Download Mutational analysis of eggplant latent viroid RNA processing in

Journal of General Virology (2009), 90, 3057–3065
DOI 10.1099/vir.0.013425-0
Mutational analysis of eggplant latent viroid RNA
processing in Chlamydomonas reinhardtii
chloroplast
Fernando Martı́nez,1 Jorge Marqués,1 Marı́a L. Salvador2
and José-Antonio Daròs1
1
Correspondence
Instituto de Biologı́a Molecular y Celular de Plantas (CSIC-UPV), Avenida de los Naranjos,
46022 Valencia, Spain
José-Antonio Daròs
[email protected]
2
Departamento de Bioquı́mica y Biologı́a Molecular, Universitat de València, Avenida Dr Moliner,
46100 Burjassot, Spain
Received 19 May 2009
Accepted 4 August 2009
Viroids of the family Avsunviroidae, such as eggplant latent viroid (ELVd), contain hammerhead
ribozymes and replicate in the chloroplasts of the host plant through an RNA-based symmetrical
rolling-circle mechanism in which oligomeric RNAs of both polarity are processed to monomeric
linear RNAs (by cleavage) and to monomeric circular RNAs (by ligation). Using an experimental
system consisting of transplastomic lines of the alga Chlamydomonas reinhardtii, a mutational
analysis of sequence and structural elements in the ELVd molecule that are involved in transcript
processing in vivo in a chloroplastic context was carried out. A collection of six insertion and three
deletion ELVd mutants was created and expressed in C. reinhardtii chloroplast. All mutants
cleaved efficiently except for the control with an insertion inside the hammerhead ribozyme
domain, supporting the prediction that this domain is necessary and sufficient to mediate
transcript cleavage in vivo. However, two deletion mutants that cleaved efficiently showed ligation
defects, indicating that during RNA circularization, other parts of the molecule are involved in
addition to the hammerhead ribozyme domain. This is probably a quasi double-stranded structure
present in the central part of the molecule which contains the ligation site in an internal loop.
However, the mutations prevented the viroid from infecting its natural host, eggplant, indicating
that they affected other essential functions in ELVd infectious cycle. The insertion in the terminal
loop of the right upper hairpin of ELVd did not have this effect; it was tolerated and partially
maintained in the progeny.
INTRODUCTION
Viroids are small circular single-stranded non-coding
RNAs (246–401 nt) that infect plants (Flores et al., 2005;
Daròs et al., 2006; Ding, 2009). Their infectious cycle
depends completely on functional elements in the viroid
RNA molecule and interacting host factors. Eggplant latent
viroid (ELVd), which produces asymptomatic infections in
eggplants, has a branched and highly base-paired secondary
structure of minimum free energy (Fig. 1) and exhibits
elevated sequence variability in vivo (Fadda et al., 2003a).
ELVd belongs to the family Avsunviroidae (Flores et al.,
2000) which also contains Avocado sunblotch viroid, Peach
latent mosaic viroid (PLMVd) and Chrysanthemum chlorotic mottle viroid. This classification is based on chloroplastic replication, the presence of hammerhead
ribozymes and the lack of the central conserved region
Two supplementary figures are available with the online version of this
paper.
013425 G 2009 SGM
characteristic of nuclear viroids (family Pospiviroidae).
Viroids replicate through a rolling-circle mechanism with
RNA intermediates characterized by the reiterative transcription of a circular RNA template to produce an
oligomeric RNA of complementary polarity (Branch &
Robertson, 1984). In viroids, positive (+) polarity is
arbitrarily assigned to the most abundant strand in vivo
and negative (2) polarity to the complementary strand. The
viroids of the family Avsunviroidae follow a symmetrical
pathway within this general mechanism, in which both
oligomeric (+) and (2) RNAs are processed by cleavage to
monomeric linear (ml) RNAs and by subsequent ligation to
monomeric circular (mc) RNAs. Both mc (+) and (2) RNAs
serve as templates to synthesize oligomeric RNAs of
complementary polarity (Hutchins et al., 1985; Daròs et al.,
1994; Navarro et al., 1999; Bussière et al., 1999).
ELVd and the other viroids in its family are highly host
specific. They only infect the species in which they were
discovered or another closely related species (Flores et al.,
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F. Martı́nez and others
Fig. 1. Library of ELVd insertion and deletion mutants. (a) Scheme of a dimeric ELVd with arrows indicating the positions where
the GCGGCCGC octanucleotide was inserted (mutants I1–I6) and double-headed arrows marking the extent of the deletions
(mutants D7–D9). (b) Scheme of the cassettes for dimeric ELVd transcription in vitro and in vivo in C. reinhardtii chloroplast.
Arrowheads indicate hammerhead ribozyme self-cleavage sites. The T3 RNA polymerase promoter (grey arrow) allows ELVd
transcription of DNA linearized with XbaI in vitro. The rbcL promoter (grey arrow) and psaB terminator (grey rectangle) direct
ELVd transcription in vivo in the C. reinhardtii chloroplast. White and black rectangles represent ELVd and non-ELVd (cloning
vector) sequences, respectively. Numbering refers to ELVd sequence variant with GenBank accession no. AJ536613. Not to
scale. (c) ELVd minimum free energy structure (sequence variant AJ536613). The positions of octanucleotide insertions
(mutants I1 to I6) and the extent of deletions (mutants D7 to D9) are indicated with straight and bent arrows, respectively. Flags,
boxes and arrowheads indicate the domains, conserved nucleotides and self-cleavage sites of both hammerhead ribozymes.
The ribozyme of the (+) RNA (represented) and the (”) RNA (complementary to the represented) are indicated by black and
white symbols (boxes/arrowheads), respectively.
2000; Fadda et al., 2003a). This makes research on the
molecular biology of these pathogens particularity difficult;
this would otherwise be very attractive as they are pure RNA
pathogens (Daròs et al., 2006) and the only class of pathogen
in which it is well established that replication is within the
chloroplast (Flores et al., 2005), an organelle of major
biotechnological interest (Bock, 2007). Recently, a heterologous experimental system has been developed to study
certain aspects of the replication in viroids of the family
Avsunviroidae (Molina-Serrano et al., 2007). This system is
based on the use of transplastomic lines of the unicellular
green alga Chlamydomonas reinhardtii (phylum Chlorophyta)
(Harris, 2001) which expresses viroid transcripts in the
chloroplast. Although these viroids do not complete their
replication cycle in the algal chloroplast, the expressed viroid
RNAs are efficiently processed to their corresponding ml and
mc forms (Molina-Serrano et al., 2007).
In the present work, a library of ELVd insertion and deletion
mutants that affect different sequence and structural elements
along the molecule was constructed to identify elements in the
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ELVd molecule that are involved in transcript processing in
vivo. These mutants have been expressed in the C. reinhardtii
chloroplast and their processing in vivo has been analysed.
The results suggest that ELVd transcript cleavage depends
exclusively on the hammerhead ribozyme, but other elements
in the molecule take part in ELVd RNA circularization,
probably including a central, highly base-paired structure
with an internal loop that contains the ligation site.
METHODS
Construction of an ELVd mutant library. The plasmid pBrmELVd
contains the promoter, the 59 untranslated region (59 UTR) and the
beginning of the open reading frame (ORF) of C. reinhardtii rbcL gene
(from position 270 to +157) followed by a monomeric ELVd cDNA
(sequence variant AJ536613, 333 nt, from nt 211 to 210) in
pBluescript II KS+ (GenBank accession no. X52327). From this
plasmid, six mutants with insertion of the octanucleotide 59GCGGCCGC-39 inserted between ELVd positions 41–42, 71–72,
138–139, 176–177, 245–246 and 305–306 (mutants I1–I6, respectively,
Fig. 1) were constructed by PCR by using couples of adjacent and
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Journal of General Virology 90
ELVd RNA processing in C. reinhardtii chloroplast
divergent primers that contained the sequence CCGC at their 59 ends.
The amplification (in 50 ml) was carried out with 5 U Tth DNA
polymerase (Biotools), 0.2 mM dNTPs, 1 pmol 59-phosphomonoester (59-P) primers ml21 and 100 ng plasmid template for 2 min at
94 uC, 30 cycles of 40 s at 94 uC, 30 s at 55 uC and 4 min at 72 uC,
plus a final extension of 10 min at 72 uC. Amplification products
were separated by electrophoresis in a 1 % agarose gel in TAE buffer
(40 mM Tris/acetic acid, 20 mM sodium acetate, 1 mM EDTA,
pH 7.2) and eluted from the gel. Products were treated with 1 U T4
DNA polymerase (Fermentas) for 20 min at 11 uC in the presence of
0.1 mM dNTPs to generate blunt ends, and circularized with T4 DNA
ligase (Fermentas). After transformation of Escherichia coli DH5a,
selected clones with plasmids containing an additional NotI site in the
appropriate position were sequenced. The monomeric mutant ELVd
cDNAs were inserted at the appropriate position on a wild-type
pBrmELVd to generate the collection of pBrdELVd plasmids with
dimeric ELVd cDNAs in which only the viroid central unit contained
the mutations (Fig. 1a). Similarly, ELVd mutants with double
insertions were created (I2–I4, I4–I6 and I5–I6). From these, clones
with sequence deletions between ELVd positions 72–176, 177–305
and 246–305 of the viroid central unit were created by NotI
(Fermentas) digestion and religation with T4 DNA ligase (mutants
D7, D8 and D9, respectively, Fig. 1).
Transcription and self-cleavage in vitro of ELVd mutants. The
pBrdELVd plasmids with dimeric ELVd cDNAs (wild-type and
mutants) were linearized with XbaI (Fermentas) (Fig. 1b) and 250 ng
of each was transcribed in 20 ml with 20 U T3 RNA polymerase
(Roche), 0.5 mM NTPs and 20 U RNase inhibitor for 1.5 h at 37 uC.
The template DNA was digested with 4 U DNase I (Fermentas) for
15 min at 37 uC and the RNA was separated by denaturing PAGE in a
5 % gel containing 8 M urea and TBE buffer (89 mM Tris, 89 mM
boric acid, 2 mM EDTA) that was stained with ethidium bromide.
Transformation of C. reinhardtii chloroplast and analysis of
transcripts. Wild-type and mutant dimeric ELVd cDNAs were
inserted between the XbaI and EcoRI sites of the vector pCrc+157
(Suay et al., 2005) to transform C. reinhardtii chloroplast by
substituting the b-glucuronidase ORF, which is preceded by the
promoter, 59 UTR and beginning of the rbcL gene ORF (from –70 to
+157), and followed by the 39 UTR of the psaB gene, both from the
C. reinhardtii chloroplast (Fig. 1b). Plasmid (5 mg), precipitated on
0.6 mm gold microprojectiles, was used to transform the C. reinhardtii
non-photosynthetic strain CC-373 (ac-au-2-21) using the biolistic
PDS-1000/He system (Bio-Rad) (Boynton et al., 1988; Blowers et al.,
1989). Stable transplastomic clones were selected that recovered
photosynthetic ability by growing in HS agar under constant light
(Suay et al., 2005). Lines with a high degree of homoplasmicity were
selected by PCR analysis. Cells from C. reinhardtii colonies were
resuspended and lysed by incubation for 15 min at 65 uC in 100 mM
Tris/HCl, pH 8.0, 10 mM EDTA, 0.5 M NaCl, 10 mM 2-mercaptoethanol, 10 % SDS. Chloroplastic DNA was purified from the lysate
using silica gel columns (Wizard plus SV minipreps DNA purification
system, Promega). The ELVd cDNA was amplified from normalized
amounts of DNA (10 ng) using primers P3 (59-TTTAAAGTCCCGACC-39, complementary to ELVd nt 57–71) and P4 (59TTCGGAGGATTCGTC-39, homologous to ELVd nt 72–86)
(Supplementary Fig. S1, available in JGV Online) and Tth DNA
polymerase (see above). The products were separated by PAGE in a 5 %
gel in TAE buffer and stained with ethidium bromide; this was taken as
an approximate estimation of the homoplasmicity degree of each clone.
Selected transplastomic lines were grown for approximately 1 week to
exponential phase in liquid HS media with constant bubbling of 2 % (v/v)
CO2 in air at 32 uC with a 12 h (light), 12 h (dark) photoperiod. RNA
was purified from 50 ml saturated cultures recovered during the dark
period. Cells were lysed in the presence of SDS, total RNA was purified
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with a mix of buffer and phenol/chloroform, and the viroid RNA fraction
was enriched by non-ionic cellulose CF11 (Whatman) chromatography
(Molina-Serrano et al., 2007). Aliquots of the RNA preparations were
separated by double PAGE, first in a 5 % gel in TAE buffer that was
stained with ethidium bromide, next a segment of the gel between the
appropriate size markers was cut (300–400 nt for wild-type ELVd and
insertion mutants and 150–300 nt for deletion mutants) and applied on
top of a second denaturing gel (5 % polyacrylamide, 8 M urea, 0.256
TBE) to continue separation. RNA from the second gel was electroblotted
to a positively charged nylon membrane (Nytran SPC, Whatman), crosslinked with 0.12 J UV light cm22 and hybridized overnight at 70 uC
(50 % formamide, 0.1 % Ficoll, 0.1 % polyvinylpyrrolidone, 100 ng
salmon sperm DNA ml21, 1 % SDS, 0.75 M NaCl, 75 mM sodium
citrate, pH 7.0) with approximately 1 million c.p.m. ELVd (–) 32Plabelled RNA obtained by transcription in vitro in the presence of
[a-32P]UTP (400 Ci mmol21). The membrane was washed three times for
10 min with 26 SSC, 0.1 % SDS at room temperature and once for
15 min at 55 uC with 0.16 SSC (150 mM NaCl, 15 mM sodium citrate,
pH 7.0), 0.1 % SDS and visualized by autoradiography. Radioactive
signals were quantified with an image analyser (Fujifilm FLA-5100).
Eggplant inoculation with ELVd mutants and progeny analysis.
Wild-type and mutant ELVd ml RNAs (except for mutant I1)
produced by transcription and self-cleavage in vitro, were eluted from
a denaturing gel and used to inoculate eggplant (Solanum melongena
L. cv. Black beauty). For each sample, three plants were inoculated in
one leaf with 2 pmol RNA in 10 ml 5 % Carborundum in 50 mM
K2HPO4. Inoculated plants were maintained for 1 month at 25 uC
with a photoperiod of 16 h (light) and 8 h (dark). Total RNA was
purified from systemic leaves of these plants with a mix of buffer and
phenol, and enriched in viroid RNAs by non-ionic cellulose CF11
chromatography (Daròs & Flores, 2004). Aliquots of the RNA
preparations were separated by denaturing PAGE in a 5 % gel
containing 8 M urea and TBE buffer. The RNAs were blotted to a
nylon membrane and hybridized as described above.
For the infected plants, the viroid progeny was amplified by RT-PCR
with two different primer pairs: P1 (59-GTGGCACACACCACCCTATGG-39, complementary to ELVd nt 329–16) and P2 (59-CCCTGATGAGACCGAAAGGTC-39, homologous to ELVd sequence 17–37) or
P3 and P4 (Supplementary Fig. S1). The RNA was reverse transcribed
with 50 U M-MuLV RT (Revertaid, Fermentas), 10 U RNase inhibitor,
0.1 mM dNTPs and 5 pmol primer P1 or P3 (in 10 ml) for 45 min at
42 uC, 10 min at 50 uC and 5 min at 60 uC. An aliquot (2 ml) of this
reaction was used for PCR amplification in 20 ml with 0.4 U Phusion
DNA polymerase (Finnzymes), 0.2 mM dNTPs, 0.5 pmol ml21 59-P
primers P1 and P2 or P3 and P4 for 30 s at 98 uC, 30 cycles of 10 s at
98 uC, 30 s at 55 uC and 30 s at 72 uC, and a final extension of 10 min
at 72 uC. Amplification products were separated by electrophoresis in a
2 % agarose gel in TAE buffer, stained with ethidium bromide and
eluted. Next, they were ligated to plasmid pUC18 (GenBank accession
no. L08752), linearized with SmaI (Fermentas) and transformed into E.
coli. For each sample (wild-type and mutant I5) 10 clones were
sequenced, using each pair of primers to amplify five clones.
Sequence analyses. ELVd RNA sequences were aligned using the
CLUSTAL W program and minimum free energy conformations were
calculated using Mfold (Zuker, 2003).
RESULTS
Construction of a library of ELVd insertion and
deletion mutants
To identify sequence and structure requirements in the
processing (cleavage and ligation) of oligomeric ELVd
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F. Martı́nez and others
RNA in vivo in a chloroplastic context, a library of
insertion and deletion mutants of this viroid was
constructed (Fig. 1a). In the plasmid pBrdELVd, which
contains a dimeric ELVd cDNA (nt 211–210) under the
control of the T3 RNA polymerase promoter (Fig. 1b), the
octanucleotide GCGGCCGC was introduced, by PCR sitedirected mutagenesis, to six positions in the central viroid
unit, which is flanked by two ribozyme self-cleavage sites
(Fig. 1a, mutants I1–I6). In the ELVd (+) RNA molecule,
these mutations affect a conserved box of the hammerhead
ribozyme, an internal loop in the left terminal hairpin, an
internal loop in the left upper hairpin, a double-stranded
region in the central part of the viroid, the terminal loop of
the right upper hairpin and a double-stranded region
within the right terminal hairpin (Fig. 1c, mutants I1–I6,
respectively). The sequence of the inserted octanucleotide
matches the recognition site of NotI and was chosen to
facilitate mutant preparation. Moreover, three deletion
mutants were also constructed in which the sequences
between ELVd positions 72–176 (105 nt), 246–305 (60 nt)
and 177–305 (129 nt) were deleted and substituted with the
GCGGCCGC octanucleotide (Fig. 1a, mutants D7–D9).
The deletions correspond to, approximately, the left third
of the viroid molecule, including the two left hairpins; the
right fifth of the molecule, including half of the right upper
hairpin and the whole terminal right hairpin; and the right
third of the molecule, including both right hairpins (Fig. 1c,
mutants D7–D9, respectively).
As a control experiment, the in vitro self-cleavage ability
of the collection of ELVd mutant transcripts was analysed
(Supplementary Fig. S2, available in JGV Online).
Dimeric ELVd (+) transcript self-cleaves efficiently in
vitro through the hammerhead ribozyme (Fadda et al.,
2003a) to produce a set of five RNAs which are products
of partial and double self-cleavage, and include the 333 nt
ml ELVd (+) RNA (Supplementary Fig. S2a). Equal
amounts of the plasmids were transcribed in vitro with T3
RNA polymerase; the products of transcription and
subsequent in vitro self-cleavage were analysed by
denaturing PAGE. In all cases, the five expected selfcleavage products, as well as the uncleaved primary
transcript, were detected (Supplementary Fig. S2b). As
expected, mutant I1, which contains the insertion
between conserved nucleotides of the first hammerhead
ribozyme, showed defective processing in the first selfcleavage site. The sizes of the ml RNA products of double
self-cleavage for the different mutants were consistent
with those expected according to the introduced mutations: 341 nt for insertion mutants I1–I6, and 236, 281
and 212 nt for deletion mutants D7, D8 and D9,
respectively (Supplementary Fig. S2b). It was noticeable
that the ml ELVd 212 nt deletion mutant migrated faster
than the 200 nt RNA employed as a marker in the present
work (Supplementary Fig. S2b, compare lanes 1 and 11).
This is possibly due to the highly compact structure that
this mutant can adopt (see below) that might partly resist
denaturation in the urea gel.
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Processing of mutant ELVd transcripts in C.
reinhardtii chloroplast.
The whole set of ELVd mutants was transferred to a plasmid
vector that allows transformation of the C. reinhardtii
chloroplast genome by homologous recombination generating an expression cassette under the control of the rbcL
promoter (Rubisco large subunit) and the psaB terminator
(photosystem I subunit B), both from C. reinhardtii (Fig.
1b). Transcription in vivo in the chloroplasts produces a
dimeric transcript preceded by a fragment of rbcL mRNA
and followed by sequences of the psaB terminator (MolinaSerrano et al., 2007). After transforming chloroplasts, C.
reinhardtii clones in which the photosynthetic ability is
restored were selected and two or three lines with a high
degree of homoplasmicity were chosen for each mutant by
detection of viroid cDNA by PCR. Subsequent analysis of
independent clones corresponding to the same mutant class
showed complete reproducibility with respect to viroid
RNA processing. Consequently, the clone expressing more
viroid RNA transcript for each mutant class was chosen for
comparative analysis. Viroid-enriched RNA was purified by
cellulose CF11 chromatography from liquid cultures of the
selected clones. ELVd RNAs were analysed by Northern blot
hybridization with an ELVd (2) RNA probe after separation
by double PAGE, first in a native gel and next in a
denaturing one, which allows a better resolution of circular
forms than single denaturing PAGE. Hybridization signals
corresponding to mc and ml RNA forms were detected for
wild-type ELVd and most of the mutants, according to their
positions and comparison with controls and RNA markers
(Fig. 2a). An exception was the negative control mutant I1,
with the insertion in a conserved box of the hammerhead
ribozyme, in which no in vivo processing product was
detected (Fig. 2a, lane 4). A similar analysis, in which the
RNA was separated by single denaturing PAGE, proved the
correct expression of this primary mutant transcript in the
selected transplastomic C. reinhardtii line (data not shown).
For the remaining octanucleotide insertion mutants (I2–
I6), both ml and mc RNA forms were detected in a ratio
similar to the wild-type control (Fig. 2a, compare lane 3
with lanes 5–9). The mc RNA was always more abundant
than the ml RNA, similar to the situation in the natural host
eggplant (Fig. 2a, lane 1). A tentative quantification of the
ligation ratio [mc/(mc+ml)] based on these hybridization
signals confirmed this observation (Fig. 2b). In the deletion
mutant D8, the corresponding 281 nt ml and mc RNAs were
also detected (Fig. 2a, lane 11); again, the ratio of mc : ml
was similar to wild-type (Fig. 2b). In the deletion mutant
D7, both 236 nt ml and mc RNA forms were detected (Fig.
2a, lane 10), but in this case, the band corresponding to mc
RNA was not significantly more intense than that of ml
RNA (Fig. 2a, lane 10 and Fig. 2b). Finally, in the deletion
mutant D9, only one band corresponding to the 212 nt ml
RNA was detected (Fig. 2a, lane 12). These results indicate
that none of the mutations (insertion or deletion) studied
in this work, apart from that which directly disturbs the
ribozyme, affect ELVd transcript cleavage in vivo in a
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Journal of General Virology 90
ELVd RNA processing in C. reinhardtii chloroplast
Fig. 2. Processing of ELVd insertion and deletion mutants in C.
reinhardtii chloroplasts in vivo. (a) Northern blot hybridization of
RNA from C. reinhardtii transplastomic clones separated by
double PAGE (native and denaturing) with an ELVd (”) RNA
probe. Lanes: 1, infected eggplant control; 2, non-transformed C.
reinhardtii control; 3, C. reinhardtii expressing a wild-type ELVd
transcript in the chloroplast; 4–12, C. reinhardtii expressing ELVd
transcripts with insertions I1–I6 and deletions D7–D9, respectively, in the chloroplast. Linear (left) and circular (right) RNA
markers are indicated. (b) Plot of the ligation ratio of the
processing products of mutant ELVd transcripts in C. reinhardtii
chloroplast in vivo. The ligation ratio was estimated from the
radioactive signals of the different samples from Northern blot
hybridization (a) and calculated as mc/(mc+ml).
chloroplastic context. The insertions, as well as deletion D8,
also have no effect on ELVd circularization in vivo.
However, deletion D7 causes a ligation defect and deletion
D9 abolishes it completely.
Infectivity of ELVd mutants in eggplant
The infectivity of the ELVd mutant collection was also
assayed in the natural host eggplant. For this purpose, ml
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ELVd RNAs from the wild-type and the different mutants
produced by self-cleavage in vitro were eluted from the gel
after separation by denaturing PAGE. Mutant I1, which
produces minor amounts of its ml RNA in vitro, was
excluded from this analysis. Three plants were inoculated
with equal molar amounts of the different RNAs and 1
month later, RNA was purified from systemic leaves.
Viroid infection was tested by Northern blot hybridization
following separation, by denaturing PAGE, of the RNA that
had been pooled from each group of three plants. Only
wild-type ELVd and mutant I5 (with an insertion in the
terminal loop of the right upper hairpin) were able to
infect eggplant (Fig. 3a, lanes 3 and 7). When analysed
separately, the three plants belonging to each group in both
cases were all infected. The progenies from wild-type and
mutant I5 ELVd were analysed 1 month post-infection.
Viroid cDNAs were amplified by RT-PCR from the RNA
preparations with two different couples of adjacent
primers: P1 and P2, and P3 and P4. The amplified
cDNAs were cloned and 10 different clones from wild-type
ELVd and 10 from mutant I5 were sequenced (five
obtained with each pair of primers). Five of the 10 clones
corresponding to mutant I5 progeny conserved an
octanucleotide in the insertion site between positions 245
and 246. However, in all cases, some mutation of the
original GCGGCCGC octanucleotide had been produced
(Fig. 3b). In the other five clones, the octanucleotide had
been simplified to a GC dinucleotide (three clones), a G
mononucleotide (one clone) or completely deleted (one
clone) (Fig. 3b). All 10 clones exhibited other point
mutations along the molecules, similar to the clones
corresponding to wild-type ELVd progeny, and consistent
with the fact that this viroid accumulates high sequence
variability during infection (Supplementary Fig. S1). These
results indicate that, apart from insertion I5, all mutations
introduced into ELVd impede the biological viability of the
viroid.
DISCUSSION
In this work, the C. reinhardtii-based experimental system
was employed in combination with expression of a library
of viroid insertion and deletion mutants to study possible
sequence and structural requirements in the processing
(cleavage and ligation) of ELVd (+) transcripts in vivo in a
chloroplastic context. This experimental approach allows
structure–function studies in the biochemical and physiological context of the subcellular compartment where the
processing of this viroid takes place, which would not
otherwise be possible since almost all assayed mutations are
lethal (Fig. 3). Moreover, this study benefits from the lack
of viroid replication in C. reinhardtii chloroplast, which
allows a more direct interpretation of the effect of
mutations in transcript processing (Molina-Serrano et al.,
2007). In the ELVd molecule, the six insertions (Fig. 1c)
distort the hammerhead ribozyme (I1), the left terminal
hairpin (I2), the left upper hairpin (I3), the central double-
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F. Martı́nez and others
part of the molecule, splitting the right upper hairpin and
completely deleting the right terminal hairpin (D8), and
the right part of the molecule, including the whole of both
terminal hairpins (D9).
Cleavage of ELVd transcripts during rolling-circle
replication
Fig. 3. Infectivity of ELVd insertion and deletion mutants. (a) RNAs
purified from eggplants inoculated with wild-type and mutant ELVd
monomeric linear RNAs were analysed by Northern blot hybridization with a complementary RNA probe after separation by
denaturing PAGE. Lanes: 1 and 2, ELVd-infected and mockinoculated eggplant controls; 3, eggplant inoculated with wild-type
ELVd; 4–11, eggplants inoculated with ELVd insertion mutants I2–
I6 and deletion mutants D7–D9, respectively. RNA markers are
indicated on the left. ELVd monomeric circular (mc) and
monomeric linear (ml ) RNAs are indicated on the right. (b)
Evolution of the insertion in mutant I5 1 month after infection of
eggplant. Sequence fragment of ELVd mutant I5 showing the
GCGGCCGC insertion (grey box) between positions 245 and
246 and the sequence observed in 10 different clones of the
progeny. Mutations in the progeny the same as the original
octanucleotide sequence are indicated in white on a black
background. When the same sequence was found in the progeny
more than once, this is indicated (grey text).
stranded region (I4), the right upper hairpin (I5) and the
right terminal hairpin (I6). These insertions affect doublestranded stretches (I1, I4 and I6) as well as loops (I2, I3 and
I5). The three deletions (Fig. 1c) encompass the left part of
the molecule, including both left hairpins (D7), the right
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ELVd strands of both polarities self-cleave efficiently in
vitro through the hammerhead ribozymes (Fadda et al.,
2003a), as occurs with the rest of viroids of the family
Avsunviroidae (Flores et al., 2000). Evidence exists that
hammerhead ribozymes are also functional in vivo; this has
been obtained mainly via the characterization of linear
RNAs purified from infected plants that are naturally
opened at the ribozyme cleavage site (Daròs et al., 1994;
Navarro & Flores, 1997) and the analysis of numerous
natural sequence variants that tend to maintain the
structure and conserved residues of the ribozymes
(Rakowski & Symons, 1989; Navarro & Flores, 1997;
Ambrós et al., 1998; Fadda et al., 2003a). However, the
finding that an RNA-binding protein from avocado
chloroplast binds Avocado sunblotch viroid RNA in vivo
and facilitates the ribozyme self-cleavage of this viroid
transcript in vitro suggests that the activity of hammerhead
ribozymes during viroid replication might be helped by
host proteins (Daròs & Flores, 2002). All mutants assayed
in this work, apart from the negative control which directly
affects the hammerhead ribozyme (I1), cleaved as efficiently as wild-type RNA in vivo (Fig. 2a). This suggests
that the function of transcript cleavage in vivo resides
exclusively in the hammerhead ribozyme domain and that
there is no other element in the molecule that participates
directly or indirectly (for example by recruiting host
auxiliary factors) in the process. Nevertheless, participation
of some domain not affected by the mutations produced in
this study cannot be disregarded.
Ligation of cleavage products of ELVd transcripts
during rolling-circle replication
Mature ELVd accumulates in the tissues of infected
eggplant mainly as circular forms (Fadda et al., 2003a)
(Fig. 2a, lane 1); nonetheless, viroid circularization
continues to be the least-known step of the viroid
replication cycle (Flores et al., 2005). This reaction might
be mediated by a host factor such as an RNA ligase or it
may be an autocatalytic process inherent to the viroid
RNA. The reaction could also be RNA-based, but assisted
by a host factor, such as an RNA chaperone, helicase or
annealer. Alternatively, the reaction may only occur
efficiently in a particular chloroplastic biochemical environment that is difficult to replicate in vitro.
Plant tRNA ligase recognizes the 59-hydroxyl (59-OH) and
29,39-cyclic phosphodiester (29,39.P) termini generated by
hammerhead ribozymes and efficiently circularizes the ml
viroid RNA by producing mc RNA (Kikuchi et al., 1982;
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Journal of General Virology 90
ELVd RNA processing in C. reinhardtii chloroplast
Branch et al., 1982). Moreover, this enzyme has been
proposed to localize in the nucleus, cytoplasm and
chloroplast of plant cells (Englert et al., 2007). On the
other hand, the PLMVd ml RNA product of ribozyme selfcleavage, which contains 59-OH and 29,39.P termini, selfligates in vitro generating a 29,59-phosphodiester bond
(Côté & Perreault, 1997). But, although the ml RNAs
resulting from ribozyme self-cleavage in most of the other
viroids of the family Avsunviroidae also self-ligate in vitro
and some PLMVd mc forms closed by a 29,59-phosphodiester bond have been detected in infected tissue (Côte et
al., 2001), this reaction is inefficient and does not seem to
be responsible for the production of most of the viroid mc
RNAs that accumulate in vivo which are closed mainly by a
39,59-phosphodiester bond (D. Molina-Serrano, R. Flores
and J.-A. Daròs, unpublished results). It has also been
reported that the hammerhead ribozyme can work in
reverse by catalysing the ligation of an RNA with 59-OH
and 29,39.P termini producing a 39,59-phosphodiester
bond (Hertel et al., 1994; Nelson et al., 2005; Canny et al.,
2007) in a manner similar to the hairpin ribozyme of plant
virus RNA satellites (Fedor, 2000). However, the ELVd
ligation product observed in vivo in the host eggplant (Fig.
2a, lane 1) or in C. reinhardtii chloroplast (Fig. 2a, lane 3)
is much larger than that produced in vitro, which is
negligible (Supplementary Fig. S2). An estimation of the
ligation ratio using the hybridization signals from the
present work (Fig. 2a) indicates a ratio of about 0.8 in
infected eggplant and about 0.9 in C. reinhardtii chloroplast (Fig. 2b), suggesting that a very efficient mechanism
operates in vivo, most probably mediated by a chloroplastic
RNA ligase or, alternatively, by some host auxiliary factor
or some chloroplast biochemical context that potentiates
ELVd RNA self-ligation.
Moreover, the effect of sequence deletions on ELVd ml
RNA ligation in the C. reinhardtii chloroplast observed
here involves other sequences, in addition to the hammerhead ribozyme domain, in the ligation function. This is
shown by deletion mutant D7, which affects the left part of
the molecule, that had a ligation defect (Fig. 2a, lane 10 and
Fig. 2b) and deletion mutant D9, which affects the right
part of the molecule, that did not ligate at all (Fig. 2a, lane
12). The ligation site in the ELVd (+) minimum free energy
conformation is located in an internal loop situated in a
quasi double-stranded structure in the centre of the
molecule (Fig. 1c and Fig. 4). This structure results from
base pairing of the sequences corresponding to both (+)
and (2) hammerhead ribozyme domains. The degree of
distortion of this central structure produced in the
different deletion mutants correlates with the ligation
defect observed in vivo (Fig. 2a, lanes 10–12, Figs 2b and 4).
While deletion D8, in which the stability of this structure is
not affected (Fig. 4), did not influence ligation (Fig. 2a,
lane 11), ligation efficiency was diminished in deletion
mutant D7 (Fig. 2a, lane 10 and Fig. 2b) in which this
central structure is partially affected, without directly
affecting the sequence of the loop, the ligation site or the
http://vir.sgmjournals.org
Fig. 4. Schematic representation of the minimum free energy
conformation of wild-type ELVd (sequence variant AJ536613) and
the deletion mutants D7, D8 and D9. The position of the viroid
processing site is indicated with an arrowhead. The parts of the
molecules that are distorted as a consequence of the deletions are
boxed in grey.
adjacent sequences (Fig. 4). Finally, in deletion mutant D9,
in which the upper strand of the quasi double-stranded
structure is affected, including the upper part of the loop
with the ligation site (Fig. 4), there is no ligation in vivo
(Fig. 2a, lane 12). In short, these results suggest that, in
addition to the hammerhead ribozyme domain, other
ELVd sequences or structural elements are essential for
ligation in vivo. It is possible that the correct folding of
ELVd ml molecule in a ligation conformation is necessary,
including a quasi double-stranded structure in the central
part of the molecule that contains the ligation site in a
loop. Furthermore, this implies that the motifs in the
viroid molecule involved in cleavage and ligation are
different.
Biological viability of an ELVd mutant with an
inserted octanucleotide in the terminal loop of the
right upper hairpin
In the tissues of viroid-infected plants, numerous sequence
variants with point mutations and small deletions or
insertions have been reported, in the case of both
chloroplast viroids of the family Avsunviroidae and nuclear
viroids of the family Pospiviroidae (Flores et al., 2005).
Despite this, viroids are very compact molecules and
biologically viable mutants with large insertion or deletions
have not been described frequently. Exceptions are the
sequence variants within block duplications of parts of the
rod-like structure in coconut cadang-cadang viroid or
citrus exocortis viroid (Haseloff et al., 1982; Semancik et al.,
1994; Fadda et al., 2003b) or the PLMVd sequence variants
with 12–13 nt insertions, some of which induce the peach
calico symptom (Malfitano et al., 2003; Rodio et al., 2006,
2007). It is notable that ELVd insertion mutant I5, with an
insertion in the terminal loop of the right upper hairpin, is
infectious (Fig. 3a, lane 7) and that this insertion is partly
conserved during a 1 month infection process (Fig. 3b).
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3063
F. Martı́nez and others
This insertion resembles, according to size and position,
those described in PLMVd variants which also fold into
hairpins and are located in the loop capping a long viroid
arm (Malfitano et al., 2003). The observation that ELVd
tolerates an 8 nt insertion in this particular position of the
molecule must contribute to understanding of the
structural requirements of this viroid during its infectious
cycle. Moreover, this position will be suitable for the
introduction of sequence tags in future experiments to
study the molecular biology of this viroid and, in
particular, the host factors interacting during the infectious
cycle.
Côte, F., Lévesque, D. & Perreault, J. P. (2001). Natural 29,59phosphodiester bonds found at the ligation sites of peach latent
mosaic viroid. J Virol 75, 19–25.
Daròs, J. A. & Flores, R. (2002). A chloroplast protein binds a viroid
RNA in vivo and facilitates its hammerhead-mediated self-cleavage.
EMBO J 21, 749–759.
Daròs, J. A. & Flores, R. (2004). Arabidopsis thaliana has the
enzymatic machinery for replicating representative viroid species of
the family Pospiviroidae. Proc Natl Acad Sci U S A 101, 6792–6797.
Daròs, J. A., Marcos, J. F., Hernández, C. & Flores, R. (1994).
Replication of avocado sunblotch viroid: evidence for a symmetric
pathway with two rolling circles and hammerhead ribozyme
processing. Proc Natl Acad Sci U S A 91, 12813–12817.
Daròs, J. A., Elena, S. F. & Flores, R. (2006). Viroids: an Ariadne’s
thread into the RNA labyrinth. EMBO Rep 7, 593–598.
ACKNOWLEDGEMENTS
This work was supported by grants AGL2004-06311-C02-01 (to
J.-A. D.) and BFU2006-07783 (to M. L. S.) from Ministerio de
Educación y Ciencia (MEC) from Spain, and grant BIO2008-01986
(to J.-A. D.) from Ministerio de Ciencia e Innovación from Spain.
F. M. and J. M. were recipients of masters and predoctoral fellowships,
respectively, from MEC. F. M. was a recipient of a predoctoral
fellowship from Universidad Politécnica de Valencia from Spain. We
thank Drs Ricardo Flores, Gustavo Gómez and Douglas Grubb for
critical reading of the manuscript.
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Phytopathol 47, 105–131.
Englert, M., Latz, A., Becker, D., Gimple, O., Beier, H. & Akama, K.
(2007). Plant pre-tRNA splicing enzymes are targeted to multiple
cellular compartments. Biochimie 89, 1351–1365.
Fadda, Z., Daròs, J. A., Fagoaga, C., Flores, R. & Duran-Vila, N.
(2003a). Eggplant latent viroid, the candidate type species for a new
genus within the family Avsunviroidae (hammerhead viroids). J Virol
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Fadda, Z., Daròs, J. A., Flores, R. & Duran-Vila, N. (2003b).
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