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Journal of Virology, August 1999, p. 6353-6360, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Subcellular Localization and Rolling Circle Replication of Peach
Latent Mosaic Viroid: Hallmarks of Group A Viroids
F.
Bussière,1
J.
Lehoux,1
D. A.
Thompson,2
L. J.
Skrzeczkowski,3 and
J.-P.
Perreault1,*
Département de biochimie, Faculté
de médecine, Université de Sherbrooke, Sherbrooke,
Québec J1H 5N4,1 and Centre
for Plant Health, CFIA, Sidney, British Columbia V8L
1H3,2 Canada, and Department of
Plant Pathology, Washington State University, Prosser, Washington
993503
Received 15 December 1998/Accepted 23 April 1999
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ABSTRACT |
We characterized the peach latent mosaic viroid (PLMVd) replication
intermediates that accumulate in infected peach leaves and determined
the tissue and subcellular localization of the RNA species. Using in
situ hybridization, we showed that PLMVd strands of both plus and minus
polarities concentrate in the cells forming the palisade parenchyma. At
the cellular level, PLMVd was found to accumulate predominantly in
chloroplasts. Northern blot analyses demonstrated that PLMVd replicates
via a symmetric mode involving the accumulation of both circular and
linear monomeric strands of both polarities. No multimeric conformer
was detected, indicating that both strands self-cleave efficiently via
their hammerhead sequences. Dot blot hybridizations revealed that PLMVd strands of both polarities accumulate equally but that the relative concentrations vary by more than 50-fold between peach cultivars. Taken
together these results establish two hallmarks for the
classification of viroids. Group A viroids (e.g., PLMVd), which possess
hammerhead structures, replicate in the chloroplasts via the symmetric
mode. By contrast, group B viroids, which share a conserved
central region, replicate in the nucleus via an asymmetric mechanism. This is an important difference between self-cleaving and
non-self-cleaving viroids, and the implications for the evolutionary
origin and replication are discussed.
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INTRODUCTION |
Viroids are small (~300-nucleotide
[nt]), single-stranded, circular RNAs that infect higher plants,
causing significant losses in the agricultural industry (see references
15 and 33 for reviews). The 26 known viroid species have been classified in two groups, A and B (see
references 6 and 15 for reviews). This classification is based primarily on whether a viroid possesses the five structural domains characteristic of a group B viroid. The
group B viroids are further subdivided on the basis of both the
sequence and the length of a highly conserved central region. Three
viroids possess no sequence or structural similarity with the group B
viroids and have been classified in group A. These three viroids
possess self-cleaving hammerhead motifs that are essential for their
replication (see below). This classification is supported by
phylogenetic reconstructions in which a group A viroid (avocado
sunblotch viroid [ASBVd]) has been proposed as an evolutionary link
between the classical group B viroids and the plant viroid-like
satellite RNAs (14).
In infected cells, viroids replicate in a DNA-independent manner via a
rolling circle mechanism that follows either a symmetric or an
asymmetric mode (15) (Fig. 1).
In the symmetric mode, the infecting circular monomer (which is
assigned plus polarity by convention) is replicated into linear
multimeric minus strands, which are then spliced and ligated, yielding
minus circular monomers. By using the latter RNA as template, the same
three steps are repeated to produce the progeny. In contrast, in the
asymmetric mode, the linear multimeric strands serve directly as the
template for the synthesis of linear multimeric plus strands.
Therefore, both the linear and circular minus monomers are produced
only in the symmetric mode. For example, the fact that minus circular monomeric strands of ASBVd are present in RNA isolated from infected avocado plants is taken as evidence that ASBVd replicates via the
symmetric mode (10, 20). Similarly, the fact that the circular minus monomer of potato spindle tuber viroid (PSTVd) has not
been found in plants infected by this viroid has been taken as evidence
that it replicates via the asymmetric mode (5).
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FIG. 1.
Schematic representation of the mechanism of viroid
rolling circle replication by either the symmetric (A) or the
asymmetric (B) mode. The polarity of the strands is indicated in
parentheses, and the small circle on the strands denotes the cleavage
site. The process begins with the infecting circular (+) strand.
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To date most of our knowledge of viroid biology comes from studies of
group B viroids (e.g., PSTVd). Three group B viroids have been shown to
accumulate in the nuclei of infected cells (4, 18, 29),
where they are believed to replicate via the asymmetric mode with host
RNA polymerase II as the replicase enzyme. The actual mechanism of
processing of the resulting multimeric strands is still a matter of
debate. It was proposed that a host endoribonuclease catalyzes the
cleavage of multimeric strands into monomers (15); however,
a recent report has shown that coconut cadang cadang viroid processing
could be mediated by a new self-cleaving motif under specific
conditions (25). In contrast, studies on ASBVd have shown
that this group A viroid is located mainly in the host chloroplasts
(3, 23). In this system, the processing of the multimeric
intermediates is mediated by self-catalytic hammerhead motifs.
Therefore, it has been proposed that the replication mode and the
viroid subcellular localization may be linked and may potentially
constitute a fundamental difference between the two major groups of
viroids (15). To confirm this analysis, it is essential to
determine whether the ASBVd features are common to other group A
viroids, more specifically to a member of the peach latent mosaic
viroid (PLMVd) subgroup.
PLMVd is an RNA species of 335-338 nucleotides (nt) which causes a
latent mosaic of peach trees (19). Both the plus and minus
multimeric PLMVd strands efficiently self-cleave in vitro by using
hammerhead structures (1, 19). Due to the presence of
self-cleavage properties and the absence of a known conserved central
region, PLMVd was proposed to be a group A viroid (19). PLMVd, ASBVd, and chrysanthemum chlorotic mottle viroid (CChMVd) are
the only hammerhead-containing species for which the group A
classification is confirmed. Unlike other viroids, which fold into
rod-like or quasi-rod-like structures, CChMVd and PLMVd adopt an
unusual branched secondary structure which makes them the only viroids
that are insoluble in 2 M lithium chloride (27). These results prompted the classification of the self-cleaving group A
viroids into two subgroups, one formed by ASBVd and one formed by PLMVd
and CChMVd (27). In this report, we characterize the PLMVd
replication intermediates which accumulate in infected peach leaves and
determine the tissue and subcellular localization of this RNA species
to strengthen the existing criteria for the classification of viroids.
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MATERIALS AND METHODS |
RNA isolation.
RNA samples were isolated from leaves
harvested from a total of nine peach cultivars, six that had been
infected by PLMVd and three healthy ones (see Table 1). Four different
procedures were used to prepare the RNA samples. The first procedure is
a modified version of the procedure involving a polyethylene glycol precipitation (PEG precipitation) (28, 32). Frozen leaf
petioles (0.6 g) were ground to a fine powder in liquid nitrogen. The
resulting powdered tissue was transferred to a 50-ml centrifuge tube,
and 5 ml of phenol mix (phenol-saturated Tris [pH 8.0], chloroform, octanol [100:100:4]), 4 ml of LG buffer (3.5 M LiCl, 0.3 M glycine [pH 9.5]), 80 µl of bentonite solution, 40 µl of
2-mercaptoethanol, and 40 µl of 20% lithium dodecyl sulfate in
water) were added. The mixture was vortexed for 30 to 60 s and
then centrifuged at 10,000 × g for 20 min. The
resulting supernatant was transferred to a fresh tube, and powdered PEG
6000 was added to a final concentration of 20% (wt/vol). After being
vortexed for 15 to 30 s, the tubes were successively incubated at
37°C for 5 min, at room temperature for 10 min, and on ice for 20 min
and were then vortexed again for 15 s. The mixture was centrifuged
at 10,000 × g for 20 min, and the pellet was
recovered, washed with 70% ethanol, and air dried. The pellet was
dissolved in 400 µl of nuclease-free water, transferred to a sterile
Eppendorf tube, and centrifuged at 12,000 × g for 5 min to remove any insoluble material. Finally, the RNA was ethanol
precipitated. The second procedure used the RNeasy Plant mini kit
(Qiagen) to prepare RNA from ~150 mg of tissue as specified by the
manufacturer. The third procedure was identical to the second, except
that 5 mM EDTA was added to all buffers. The fourth procedure was the
Tris-EDTA extraction method as previously described (17).
All RNA samples were quantified by UV spectrophotometry and
electrophoresed on 1.3% agarose gels to assess the quality of the
preparation. Dried RNA samples were stored at 
70°C.
Preparation of RNA probes.
Both the plus and minus
strand-specific riboprobes were synthesized and purified with plasmid
pPD1 as the template (1). Briefly, this construction
possesses two tandemly repeated PLMVd sequences cloned into the
PstI restriction site of pBluescript II KS. The insert is
flanked by T3 and T7 promoters for the production of plus- and
minus-polarity transcripts, respectively. For Northern blot
hybridization, we used the StripEZ transcription kit (Ambion) to obtain
probes that can be stripped under mild washing conditions to permit
multiple probings of the same membrane. The transcription reaction was
performed in the presence of 50 µCi of [
-32P]UTP (or
[-32P]GTP) (3,000 Ci/mmol; Amersham Life Science). For
in situ hybridizations, in vitro transcription was performed in the
presence of 3.5 mM digoxigenin-11-UTP (DIG-UTP; Boehringer Mannheim)
under the conditions recommended by the manufacturer. During
transcription, RNAs of both polarities possessing hammerhead sequences
are produced and self-cleave efficiently, producing 338-nt linear
monomeric transcripts. After transcription, treatment with DNase (RNase
free; Pharmacia) was performed and the nucleic acids were ethanol
precipitated. In general, the transcripts were resuspended in 20 µl
water, 10 µl of stop buffer (0.3% [wt/vol] each bromophenol blue
and xylene cyanol, 10 mM EDTA [pH 7.5], 97.5% [vol/vol] deionized
formamide) was added, and the resulting mixture was denatured for 2 min
at 65°C prior to polyacrylamide gel electrophoresis (PAGE) through a
5% (wt/vol) polyacrylamide gel in 100 mM Tris-borate (pH 8.3)-1 mM
EDTA-7 M urea buffer. Transcripts were detected by UV shadowing, excised, eluted, precipitated, purified by passage through Sephadex G-50 spun columns (Pharmacia), lyophilized, quantitated by absorbance spectrophotometry at 260 nm, and stored dry at 70°C. Incorporation of the DIG label into the transcripts was monitored by using dot blots
probed with alkaline phosphatase-conjugated anti-DIG antibody (Boehringer Mannheim) as specified by the manufacturer for DIG-labelled transcripts, while Cerenkov counting was used for the
32P-labeled transcripts.
PSTVd probes labeled with DIG were prepared by in vitro transcription
from plasmid pHa106 (kindly provided by Martin Tabler [34]). EcoRI-linearized pHa106 was
transcribed with SP6 RNA polymerase, producing a plus-polarity 406-nt
PSTVd riboprobe, which is slightly longer than the PSTVd monomer (i.e.,
359 nt).
In situ hybridization for electron and light microscopy.
Fixation and embedding of peach samples were performed as previously
described (23) with minimal modifications. Briefly, leaf
pieces from both healthy and PLMVd-infected peach plants (Siberian C
cultivar) were fixed with a modified Karnovsky fixation (1%
glutaraldehyde and 2% paraformaldehyde in 100 mM cacodylate buffer
[pH 7.2] containing 5 mM CaCl2) for 2 to 48 h,
dehydrated through an ascending ethanol series at room temperature, and
embedded in LR Gold (Electron Microscopy Science) at 20°C under UV light.
In situ hybridizations were performed as previously described
(
13). For light microscopy, 0.5-µm sections were collected
on poly-
L-lysine-coated Superfrost glass slides (dried for
3 h
at 42°C) and acetylated. Prehybridization (1 h) and
hybridization
(overnight) were performed as described for Northern
blots, except
that the salmon sperm DNA was replaced by 0.5 mg of yeast
tRNA
per ml, the temperature was held constant at 55°C, and both
steps
were performed in a petri dish humidified with 50%
formamide-4×
SSC (20× SSC is 3 M NaCl plus 0.3 M sodium citrate [pH
7.0]).
After hybridization, the slides were washed three times in 2×
SSC at 37°C for 15 min and once in 1× SSC for 15 min at 37°C.
Identical results were obtained when the slides were washed after
the
first 2× SSC wash with 2× SSC plus 1 µg of RNase per ml for
10 min
at room temperature. DIG-labeled hybrids were detected
as recommended
by the manufacturer (Boehringer Mannheim) by using
the reaction between
alkaline phosphatase-coupled anti-DIG antibody
and indoxyl-nitroblue
tetrazolium in the presence of polyvinyl
alcohol (
11). For
electron microscopy, ultrathin sections were
mounted on 150-mesh nickel
grids covered with Parlodion-carbon
coating and were then acetylated
and washed twice with 2× SSC
for 5 min. The grids were placed, section
down, on 200 to 400
µl of hybridization buffer and incubated
overnight at 60°C as
described for light microscopy. After
hybridization, the sections
were washed at 37°C successively once for
20 min and four times
for 5 min each with 2× SSC solution and finally
twice for 5 min
each with phosphate-buffered saline (0.34 M NaCl, 0.007 M KCl,
0.019 M KH
2PO
4, 0.004 M
Na
2HPO
4) plus 0.1% Tween 20. For indirect
immunodetection, the sections were incubated for 60 min at 37°C
with
mouse anti-DIG gold-labeled antibodies (Boehringer Mannheim)
diluted
1:25 in phosphate-buffered saline-0.1% Tween 20-1% casein.
After
two washes in PBS buffer, sections were incubated for 60
min at 37°C
on gold-labeled mouse anti-DIG (10-nm gold particles
[Cedar Lane])
and then briefly rinsed with distilled water. The
grids were examined
in a Philips 300 electron
microscope.
Northern and dot blot hybridizations.
Northern blot
hybridizations were performed as described previously (16)
with probes radiolabeled with [-32P]UTP. Isolated RNA
(2 µg) or PLMVd transcript (0.5 ng) was either resuspended in
formaldehyde gel running buffer, denatured for 3 min at 80°C, and
fractionated on 1.3% agarose gels containing formaldehyde and ethidium
bromide or resuspended in formamide-dye mixture (95% formamide, 10 mM
EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol), denatured for 2 min at 70°C, and subjected to PAGE (5% polyacrylamide) on gels in 50 mM Tris-borate (pH 8.3)-1 mM EDTA-7 M urea running buffer as
previously described (1). Nucleic acids were transferred by
capillary overnight to nylon filters (Hybond N+; Amersham
Life Science) in either 10× SSC (acrylamide gels) or 20× SSC (agarose
gels). The filters were then UV cross-linked for 5 min and baked at
80°C for 90 min before being prehybridized for 4 h at 60°C in
a solution containing 50% formamide, 5× SSC, 1% sodium dodecyl
sulfate (SDS), 5% Denhardt's solution, and 100 µg of salmon sperm
DNA per ml. Hybridizations were performed overnight (~16 h) at 60°C
with fresh prehybridization buffer containing the probe. After
hybridization, the filters were successively washed three times for 15 min each in 1× SSC-0.1% SDS at 65°C and once for 15 min in 0.1×
SSC-0.1% SDS at 60°C (or 65°C). The filters were analyzed by
either autoradiography or with a PhosphorImager (Molecular Dynamics).
All Northern blot hybridizations were performed as described above,
with the exception of a few controls, which are indicated Results. All
blots were successively hybridized with the plus- and the minus-strand
probes. Linear PLMVd transcripts used as controls were synthesized as
described previously (1). Circular PLMVd transcripts having
only 3',5'-phosphodiester bonds were synthesized as reported previously
(2).
Dot blot hybridizations were performed as described for Northern blots,
except that serial dilutions of the RNA samples were
applied to the
filter under vacuum. In addition, quantities ranging
from 0.01 to 32 ng
of gel-purified monomeric PLMVd transcripts
of both polarities
(synthesized by runoff transcription) were
used as standards.
Gel-purified monomeric (338-nt) PLMVd transcripts
of both polarities
were used as probes, and the filters were quantified
with a
PhosphorImager.
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RESULTS |
In situ hybridization of PLMVd strands.
To determine the
localization of PLMVd in tissue, leaf pieces from both healthy and
PLMVd-infected Siberian C peach plants were harvested, fixed, and
studied by in situ hybridization with DIG-UTP-labeled riboprobes (Fig.
2). The PLMVd infection was confirmed by
Northern blot hybridization (see below). In the first set of experiments, the DIG-labeled hybrids were detected with alkaline phosphatase-conjugated anti-DIG antibodies (e.g., Fig. 2A to E). No
hybridization signal was detected when PLMVd-infected samples were
incubated in the absence of a probe or with a DIG-PSTVd probe (Fig. 2A
and B). Similarly, no DIG-labeled hybrids were detected in healthy
peach leaves (Fig. 2C). In contrast, riboprobes of both polarities of
PLMVd reacted with PLMVd-infected samples (Fig. 2D and E). These
results were consistent within the various cultivars examined, with the
hybridization signals being limited to PLMVd-infected peach leaves
(data not shown). At the tissue level, the DIG-labeled hybrids were
observed to concentrate in the cells forming the palisade parenchyma.
Several DIG-labeled hybrids appeared in each cell; unfortunately, the
low resolution of light microscopy could not discern the specific
subcellular localization. Variations in the hybridization conditions,
washings, and DIG detection revealed different amounts of DIG-labeled
hybrids, but in all cases the proportion of the plus and minus strands
remained identical.
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FIG. 2.
Detection of PLMVd by in situ hybridizations with
DIG-labeled riboprobes. (A to E) Observations of the DIG-labeled
hybrids by light microscopy. The micrographs show sections of
PLMVd-infected leaves hybridized in either the absence (A) or the
presence (B) of DIG-PSTVd riboprobe, a section of a healthy peach leaf
probed with a minus-polarity PLMVd riboprobe (C), and sections of
PLMVd-infected peach leaves probed with either the plus (D) or minus
(E) PLMVd riboprobes. Control panels (A to C) were overstained to
ensure the detection of trace amounts of PLMVd strands. (F) Typical
electron micrograph of the hybridization of PLMVd-infected peach leaves
with the minus-strand PLMVd riboprobe. The arrows point to clusters of
grains representing PLMVd accumulated strands.
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In the second set of experiments, the target-probe hybrids were
revealed by using anti-DIG antibodies coupled to gold particles
and
were visualized by electron microscopy to determine the subcellular
localization of PLMVd. Figure
2F shows a typical result when probing
with the PLMVd riboprobe of minus polarity. In situ hybridization
revealed clusters of PLMVd strands of both polarities in the
chloroplasts
of infected leaves. In contrast, only negligible numbers
of gold
particles were found in healthy peach leaves due to the
nonspecific
binding of the labeled antibody (data not shown).
Statistical
analysis involved counting the colloidal gold particles in
15
to 20 cells per experiment. In some experiments more than
10
3 particles were counted in PLMVd-infected samples. These
analyses
showed an increase of at least 8-fold (with an average of
11.3-fold)
in the number of gold particles detected in the chloroplasts
of
PLMVd-infected peach leaves as compared to the chloroplasts of
healthy ones. No corresponding increase was observed in other
organelles. Similar increases in chloroplast labeling was observed
with
both plus and minus PLMVd riboprobes on PLMVd-infected samples
but not
with PSTVd riboprobes (data not shown). These investigations
revealed
that more than 80% (81 to 96%) of the total grains observed
in the
cell were located in the chloroplasts. The chloroplasts
in
PLMVd-infected tissues contained an average of 10 to 14 grains
depending on the experiment. The remaining grains appeared in
the
cytoplasm, the vacuoles, and the nuclei, while other cellular
structures such as the mitochondria and the cell wall had negligible
counts. Finally, approximately the same numbers of grains were
detected
irrespective of the polarity of the PLMVd riboprobe used
(i.e. ~10 to
14 grains per chloroplast), suggesting that both
PLMVd strands
accumulate to the same degree. These results clearly
show that PLMVd is
located predominantly in the
chloroplasts.
Detection of PLMVd replication intermediates.
To identify the
PLMVd replication intermediates which accumulate in cells, RNA samples
from both healthy and PLMVd-infected leaves were isolated and analyzed
by Northern blot hybridization. Because PLMVd strands of both
polarities have the ability to self-cleave in the presence of
magnesium, RNA samples were isolated by various procedures (see
Materials and Methods). Two of the four methods used included 5 mM EDTA
in their extraction buffers to chelate any cations and thereby prevent
any self-cleavage of the multimeric strands during purification. All
RNA samples were electrophoresed through both agarose and
polyacrylamide gels, transferred to filters and successively probed
with plus and minus PLMVd riboprobes (Fig. 3).
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FIG. 3.
Autoradiograms of Northern blot hybridizations of RNA
samples isolated from both healthy and PLMVd-infected peach leaves. RNA
samples were fractionated on either agarose (A to C) or polyacrylamide
(D and E) gels and then blotted onto nylon filters. The polarity of the
PLMVd riboprobe is indicated by the symbol (+) and () at the top of
the panel. (A) RNA samples were isolated by various extraction
procedures: RNeasy Plant mini kit (Qiagen) in the presence of EDTA
(lanes 3 to 5); Tris-EDTA isolation (lanes 6 and 7); RNeasy Plant mini
kit in the absence of additional EDTA (lane 8); and the PEG
precipitation procedure (lanes 9 to 12). In lanes 1 and 2, nonradioactive synthetic PLMVd transcripts of plus (761 and 588 nt) and
minus (745 and 462 nt) polarity, respectively, were loaded as controls.
Lane 3 contains a sample of healthy GF-305 peach cultivar; lanes 4, 6, 8, 9, 11, and 12 contain RNA samples isolated from a PLMVd-infected
RedGold peach cultivar. The samples in lanes 11 and 12 were subjected
to either DNase treatment or alkaline hydrolysis prior electrophoresis.
Lanes 5 and 7 contain to samples from a PLMVd-infected Redhaven
cultivar, while lane 10 contains a sample from the leaves of the Agua
cultivar. (B and C) The same filter following hybridization with either
the plus- or minus-polarity riboprobe. All samples were isolated by the
PEG precipitation procedure. Lanes 1 and 2 contain samples isolated
from different leaves of a Redhaven peach. Lane 3 contains an RNA
sample from a healthy Bailey cultivar. Lanes 4 and 5 contain RNA
samples of PLMVd-infected Redhaven and Siberian C cultivars. (D and E)
Filter following PAGE and blotting that was probed with both the plus-
and minus-polarity riboprobes, respectively. In panel D, lanes 1 and 2 contain synthetic circular and linear PLMVd controls, while lanes 3 to
5 (which correspond to lanes 1 to 3 of panel E) contain samples from
PLMVd-infected Redgold, Hardired, and Siberian C cultivars and lanes 6 and 7 (which correspond to lanes 4 and 5 of panel E) contain samples
from healthy GF-305 and Bailey cultivars. Adjacent to the gel, the
positions of several PLMVd transcripts are indicated as size references
including both the circular (338C) and linear (338L) strands and the
mixture of circular and linear molecules (PLMVd). The position of the
unknown ~1-kb species is also indicated in panels A and B.
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Initially, the RNA samples were analyzed on 1.3% agarose gels to
verify whether PLMVd multimeric strands were accumulated.
Nucleic acids
were hybridized with radioactive PLMVd-riboprobes
synthesized by runoff
transcription (see Materials and Methods).
Figure
3A shows a typical
autoradiogram of a hybridization performed
with the plus riboprobe to
detect minus-polarity PLMVd strands.
Lanes 1 and 2 are controls
containing synthetic PLMVd transcripts
of plus and minus polarities,
respectively. Cross-hybridization
between the probe and RNA strands of
the same polarity was detectable
only after overexposure of the gel
(i.e., <2% cross-hybridization).
Irrespective of the RNA isolation
procedure used, a band corresponding
to the PLMVd monomers was
consistently detected in the infected
samples but not in the healthy
ones (compare lanes 4 to 10 with
lane 3). The position of this band
varied slightly depending on
the RNA isolation procedure, most probably
because the various
RNA isolation methods yielded RNA samples which
contained different
salts in different concentrations. In addition, the
intensity
of the band varied depending the extraction procedure used
and
on the particular cultivar from which the sample was isolated.
The
RNA nature of the species was confirmed by its resistance
to DNase
treatment (lane 11) and susceptibility to alkaline hydrolysis
(lane
12). An additional nonspecific RNA band of ~1 kb was detected
in both
infected and healthy samples and appeared to be more abundant
when the
Qiagen RNA extraction procedure was used. Regardless
of the RNA
extraction method used, overexposure of the Northern
blots resulted in
the appearance of this band (data not shown).
However, since the
greatest amount was observed in samples from
healthy leaves (lane 3),
we believe that it is not a PLMVd replication
intermediate.
Both the plus and minus PLMVd riboprobes were used to analyze the PLMVd
replication intermediates in several peach cultivars.
The plus
PLMVd-riboprobe (Fig.
3B) detected a band corresponding
to monomer
minus-strand PLMVd (the nonspecific band of ~1 kb was
detected in one
sample), while probing of the same filter (after
being stripped) with
the minus PLMVd riboprobe detected only monomeric
PLMVd of plus
polarity (Fig.
3C). These results suggests that
both strands of PLMVd
accumulate as
monomers.
Regardless of the extraction method used, no multimeric strands of
either polarity were detected except after overexposure.
The presence
of 5 mM EDTA in the buffers, which chelates the divalent
cations
necessary for self-cleavage, did not increase the concentration
of
multimeric strands. To rule out the possibility that both the
linear
multimers and circular monomers self-cleaved to yield mainly
linear
monomers, we performed a set of control extractions. Synthetic
radiolabeled PLMVd transcripts were synthesized as described previously
(i.e., linear dimers and circular monomers [
8]) and
added to
the ground leaf powder. RNAs of these mixtures were extracted
with the RNeasy plant mini kit (Qiagen), either with or without
5 mM
EDTA, and were subsequently fractionated on polyacrylamide
gels.
Radiolabeled transcripts were revealed by autoradiography
(data not
shown). Independent of the presence of EDTA, only trace
amounts of
linear monomer strands were detected, suggesting that
self-cleavage was
limited during these extractions. Therefore,
self-cleavage during
extraction is not responsible for either
the absence of multimeric
strands or the low concentration of
circular conformers
observed.
PAGE followed by Northern blot hybridization was used to resolve
circular and linear PLMVd conformers (Fig.
3D and E). Only
the two
bands corresponding to the linear and circular PLMVd monomers
were
detected in RNA samples isolated from infected peach leaves.
Multimeric
strands of either polarity were not observed. The proportion
of linear
versus circular monomeric molecules was estimated for
both the plus and
minus strands of several PLMVd cultivars (Table
1). This proportion varied from 8 to 16 (average, 11) linear
conformers per circular molecule for the plus
strands and from
12 to 16 (average, 14) linear conformers per circular
molecule
for the minus strand. These results show that PLMVd strands of
both polarities accumulate predominantly as linear conformers.
Quantity of PLMVd strands.
To establish the quantity of PLMVd
strands (expressed as nanograms of PLMVd per milligram of tissue), we
performed dot blot hybridizations. This technique does not
differentiate between the linear and circular conformers; however,
since we know from the Northern blot hybridizations that the linear
conformer predominates, this is not a concern. Only RNA samples
isolated by PEG precipitation including the 3.5 M LiCl, known to favor
PLMVd extraction (27), were used. Several known
concentrations of linear monomeric (338-nt) PLMVd transcripts of both
polarities were used as standards. Serial dilutions of RNA from both
healthy and infected leaves were spotted onto filters and analyzed with
PLMVd riboprobes of both polarities. The results are reported in Table
1. PLMVd was detected only in RNA samples from infected leaves and not
in those from healthy ones (<0.01 ng/mg of tissue). The concentrations
of both the plus and minus PLMVd strands were relatively constant
within each cultivar except the Redgold cultivar, in which slightly
more minus strands were detected. In contrast, the concentrations
varied significantly between cultivars, ranging from 0.1 to 5.5 ng of
PLMVd strands per mg of tissue. PLMVd was more concentrated in Siberian
C, Redhaven, and Harrow Beauty and less concentrated in Hardired,
Redgold, and Agua peach trees. The observation that the strands of both polarities accumulate at approximately similar concentrations within a
cultivar but that these concentrations varied between the cultivars
correlates with the results of the Northern blot analyses. Total PLMVd
strands accumulate to levels comparable to those PSTVd, which
accumulates to a concentration of 1 ng per mg of tissue (7).
| |
DISCUSSION |
Subcellular localization is a trademark of viroid groups.
Using in situ hybridization, we have shown that PLMVd replicates
predominantly in the palisade parenchyma cells of infected peach leaves
(Fig. 2). However, it remains unknown if this cellular localization is
a result of the high metabolic activity occurring in these cells or of
a PLMVd molecular determinant to either enter or replicate primarily in
these cells. Within these cells, PLMVd strands of both polarities
accumulate in the chloroplasts (>80% of the strands are found in this
organelle). The similar localization of PLMVd and ASBVd, even though
they are members of different subgroups (the PLMVd and ASBVd subgroups,
respectively), suggests that the subcellular location of a viroid is a
fundamental characteristic of their group classification (Fig.
4). Group A viroids are found in
chloroplasts, while group B viroids are found in nuclei (see the
introduction). Clearly, this fundamental difference has important implications in the molecular biology and evolutionary origin of both
viroid groups.
View larger version (19K):
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|
FIG. 4.
Schematic phylogenetic reconstruction of viroids and
viroid-like satellite RNAs based on physical characteristics. The
dotted lines for the satellite RNAs indicate their precise relationship
to viroid group A and B.
|
|
Because of the difference in subcellular location, nuclear and
chloroplast viroids are likely to use different mechanisms
to ensure
their replication. For example, host nuclear RNA polymerase
II has been
proposed to replicate group B viroids (
15) but has
been
shown to be unable to replicate both ASBVd (
26) and PLMVd
(
22). In fact, the host polymerase supporting the
replication
of group A viroids has yet to be identified. Indeed, two
different
RNA polymerases transcribe chloroplastic genes: one is a
bacteriophage-like
RNA polymerase, while the other appears to be
related to
Escherichia coli RNA polymerase (see reference
24 for a review). One of
these RNA polymerases
probably replicates group A
viroids.
Viroids of both groups also use different strategies in the processing
of their multimeric strands. This step is mediated
by the hammerhead
self-catalytic motif in group A viroids, while
group B members are
thought to use either a host endoribonuclease
or some other catalytic
motif. The use of a self-cleavage motif
by the chloroplastic viroids
might be the result of the absence
of an appropriate endoribonuclease
in the chloroplast. In contrast,
plant viroid-like satellite RNAs are
most probably replicated
in the cytoplasm by their helper virus
replicase (
15). The cytoplasm
may also lack an appropriate
specific endoribonuclease; hence,
small satellite RNAs, which are
believed to replicate by a rolling
circle mechanism, either use
hammerhead self-cleavage or have
evolved other catalytic motifs (e.g.,
hairpin self-catalytic motif)
for the cleavage of their multimeric
strands. If we agree with
the hypothesis of a monophyletic origin for
both the group A and
group B viroids and for the related satellite RNAs
(
14) (Fig.
4), we conclude that an essential factor for
their divergence
over time is their cellular localization. The recent
observation
that the group B viroids may use a new self-cleavage motif
(
25)
supports the belief that the ancestor of today's
viroids possessed
a catalytic motif which diverged depending on the
cellular location.
If satellite RNA and viroids have a common ancestor,
the ancestral
catalytic motif of both cytoplasmic and chloroplastic
plant small
RNA pathogens could be a
hammerhead.
The PLMVd rolling circle mechanism.
The recent discovery of
retroviroid-like elements such as carnation stunt-associated small
circular RNA (9) prompted us to investigate whether PLMVd
was integrated into the host genome. Southern blot hybridization and
PCR amplification with various primers and DNA from infected peach
leaves as the template indicated that there is no PLMVd counterpart in
either the host genome or the extrachromosomal DNA (22a).
Thus, the replication cycle of PLMVd depends solely on RNA
intermediates, as is observed for all other viroids. Northern blot
hybridizations demonstrated that PLMVd replication follows a symmetric
pathway involving predominantly, if not exclusively, accumulation of
circular and linear monomeric RNAs of both polarities (summarized in
Fig. 1A). Clearly, the linear conformation was the most abundant form,
accumulating to levels at least eight times higher than those of
circular molecules (Table 1). PLMVd is the first viroid for which
replicational intermediates of both polarities are found to be
identical and are produced at the same level.
Apart from the type of rolling circle (symmetric versus asymmetric),
two features seem to differ significantly between the
replication of
PLMVd and other viroids. First, group B viroids,
as well as ASBVd,
accumulate the minus-polarity strand in very
low abundance compared to
the plus counterpart (
15,
20). By
contrast, PLMVd accumulate
both polarities to the same levels.
Therefore, the attribution of a
given polarity for PLMVd is not
based on biological observation, since
both strands are identical
with respect to their nature (e.g., linear
and circular), concentration,
and infectivity. Second, group B viroids
accumulate a series of
various multimeric linear RNAs (i.e. 1 to 8 units in length),
probably as a consequence of a poor cleavage
efficiency (
15,
20). By contrast, multimeric forms of PLMVd
are cleaved to completion
and then circularized. Accumulation of a high
level of monomeric
linear RNA might be the result of a rather
inefficient ligation
step. The synthesis of multimeric PLMVd strands,
which is a prerequisite
for a rolling circle mechanism, is supported by
reverse transcription
experiments allowing the detection of products
longer than 1 unit
(data not shown). Interestingly, the aforementioned
peculiarities
of the PLMVd rolling circle are also shared by the newly
discovered
group A viroid CChMVd (
27) and by a small
circular viroid-like
RNA (csc RNA 1) associated with a cherry disease
(
12). More
importantly, the PLMVd-like rolling circle
mechanism implies efficient
self-cleavage reactions, rather inefficient
ligation steps, and
a good stability of the accumulated monomeric RNAs
(discussed
below).
(i) Self-cleavage.
PLMVd, CChMVd, and csc RNA 1 appear to
self-cleave to completion in vivo, although their in vitro
self-cleavage efficiency ranges from 10 to 60% (1, 12, 27).
However, we have previously shown that the self-cleavage accuracy of
PLMVd can reach almost 100% in vitro (1). When PLMVd was
transcribed under conditions of slow polymerase activity, which favors
sequential production and folding of the hammerhead sequences,
self-cleavage of the resulting transcripts was greatly increased
(>95%) compared to that under the standard conditions (50 to 60%)
(1). Most probably, polymerase processivity or an unknown
molecular adaptation (e.g., a cofactor or an interaction with a
cellular component mediating structural changes) will explain the high
level of self-cleavage of the PLMVd, CChMVd, and csc RNA 1 RNAs in
vivo. Interestingly these three RNA species have a common organization
of their plus and minus hammerhead sequences that are included within a
long hairpin. Therefore, we can expect a similar mode of regulation of
self-cleavage for these RNAs that could be responsible for the
efficient self-cleavage activity in vivo. Other hammerhead self-cleaving viroids (e.g., ASBVd) and viroid-like RNAs differ by the
accumulation of a set of plus multimeric strands (1- to 11-mer) and
smaller minus multimeric strands (1- to 3-mer) (20, 30, 31).
It has been proposed that these RNA species have reduced self-cleavage
activity. For example, the catalytic sequences in ASBVd have been
proposed to fold into double-hammerhead structures, which is less
favored than the single-hammerhead structures (1).
(ii) Ligation.
The apparently efficient in vivo self-cleavage
of multimeric PLMVd strands of both polarities leads exclusively to the
accumulation of monomeric conformers. However, it remains unknown why
the monomeric linear conformers are the most abundant RNA
intermediates. One possible explanation is an inefficient
circularization step. Self-ligation has been demonstrated for the
circularization of PLMVd strands in vitro (8, 21). In vitro
self-ligation is relatively inefficient (i.e., ~10%), and thus the
accumulation of linear monomers is favored. This correlates with the
ratio of linear to circular PLMVd strands estimated in this report
(11:1 and 14:1 for the plus and minus polarities, respectively).
However, the existence of the self-ligation activity in vivo remains to
be proved. Whatever the mechanism responsible for the accumulation of
monomeric linear PLMVd strands, it is not known if there is only a pool
of RNA molecules to be ligated or an RNA population playing a direct role in the life cycle of the viroid.
(iii) Monomeric linear-RNA stability.
The high level of PLMVd
linear monomers suggests that this conformation is as stable as the
circular conformers, although the circular conformers are proposed to
be stabilized by the absence of termini. To prevent degradation by host
exonucleases, both the 5' and 3' extremities of the linear conformers
must be embedded within the RNA structure. This hypothesis is supported
by the observation that PLMVd linear monomeric strands are difficult to
end label at both termini (28a). Thus, it seems possible
that PLMVd linear monomers can accumulate because their termini are located within the RNA structure and are protected from the host exonuclease.
In conclusion, the three group A viroids (PLMVd, ASBVd, and CChMVd)
replicate via the symmetric mode, involving self-cleavage
activity, and
at least two of them are located in the chloroplasts
of infected cells.
These two characteristics appear to be hallmarks
of viroids belonging
to this group. However, this does not completely
exclude the
possibility of finding in the future a group A viroid
that will
replicate by the asymmetric mechanism. Moreover, the
presented data
strengthen the relationship between PLMVd and CChMVd
regarding their
rolling circle mechanism and accumulated intermediates.
These two
viroids share LiCl solubility (
27), a similar hammerhead
organization, and surprising structural features (
6a). Thus,
PLMVd and CChMVd appear to be closely related even though no obvious
sequence similarity is observed beside their hammerhead motifs.
These
observations support the proposed subdivision of group A
into ASBVd and
PLMVd subgroups (
27). Furthermore, PLMVd subgroup
viroids
show some interesting similarities to the newly discovered
satellite
RNA csc RNA 1 that extend beyond the presence of a hammerhead
sequence
on strands of both polarities (see above). These results
emphasize the
possible evolutionary link between viroids and plant
viroid-like
satellite
RNA.
| |
ACKNOWLEDGMENTS |
This work was sponsored by a grant from Natural Sciences and
Engineering Research Council (NSERC) of Canada to J.-P.P. F.B. was
the recipient of an NSERC studentship. J.-P.P. is a Medical Research
Council (MRC, Canada) scholar.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Département de biochimie, Université de Sherbrooke, 3001 12e Ave., Sherbrooke, Québec J1H 5N4, Canada. Phone: (819)
564-5310. Fax: (819) 564-5340. E-mail: jperre01{at}courrier.usherb.ca.
| |
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Journal of Virology, August 1999, p. 6353-6360, Vol. 73, No. 8
0022-538X/99/$04.00+0
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Top
Abstract
Introduction
