plants. The position of U6 snRNA is indicated as well as the
siRNA zone, which was de®ned by an unlabeled marker on a separate lane
prior to hybridization. The two lower panels show the siRNA after probing
for PSTVd (‡) and ( ) RNA, respectively. The double band represents the
size of 21/22 nucleotides. Between hybridizations, the membrane was
stringently stripped; control exposures con®rmed the removal of the hybridization probe.
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 762±769
765
complete duplex RNA from which siRNA originates. In
accordance with the different substrate RNAs, different
enzymes are responsible for the processing reaction,
and DCL1 was found associated with miRNA production
(Finnegan et al., 2003). We, therefore, also tested the total,
cytoplasmic, and nuclear fractions for miR167, which have
been detected not only in A. thaliana (Reinhart et al., 2002;
Rhoades et al., 2002) but also in tobacco (Mallory et al.,
2002), so that there was a good chance that would be
present in tomato as well. We used the same membrane
as shown in Figure 2 after stripping, and the hybridization
with the miR167-speci®c probe is shown in Figure 3. We
could detect the precursor RNA in the nucleus, however,
only in the PSTVd-infected nuclei fraction (Figure 3, lane 4).
Overlaying the original ®lms of Figures 2 and 3 showed that
the position of the pre-miR167 signal was almost identical
to that of the U6 snRNA signal (102±104 nts), which is
consistent with the length of 101 nts for the precursor of
miR167a. We could not detect the precursor in any other
fraction, neither in the total fraction nor in the nuclear
fraction of the healthy control, possibly because of a hybridization artifact in lane 7. In general, it seems to be dif®cult
to detect pre-miRNAs in plants, and we do not know about
any other successful example described. The mature
miR167 could be detected in the total and in the cytoplasmic
fractions of PSTVd-infected and healthy tomato (Figure 3,
Figure 3. Detection of miR167 and its precursor in different cell fractions.
The membrane of Figure 2 was re-hybridized with a radiolabeled DNA
oligonucleotide of antisense polarity for miR167. Loading was like in
Figure 2. Lane 7 contained a hybridization artifact, which most likely prevented detection of the pre-mi167 RNA that is visible in lane 4.
766 Michela Alessandra Denti et al.
lanes 2, 3, 5, and 6) but not in the nuclear fractions. As it was
recently concluded that the processing of miRNA proceeds
in the nucleus (Papp et al., 2003), this would suggest a rapid
exportation of miRNAs, possibly in a concerted reaction
following the processing step.
Discussion
Potato spindle tuber viroid replicates in the nucleus via
dsRNA intermediates that are subject to cleavage by one
of the Dicer nucleases. Our biochemical analysis has shown
that viroid-speci®c siRNAs are accumulating in the cytoplasm but are not detectable by conventional hybridization
methods in the nucleus. Although other small RNAs, such
as incomplete fragments of U6 RNA, are retained in the
nucleus, possibly because they are associated with protein
complexes, it cannot be ruled out that some leakage of
siRNAs from the nucleus might occur during the puri®cation procedure. However, regardless of whether more sensitive detection methods might reveal traces of nuclear
siRNAs, it is hard to envisage that solely leakage is responsible for the predominantly cytoplasmic accumulation of
the PSTVd-speci®c siRNAs. Although viroids are not
derived from nuclear DNA sequences, they do not represent the only example of nuclear dsRNA because duplex
RNA can be produced from transposons and arti®cially
introduced chromosomal transgenes. In case of `hairpin
genes', there is indirect evidence that a substantial part of
the siRNAs accumulate in the cytoplasm. For example,
transgenic tobacco generating substantial amounts of hairpin-derived siRNA speci®c for cucumber mosaic virus
(CMV) is resistant to viral infection (Kalantidis et al.,
2002), which necessitates a cytoplasmic localization of
the siRNAs. There is also evidence for transposon-derived
dsRNA in Caenorhabiditis elegans (Sijen and Plasterk,
2003), and for the MuDR/Mu elements that are responsible
for the Mutator activity in maize, siRNAs could be detected
primarily in the cytoplasmic fraction (Rudenko et al., 2003).
However, some short RNAs of antisense polarity could be
detected as well in the nuclear fraction, especially in an
inactive Mutator line.
The cytoplasmic localization of siRNAs could be the result
of two pathways. Either the dsRNAs are exported and then
cleaved by Dicer, or alternatively, Dicer cleavage occurs in
the nucleus and siRNAs are exported. The latter pathway
would be similar to the nuclear processing of plant miRNAs
(Papp et al., 2003), which is different from the mammalian
pathway (Lee et al., 2002). Assuming that PSTVd-speci®c
siRNA is indeed generated in the nucleus, cleavage should
be performed by DCL4, the only other Dicer enzyme with an
NLS besides DCL1, which however seems to be exclusively
involved in miRNA processing (Finnegan et al., 2003).
Regardless of the site of enzymatic cleavage, the accumulation of PSTVd-speci®c siRNAs in the cytoplasm is
puzzling with regard to their role in viroid replication.
PSTVd replicates and accumulates in the nucleus and, in
particular, in the nucleolus (Harders et al., 1989), and a
recent detailed analysis has shown that the (‡) and ( )
strands accumulate in different nuclear sites (Qi and Ding,
2003). This results in the paradox situation that the majority
of genomic PSTVd RNAs is nuclear, while PSTVd-speci®c
siRNAs are cytoplasmic, leaving them without an actual
target for RISC-mediated cleavage. One interpretation of
our observation is that unlike what has been assumed so
far, a substantial part of either single-stranded and/or ds
PSTVd RNAs is actually exported from the nucleus to the
cytoplasm, but they are not detectable because the dsRNAs
are cleaved by a cytoplasmic Dicer and the single-stranded
RNAs of both polarities are cleaved by cytoplasmic RISC, so
that they are apparently absent from the cytoplasm. As a
consequence of this, the only detectable viroid-speci®c
RNAs in the cytoplasm are siRNAs. This is reminiscent to
the observation we described for two transgenic tobacco
lines (28 and 65) where no transgene-derived RNA transcript of a ds CMV RNA was detectable, but large amounts
of siRNAs (Kalantidis et al., 2002). Further support for RNA
turnover in the cytoplasm is provided by Figure 1(a), which
shows that PSTVd-speci®c siRNAs actually represent a
substantial portion of the total PSTVd (‡) RNA present in
an infected cell. Moreover, there is much more PSTVd ( )
siRNA than expected, given the low concentration of PSTVd
( ) RNA in an infected cell (Spiesmacher et al., 1985). It
should be added that PSTVd-speci®c siRNAs can only be
detected if viroid concentration has also reached detectable
levels, which is about 1 to 2 days before symptom development (own unpublished results). The presence of cytoplasmic viroid-speci®c siRNAs (and an activated RISC)
would restrict PSTVd and related viroids to the nucleus,
a `safe' zone in the cell free of RISC or at least of an activated
RISC (for discussion of whether or not there is a nuclear
RISC, see Cerutti, 2003). The monomeric covalently closed
circular form is not very sensitive to Dicer cleavage (Chang
et al., 2003). Because of the circularity and the resulting rodshaped secondary structure, the monomer may be also less
sensitive to RISC-mediated cleavage so that this ®nally
accumulating form of the viroid RNA can pass the cytoplasm. The concept of retreating to a certain cellular compartment would also be in accordance with the occurrence
of siRNAs of two chloroplastic viroids (Martinez de Alba
et al., 2002). They just prefer another organelle that is not
able for RNAi.
An alternative explanation for the occurrence of cytoplasmic viroid-speci®c siRNA is the possibility that monomeric
circular RNA is actually converted in the cytoplasm by RNAdirected RNA polymerase (RdRp) with the aid of siRNAs
into cytoplasmic dsRNA, which is then cleaved by Dicer to
secondary siRNA. Further, one could envisage that viroidspeci®c siRNAs are actually generated preferentially in
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 762±769
Cellular localization of viroid-specific siRNA
some specialized cell types. Tissue-print hybridizations
have shown that PSTVd is preferentially accumulating
around the vascular system (Stark-Lorenzen et al., 1997;
Zhu et al., 2001). The viroid RNA has to pass this zone for
long-distance transport, and thus its titer is high in cells of
the vein complex, including companion cells. The mechanisms of transport through the vascular system are supposed to be the same for both families of viroids,
regardless of whether they replicate in the nucleus or in
the chloroplast. Especially in these cell types, the monomeric circular PSTVd RNA may be a target for RdRp so that
both families of viroids will generate siRNAs.
Although detailed mechanisms have not yet been
established, dsRNAs and siRNAs are implicated in regulating the `epigenome', i.e. they in¯uence heterochromatin
formation and/or RNA-directed DNA methylation (Hall
et al., 2002; Jenuwein, 2002; Martienssen, 2003; Rudenko
et al., 2003; Schramke and Allshire, 2003; Sijen and
Plasterk, 2003; Volpe et al., 2002; reviewed by Matzke
and Matzke, 2003). In pioneering work, Wassenegger et al.
(1994) could show that viroid RNA can direct DNA methylation of homologous sequences, and later it has been shown
that the expression of dsRNA may also result in the methylation and transcriptional inactivation of homologous promoter sequences (Mette et al., 2000) and that a DNA target
of 30 bp is suf®cient for RNA-directed DNA methylation
(PeÂlissier and Wassenegger, 2000). It is likely that siRNA (in
this context also called short heterochromatic (sh) RNA)
plays a role in this process (for discussion, see Jenuwein,
2002; Martienssen, 2003; Matzke and Matzke, 2003). The
observed absence of detectable viroid-speci®c siRNA in the
nucleus suggests that only small amounts of siRNAs are
required to induce DNA methylation, irrespective of
whether the trace amounts are generated in the nucleus
or re-imported from the cytoplasm.
The observation that virally expressed sequences can
direct DNA methylation (Jones et al., 2001) indicates that
the methylation signal indeed can be imported from the
cytoplasm.
Experimental procedures
Plant infection and growth conditions
Tomato plants (four-leaf stage, cultivar `Rentita') were inoculated
with in vitro synthesized RNA transcripts of the severe isolate
KF440-2 (Tsagris et al., 1991; Accession number X58388) as previously described by Tabler and SaÈnger (1984). The plants were
kept under greenhouse conditions; they developed PSTVd disease
symptoms about 3 weeks after inoculation.
Nuclei purification and cytoplasmic fraction separation
About 6 weeks after inoculation, tomato nuclei were puri®ed from
leaves as described by Schumacher et al. (1983), with some modß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 762±769
767
i®cations. Brie¯y, 150 g of leaves were immersed in 1.5 l of
Buffer A (600 mM mannitol, 25 mM 2-(N-morpholino)ethanesulfonic acid (MES)/NaOH, pH 6.5, 10 mM KCl, 5 mM MgCl2, 0.1%
BSA, 1 mM DTT), in®ltrated under vacuum, and incubated 30 min
on ice. Leaves were then removed from Buffer A, immersed in
450 ml of Buffer B (600 mM sucrose, 25 mM MES/NaOH, pH 6.5,
10 mM KCl, 5 mM MgCl2, 0.1% BSA, 1 mM DTT, 40% glycerol) and
homogenized using a blender. The resulting suspension was in
part used for RNA extraction (total fraction) or processed further to
separate cytoplasmic and nuclear fractions. The ®ltrated suspension was adjusted to 1% Nonidet P40, and nuclei were sedimented
by centrifuging for 10 min at 4000 g. The supernatant was decanted and kept as `cytoplasmic fraction'. The pellet, containing the
nuclei, was re-suspended in 5 ml of Buffer D (250 mM mannitol,
25 mM MES/NaOH, pH 6.5, 10 mM KCl, 5 mM MgCl2, 0.1% BSA,
1% Nonidet P40, 95% Percoll). After adding 380 ml of Buffer C
(600 mM sucrose, 25 mM MES/NaOH, pH 6.5, 10 mM KCl, 5 mM
MgCl2, 0.1% BSA, 1% Nonidet P40, 40% glycerol, 1 mM DTT), the
suspension was centrifuged for 10 min at 4000 g. The chloroplastcontaining dark green layer on the top of the supernatant was
sucked off, and the supernatant was discarded. The pellet was resuspended in 3 ml of Buffer D and 150 ml of Buffer C, and the
operation was repeated once. The nuclear pellet was re-suspended
in 45 ml of Buffer D and centrifuged for 10 min at 4000 g. The
resulting grayish thin layer ¯oating on the top of the suspension,
containing the nuclei, was collected, brought to 36% Percoll, and
centrifuged for 5 min at 4000 g. The pelleted nuclei were re-suspended in Buffer E (250 mM mannitol, 25 mM MES/NaOH, pH 6.5,
10 mM KCl, 5 mM MgCl2, 0.1% BSA, 1 mM DTT) and stored at
808C (nuclear fraction).
RNA extraction
For this study, we prepared RNA extracts from four different
starting materials. Total leaf RNA extracts were prepared as
described by Papaefthimiou et al. (2001). In brief, approximately
2±3 g of leaf material was harvested, frozen in liquid nitrogen, and
homogenized 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 addition of 10 ml
of extraction phenol (1 kg phenol, 300 ml of TEMS buffer, 1 g 8hydroxyquinoline and 250 ml of chloroform). The mixture was
vortexed and centrifuged at 48C, 4000 g for 30 min. The aqueous
phase was extracted once more with phenol and 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 volumes of ethanol. After collecting the precipitate by centrifugation, the samples were washed
with 70% ethanol and dried.
For RNA extraction of the three fractions (total, cytoplasmic, and
nuclear), the volume was measured and half a volume of TEMS
buffer was added, followed by an extraction with an equal volume
of chloroform±isoamyl alcohol (24 : 1, v/v). Subsequent phenol
extraction and RNA precipitation was as described in the procedure above.
All samples were quanti®ed spectrophotometrically and by
loading on ethidium-bromide-stained agarose gels.
Northern blot analysis
Hybridizations were performed using in vitro synthesized
32
P-labeled RNA transcripts. The transcripts were generated as
previously described using pHa106 as template (Tsagris et al.,
768 Michela Alessandra Denti et al.
1991) linearized with HindIII or EcoRI and transcribed with T7 or
SP6 RNA polymerase, respectively, providing longer-than-unit
length PSTVd (‡) or ( ) probes. The control U6 snRNA antisense
probe was obtained by transcribing an EcoRI-linearized plasmid
containing the mouse U6 snRNA gene with T7 RNA polymerase,
and U1 antisense probe (potato) was obtained by transcribing the
EcoRI-linearized plasmid pU1EH with SP6 RNA polymerase (Vaux
et al., 1992).
Northern blot analysis was performed as described by Papaefthimiou et al. (2001). Brie¯y, samples equivalent to approximately
100 mg of plant tissue were heat-treated in formamide buffer and
loaded onto a 12 or 8% polyacrylamide slab gel (acrylamide,
bisacrylamide, 20 : 1) 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 electro-blotted to NytranjN
membrane (Schleicher and Schuell, Dassel, Germany) and ®xed by
UV cross-linking. Pre-hybridization and hybridization, which
included the RNA probe at approximately 106 c.p.m. ml 1, was
carried out in 5 SSC, 1 Denhardt solution (Sambrook et al.,
1989), 1% SDS, 0.25 mg ml 1 tRNA carrier at 588C for 2 and 16 h,
respectively. When the 32P-labeled DNA oligonucleotide was used
as probe (Figure 3), the hybridization temperature was 458C. After
hybridization, the ®lter was washed with 2 SSC, 0.3% SDS for
5 min at room temperature followed by two washes for 20 min at
538C. In the case of the control hybridization, the membranes were
hybridized with 300 000 c.p.m. ml 1 U6 antisense probe in the
presence of 50% formamide at 658C and an additional stringent
wash with 0.1 SSC, 0.3% SDS for 15 min at the hybridization
temperature was included. Signals were visualized by autoradiography. Stripping of the probe from the membranes was performed by immersing them in boiling buffer (0.1 SSC, 0.5%
SDS) for 15 min.
Acknowledgements
We thank Sergia Tzortzakaki for excellent technical assistance and
Drs Kriton Kalantidis and BartheÂleÂmy Tournier for critical reading
of the manuscript. This work was supported in part by a grant from
the General Secretariat for Research and Technology of the
Hellenic Ministry of Development (contract PENED 01ED325)
and by the European Union (contract QLG2-CT-2002-01673 VIS).
Supplementary Material
The following material is available from http://www.blackwellpub
lishing.com/products/journals/suppmat/TPJ/TPJ2001/TPJ2001sm.
htm
Figure S1. Preparation of nuclei from tomato leaves.
(a) A multicellular trichome of a tomato leaf to demonstrate nuclei
(bright dots) in intact cells.
(b) Leaf tissue after homogenization.
(c) Cytoplasmic fraction after removal of nuclei; the chloroplasts
are visible as red dots.
(d) Puri®ed nuclei. The bars indicate different scales of magni®cation.
The samples were stained with ethidium to visualize nuclei.
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