Involvement of the chloroplastic isoform of tRNA ligase in the replication of viroids belonging to the family Avsunviroidae.
ABSTRACT Avocado sunblotch viroid, peach latent mosaic viroid, chrysanthemum chlorotic mottle viroid, and eggplant latent viroid (ELVd), the four recognized members of the family Avsunviroidae, replicate through the symmetric pathway of an RNA-to-RNA rolling-circle mechanism in chloroplasts of infected cells. Viroid oligomeric transcripts of both polarities contain embedded hammerhead ribozymes that, during replication, mediate their self-cleavage to monomeric-length RNAs with 5'-hydroxyl and 2',3'-phosphodiester termini that are subsequently circularized. We report that a recombinant version of the chloroplastic isoform of the tRNA ligase from eggplant (Solanum melongena L.) efficiently catalyzes in vitro circularization of the plus [(+)] and minus [(-)] monomeric linear replication intermediates from the four Avsunviroidae. We also show that while this RNA ligase specifically recognizes the genuine monomeric linear (+) ELVd replication intermediate, it does not do so with five other monomeric linear (+) ELVd RNAs with their ends mapping at different sites along the molecule, despite containing the same 5'-hydroxyl and 2',3'-phosphodiester terminal groups. Moreover, experiments involving transient expression of a dimeric (+) ELVd transcript in Nicotiana benthamiana Domin plants preinoculated with a tobacco rattle virus-derived vector to induce silencing of the plant endogenous tRNA ligase show a significant reduction of ELVd circularization. In contrast, circularization of a viroid replicating in the nucleus occurring through a different pathway is unaffected. Together, these results support the conclusion that the chloroplastic isoform of the plant tRNA ligase is the host enzyme mediating circularization of both (+) and (-) monomeric linear intermediates during replication of the viroids belonging to the family Avsunviroidae.
- SourceAvailable from: PubMed Central[show abstract] [hide abstract]
ABSTRACT: Viroids are plant-pathogenic non-coding RNAs able to interfere with as yet poorly known host-regulatory pathways and to cause alterations recognized as diseases. The way in which these RNAs coerce the host to express symptoms remains to be totally deciphered. In recent years, diverse studies have proposed a close interplay between viroid-induced pathogenesis and RNA silencing, supporting the belief that viroid-derived small RNAs mediate the post-transcriptional cleavage of endogenous mRNAs by acting as elicitors of symptoms expression. Although the evidence supporting the role of viroid-derived small RNAs in pathogenesis is robust, the possibility that this phenomenon can be a more complex process, also involving viroid-induced alterations in plant gene expression at transcriptional levels, has been considered. Here we show that plants infected with the 'Hop stunt viroid' accumulate high levels of sRNAs derived from ribosomal transcripts. This effect was correlated with an increase in the transcription of ribosomal RNA (rRNA) precursors during infection. We observed that the transcriptional reactivation of rRNA genes correlates with a modification of DNA methylation in their promoter region and revealed that some rRNA genes are demethylated and transcriptionally reactivated during infection. This study reports a previously unknown mechanism associated with viroid (or any other pathogenic RNA) infection in plants providing new insights into aspects of host alterations induced by the viroid infectious cycle.Nucleic Acids Research 10/2013; · 8.28 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Viroids are a unique class of noncoding RNAs: composed of only a circular, single-stranded molecule of 246-401 nt, they manage to replicate, move, circumvent host defenses, and frequently induce disease in higher plants. Viroids replicate through an RNA-to-RNA rolling-circle mechanism consisting of transcription of oligomeric viroid RNA intermediates, cleavage to unit-length strands, and circularization. Though the host RNA polymerase II (redirected to accept RNA templates) mediates RNA synthesis and a type-III RNase presumably cleavage of Potato spindle tuber viroid (PSTVd) and closely related members of the family Pospiviroidae, the host enzyme catalyzing the final circularization step, has remained elusive. In this study we propose that PSTVd subverts host DNA ligase 1, converting it to an RNA ligase, for the final step. To support this hypothesis, we show that the tomato (Solanum lycopersicum L.) DNA ligase 1 specifically and efficiently catalyzes circularization of the genuine PSTVd monomeric linear replication intermediate opened at position G95-G96 and containing 5'-phosphomonoester and 3'-hydroxyl terminal groups. Moreover, we also show a decreased PSTVd accumulation and a reduced ratio of monomeric circular to total monomeric PSTVd forms in Nicotiana benthamiana Domin plants in which the endogenous DNA ligase 1 was silenced. Thus, in a remarkable example of parasitic strategy, viroids reprogram for their replication the template and substrate specificity of a DNA-dependent RNA polymerase and a DNA ligase to act as RNA-dependent RNA polymerase and RNA ligase, respectively.Proceedings of the National Academy of Sciences 08/2012; 109(34):13805-10. · 9.74 Impact Factor
- New Phytologist 02/2013; · 6.74 Impact Factor
Involvement of the Chloroplastic Isoform of tRNA Ligase in the
Replication of Viroids Belonging to the Family Avsunviroidae
María-Ángeles Nohales, Diego Molina-Serrano, Ricardo Flores, and José-Antonio Daròs
Instituto de Biología Molecular y Celular de Plantas (Consejo Superior de Investigaciones Científicas—Universidad Politécnica de Valencia), Valencia, Spain
form of the tRNA ligase from eggplant (Solanum melongena L.) efficiently catalyzes in vitro circularization of the plus [(?)] and
transcript in Nicotiana benthamiana Domin plants preinoculated with a tobacco rattle virus-derived vector to induce silencing
within the family Pospiviroidae and characteristically contain a
central conserved region (CCR) in the middle of their molecules,
which are predicted to fold in rod- or quasi-rod-like minimum
free-energy conformations, and replicate in the nuclei of infected
cells. However, four viroid species—Avocado sunblotch viroid
(ASBVd), Peach latent mosaic viroid (PLMVd), Chrysanthemum
chlorotic mottle viroid (CChMVd), and Eggplant latent viroid
(ELVd)—that do not contain a CCR in their molecules are
contain hammerhead ribozymes embedded in both polarities of
their RNA strands that catalyze self-cleavage of the oligomeric
viroid RNA intermediates resulting from replication that occurs
follows the symmetric variant of an RNA-based rolling-circle
mechanism (4, 9, 25). In this variant, the circular viroid strand of
plus [(?)] polarity—which is arbitrarily assigned to the viroid
RNA strand most abundant in the infected tissue—is reiteratively
of complementary or minus [(?)] polarity. These RNAs are self-
are circularized. Then, in a second and symmetrical part of the
cycle, the monomeric (?) circular RNA is transcribed to oligom-
ers that are self-cleaved and the resulting linear monomers subse-
quently circularized to finally produce the monomeric (?) circu-
The effect of the inhibitor tagetitoxin on RNA synthesis in
chloroplastic preparations of ASBVd-infected avocado (Persea
in the infected tissue (37). This notion is sustained by the intense
coding RNA (12, 18, 19, 45). Most viroid species are gathered
PLMVd replication in peach [Prunus persica (L.) Batsch] leaves
expressing a PLMVd-incited albinism in which PEP-dependent
transcription is basically absent (41). Consistent with this view,
ASBVd double-stranded RNAs, regarded as the replication inter-
Concerning the second replication step, characterization of the
termini of linear ASBVd and CChMVd strands accumulating in
infected tissues supports the hypothesis that hammerhead ri-
bozymes mediate the self-cleavage in vivo of the oligomeric RNAs
hammerhead structures are frequently found in natural sequence
evidence regarding the host factor mediating the subsequent cir-
cularization of monomeric linear viroid RNA intermediates.
The RNA circularizing activity during the replication of the
Avsunviroidae could also reside in the hammerhead ribozyme,
which can catalyze not only RNA cleavage but also ligation (24,
38), especially when their tertiary stabilizing motifs (10, 27) are
preserved (5). However, the low efficiency of the ligation reaction
monomeric viroid RNA accumulating in ASBVd-infected avo-
cado and ELVd-infected eggplant (Solanum melongena L.) tissues
(9, 15). Also, our previous study about ELVd processing in trans-
Received 12 March 2012 Accepted 15 May 2012
Published ahead of print 23 May 2012
Address correspondence to José-Antonio Daròs, email@example.com.
Supplemental material for this article may be found at http://jvi.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
August 2012 Volume 86 Number 15Journal of Virologyp. 8269–8276jvi.asm.org
plastomic lines of the green alga Chlamydomonas reinhardtii P.A.
Dangeard expressing different ELVd mutant forms indicates that
the viroid sequence requirements for cleavage and ligation are
different. More specifically, a quasi-double-stranded structure in
the central part of the ELVd molecule (folded in the predicted
minimum free-energy conformation) containing the ligation site
in an internal loop motif seems involved in ELVd circularization
in a chloroplastic context (33). This result suggested that an RNA
ligase activity of chloroplastic localization, able to recognize the
viroid ligation conformation, likely mediates ELVd circulariza-
tion (33). A candidate for providing this activity is tRNA ligase, a
conserved enzyme involved in maturation of nuclear tRNAs (1,
13, 31, 46) that in plants is targeted to chloroplast, in addition to
duced by the hammerhead ribozymes (26, 40).
In this study, we have examined the ability of the chloroplastic
isoform of the plant tRNA ligase to mediate circularization of
viroid RNA during the replication of members of the family Av-
purified a recombinant version of this protein, and analyzed its
ability to circularize in vitro different monomeric linear ELVd
RNAs, as well as the monomeric linear replication intermediates
of the other Avsunviroidae. The effects of silencing the endoge-
nous tRNA ligase in planta provide additional evidence support-
MATERIALS AND METHODS
RT and PCR amplification of tRNA ligase cDNAs. Eggplant (cv. Black
Beauty) leaf tissue was homogenized with 5 volumes of buffer consisting
toethanol, and 5 M urea. The clarified extract was fractionated with buff-
ered (pH 8.0) phenol-chloroform (1:1), and RNAs in the aqueous phase
were precipitated with isopropanol. RNAs were further purified by chro-
scription (RT) using 5 pmol of complementary primers in 10-?l reaction
mixtures containing 50 mM Tris-HCl (pH 8.3), 50 mM KCl, 4 mM
MgCl2, 10 mM dithiothreitol (DTT), 0.5 mM each deoxynucleoside
leukemia virus (M-MuLV) reverse transcriptase (Fermentas) for 45 min
at 42°C, followed by 10 min at 50°C and 5 min at 60°C.
Aliquots of 1 ?l of the RT reaction products were subjected to PCR
amplification in 20 ?l with 0.4 U of the high-fidelity Phusion DNA poly-
merase (Finnzymes) in the presence of HF buffer (Finnzymes), 3% di-
the expected product (15 s per kbp), with a final incubation of 10 min at
72°C. In most cases, 1 ?l of the PCR products was subjected to a second
nested PCR under the same conditions but with a new pair of primers.
poly(dT) primer I in the RT reaction and primers II and III in the PCR.
Primers II and III were designed according to nucleotide sequences con-
served in tRNA ligases from other higher plant species. Primer sequences
and some description of their use are in Table S1 and Fig. S1 in the sup-
plemental material. After cloning and sequencing of the first eggplant
sequent amplifications. A cDNA corresponding to the 3= end of the egg-
primers II and IV in the first PCR, and primers V and VI in the second
was amplified by following a version of a protocol for rapid amplification
primer VII phosphorylated at the 5= end. The reaction product was then
treated with RNase H (Fermentas) and circularized with T4 RNA ligase
(Fermentas). The resulting circular cDNA was used as a template for two
X and XI in the second). A further 5= cDNA fragment was obtained
and XII and XIV in the second, heminested reaction. Primer XII was
designed from conserved sequences of plant tRNA ligases. A final cDNA
using 5=-phosphorylated primer XV in the RT reaction, primers XVI and
XVII in the first PCR, and primers XVIII and XIX in the second nested
amplification. All the cDNAs were cloned and sequenced.
Expression and purification of a recombinant version of eggplant
tRNA ligase. A cDNA corresponding to the full open reading frame of
eggplant tRNA ligase was finally amplified from the eggplant RNA prep-
aration using primer XX in the RT reaction, primers XXI and XXII in the
first PCR, and primers XXIII and XXIV in the second nested PCR. The
cDNA product was inserted between the NcoI and XhoI sites of plasmid
pET-23d(?) (Novagen). The resulting plasmid was used as a template to
generate plasmid pESmtRnl by two rounds of PCR to delete sequences
coding for extra amino acids at the amino terminus (primers XXV and
XXVI) and between the carboxyl terminus of the native eggplant protein
A recombinant version of eggplant tRNA ligase, including a carboxy-
terminal His6tail (Fig. 1), was expressed in Escherichia coli Rosetta
2(DE3)pLysS (Novagen) transformed with pESmtRnl. A 250-ml culture
at 0.6 U of optical density (OD) at 600 nm was induced with 400 ?M
isopropyl ?-D-1-thiogalactopyranoside for 3 h at 28°C. Cells were har-
vested by centrifugation, washed, resuspended in 5 ml of H2O containing
a cocktail of proteinase inhibitors (Complete; Roche), and frozen. Once
FIG 1 Amino acid sequence of eggplant tRNA ligase including the carboxy-
terminal His6tag (gray background) of the expressed recombinant version.
The chloroplast transit peptide predicted by the ChloroP algorithm is high-
PNK, and CPD) are boxed.
Nohales et al.
jvi.asm.orgJournal of Virology
the cell suspension was thawed, 0.5 ml of 1 M Tris-HCl (pH 7.5), 1 ml of
10% Nonidet P-40, 25 ?l of 1 M MgCl2, 7 ?l of 2-mercaptoethanol, and
125 U of Benzonase (Novagen) were added to the cell suspension and the
for 15 min more. The extract was clarified by centrifugation at 100,000 ?
g for 30 min and the supernatant brought to 20 mM imidazole. The re-
combinant protein was purified by chromatography using a 1-ml Ni-
Sepharose column (Histrap HP; GE Healthcare) with an A¨KTA Prime
Plus liquid chromatography system operated at 4°C with a flow rate of 1
ml/min. The column was equilibrated with 10 ml of buffer (50 mM Tris-
20 mM imidazole) and the extract loaded. The column was washed with
20 ml of buffer and the protein eluted with a 1:1 mix of equilibration
ligase was analyzed in the eluted fractions by electrophoresis in parallel
denaturing (0.05% sodium dodecyl sulfate [SDS]) 12.5% polyacrylamide
gels. One gel was stained with Coomassie blue and the other subjected to
Western blot analysis with an anti-His6antibody (Clontech).
Viroid RNA transcription and purification. Dimeric (?) and (?)
CChMVd (AJ878085, from 295 to 294), and ELVd (AJ536613, from 210 to
including 8 M urea in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM
EDTA). Gels were stained with ethidium bromide, and the products corre-
sponding to the full-length monomeric linear viroid RNAs (resulting from
hammerhead-mediated self-cleavage and consequently containing 5=-hy-
cipitated, and quantified spectrophotometrically. RNAs corresponding to
full-length monomeric linear (?) ELVd RNAs from position U90 to U89,
sponding monomeric ELVd cDNAs flanked by a modified version of the
hammerhead ribozyme from tobacco ringspot virus satellite (sTRSV) RNA
(16) and a modified version of the hepatitis delta virus (?) strand ribozyme
(43). To produce32P-labeled (?) ELVd RNA probes, 0.5 mM UTP was re-
placed by 40 ?Ci of [?-32P]UTP (800 Ci/mmol) during the transcription
ization reactions were done in a 20-?l volume in buffer consisting of 50
ATP, including 25 ng of RNA substrate and 4 ?l of the purified protein;
reaction mixtures were incubated for 30 min at 30°C. Reactions were
stopped by adding 20 ?g (1 ?l) of proteinase K (New England BioLabs)
10 mM 2-mercaptoethanol, and 0.5% SDS and incubating the reaction
mixtures successively for 15 min at 42°C and 15 min at 55°C. Reaction
products were mixed with 1 volume of loading buffer (98% formamide,
bromophenol blue), denatured by heating for 2 min at 95°C, and sepa-
rated by denaturing PAGE. ELVd (?) RNAs were detected by Northern
linear self-cleavage forms of all viroids (ASBVd, PLMVd, CChMVd, and
and the polyacrylamide gels used to separate the reaction products fixed
for 30 min in 10% acetic acid–20% methanol, dried under vacuum, and
imaged by autoradiography.
VIGS of tRNA ligase from Nicotiana benthamiana Domin. Plasmid
position 983 to 1282 of eggplant tRNA ligase cDNA (see Fig. S1 in the
and XhoI sites of the virus-induced gene silencing (VIGS) vector pTRV2
(GenBank accession no. AF406991) (30). Control plasmid pTRV2-GFP,
designed to silence Aequorea victoria green fluorescent protein (GFP),
contains an insertion corresponding to position 219 to 480 of the cDNA
from GFP variant mgfp5 (GenBank accession no. U87973) (42) also
cloned between the EcoRI and XhoI sites of pTRV2 in the sense orienta-
tion. Plasmids pCdELVd? and pCdCCCVd? contain dimeric cDNAs of
ELVd (from position 210 to 209) and coconut cadang-cadang viroid
the control of the 35S promoter from cauliflower mosaic virus and nos
terminator from Agrobacterium tumefaciens. A. tumefaciens C58C1 cells
transformed with plasmids pTRV1 (AF406990) (30), pTRV2-tRnl,
pTRV2-GFP, pCdELVd?, and pCdCCCVd? were grown to approxi-
mately 0.5 U of OD at 600 nm, recovered by centrifugation, resuspended
at 0.5 U of OD in 10 mM MES-NaOH (pH 5.6), 10 mM MgCl2, and 150
?M acetosyringone, and induced for 2 h at 28°C. Transgenic N. bentha-
miana plants constitutively expressing the GFP variant mgfp5 (line 16c)
(42) were agroinoculated at the four-leaf stage with a 1:1 mix of the A.
tumefaciens C58C1 cultures transformed with plasmids pTRV1 and
pTRV2-tRnl or pTRV1 and pTRV2-GFP. Three to 4 weeks after agroin-
oculation, GFP silencing was confirmed in the plants inoculated with the
A. tumefaciens mix containing pTRV2-GFP by direct observation using a
UV lamp. Plants were then infiltrated with A. tumefaciens cultures trans-
formed with pCdELVd? or pCdCCCVd?. These cultures were also
grown and induced at an OD (600 nm) of 0.5 but infiltrated at an OD of
0.05. At different time points, infiltrated tissues were harvested, RNAs
Northern blot hybridization.
Cloning and expression of eggplant tRNA ligase and ligation of
ELVd RNA. Using primers derived from conserved sequences in
eggplant, we amplified an 841-bp cDNA by RT-PCR that was
cloned and sequenced. When translated in silico, the amino acid
sequence of one of the three possible reading frames showed high
similarities with the sequences of tRNA ligases from wheat (Trit-
icum aestivum L.), Arabidopsis thaliana L., rice (Oryza sativa L.),
and grapevine (Vitis vinifera L.), indicating that this cDNA most
likely represented a fragment of the tRNA ligase orthologue from
to amplify new cDNAs corresponding to the remaining mRNA
material) predicted an amino acid sequence with three conserved
catalytic motifs (RNA ligase, polynucleotide kinase, and cyclic
1). Further analysis of this sequence with the ChloroP algorithm
predicted an amino-terminal transit peptide to the chloroplast
that A. thaliana and rice tRNA ligases contain functional amino-
terminal transit peptides directing these proteins to the chloro-
plast (14). These authors proposed a mechanism of alternative
translation initiation to produce in plants two different tRNA li-
gase isoforms, with or without the transit peptide, targeted to the
chloroplasts or nuclei, respectively.
The sequence corresponding to the complete open reading
frame of eggplant tRNA ligase, including the predicted transit
peptide, was cloned in a vector for expressing in E. coli a recom-
binant version of this protein tagged at its carboxyl terminus with
a His6tail (Fig. 1). The expressed protein was purified under na-
Chloroplastic tRNA Ligase Circularizes Avsunviroidae
August 2012 Volume 86 Number 15jvi.asm.org 8271
tive conditions by affinity chromatography using a Ni-Sepharose
linear ELVd RNA of (?) polarity with 5=-hydroxyl and 2=,3=-cy-
a dimeric precursor transcript. Analysis of the reaction products,
separated by denaturing PAGE and revealed by Northern blot
catalyzed efficiently the circularization of the ELVd (?) RNA,
similarly to a control preparation of recombinant A. thaliana
tRNA ligase (14, 21) (Fig. 2B).
circularization in our in vitro reaction system using the recom-
binant eggplant tRNA ligase. For this purpose, an aliquot of the
tRNA ligase preparation was dialyzed and assayed for ELVd
circularization under different reaction conditions. Assays in
pair combinations showed a strict dependence on ATP (Fig.
2C). The optimal pH determined for eggplant tRNA ligase was
quite narrow, since activity was only detected at pHs 7 and 8
(Fig. 2D). The reaction also showed stringent dependence on
MgCl2, since no circularization occurred in the absence of this
salt (Fig. 2E; compare lane 1 with lanes 2 to 7). Finally, the
reaction was inhibited at KCl concentrations above 200 mM
(Fig. 2F). From this assay, a standard ligation buffer consisting
of 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 4 mM MgCl2, 5 mM
dithiothreitol, and 1 mM ATP was used in subsequent experi-
Circularization of different viroid RNAs by eggplant tRNA
ligase. To gain support for the hypothesis that the chloroplastic
the replication of viroids of the family Avsunviroidae, we assayed
ligation using different forms of the monomeric linear ELVd (?)
RNA opened at distinct positions in the circular molecule. These
substrates were obtained by self-cleavage of dimeric precursors
in monomeric-length RNAs with 5=-hydroxyl and 2=,3=-cyclic
phosphodiester termini. Together with the bona fide monomeric
linear (?) ELVd replication intermediate opened between posi-
tions A333 and G1, we tested five additional RNAs opened be-
tween positions U89 and U90, A103 and A104, U176 and C177,
conformation (Fig. 3A). Interestingly, eggplant tRNA ligase only
circularized the physiological monomeric linear (?) ELVd
(A333-G1) (Fig. 3B). This result indicates a high specificity of the
eggplant enzyme for the genuine ELVd replication intermediate
that may reflect the situation existing in vivo.
Next, we examined the ability of eggplant tRNA ligase to me-
the embedded hammerhead ribozymes) from the other viroid
the monomeric linear (?) and (?) ELVd RNAs, the latter as a
control for comparative purposes. In these ligation assays, we re-
FIG2 Characterization of the ELVd RNA circularization activity of recombinant eggplant tRNA ligase. (A) Western blot analysis of an aliquot eluting from the
arrow points the band likely corresponding to the full-length recombinant version of eggplant tRNA ligase. The positions and molecular masses of protein
tRNA ligases. (C) ELVd circularization assay of eggplant tRNA ligase in the presence of ATP, GTP, CTP, and UTP (lanes 2 to 5), with no NTP (lane 6), or in the
presence of all possible combinations of two different NTPs (lanes 7 to 12). (D) ELVd circularization assay of eggplant tRNA ligase with buffers at pHs 5, 6, 7, 8,
2 to 7). (F) ELVd circularization assay of eggplant tRNA ligase in the presence of increasing concentrations of KCl (lanes 2 to 10). In panels B, C, D, and F, lane
1 contains a negative ligation control with no protein added. The positions of monomeric circular (mc) and linear (ml) ELVd forms are indicated on the left of
panels B to F.
Nohales et al.
jvi.asm.orgJournal of Virology
placed ATP with [?-32P]ATP. The rationale for this approach is
group of an ATP molecule is first transferred to the 5=-hydroxyl
phodiester linkage resulting from ligation. The linkage also
includes a 2=-phosphomonoester resulting from opening the
2=,3=-cyclic phosphodiester termini. The important point is that
under these assay conditions, both the monomeric linear and cir-
cular forms of all viroid RNAs may become labeled and be easily
identified and compared.
ear and circular RNAs of all viroids became labeled for both po-
larities (Fig. 4). Comparison of the intensities of the different
and 8 with lane 7). Even if some differences could be appreciated
depending on the particular viroid and the polarity of the strand
roplastic isoform of tRNA ligase catalyzing the circularization of
both (?) and (?) monomeric linear intermediates during repli-
cation of the Avsunviroidae occurring through a symmetric roll-
in ELVd circularization. To provide further support for the role
plants transiently expressing dimeric ELVd transcripts. Initial at-
tempts to adapt a VIGS strategy to eggplant were unsuccessful,
and although N. benthamiana is not a host for ELVd or any other
known viroid within the family Avsunviroidae, ELVd dimeric
transcripts are processed properly when expressed transiently in
this plant. Moreover, an ELVd-derived RNA fused to a GFP
mRNA has been shown to traffic into the chloroplast when tran-
viroid replicating in the nucleus (CCCVd, family Pospiviroidae)
served as a control in this experiment because when transiently
expressed in N. benthamiana, they are also processed into mono-
meric linear and circular RNAs (J. Marqués and J.-A. Daròs, un-
published results), but through a nuclear pathway involving dif-
ferent enzymes (20, M.-A. Nohales, R. Flores, and J.-A. Daròs,
submitted for publication). Moreover, similarly to what occurs
with ELVd, N. benthamiana is a nonhost for CCCVd. In these
experiments, we used an N. benthamiana transgenic line (16c)
constitutively expressing GFP (42) and a vector based on tobacco
the transgenic GFP, the latter serving as a control and silencing
marker. Plants preinoculated with the TRV-derived VIGS vectors
eral days postinfiltration, were analyzed by denaturing PAGE fol-
lowed by Northern blot hybridization (see Fig. S2 in the supple-
mental material). Time course analysis of ELVd processing
showed that the ratio of monomeric circular to total monomeric
(circular plus linear) ELVd (?) RNA was significantly lower in
tissues preinoculated with the VIGS vector to silence the tRNA
ligase than in tissues preinoculated with the control VIGS vector
to silence GFP (Fig. 5A). This difference was not observed follow-
ing transient expression of dimeric CCCVd (?) RNA in the same
plants preinoculated with the same VIGS vectors (Fig. 5B). This
by tRNA ligase occurs not only in vitro but also in vivo.
FIG 3 Circularization by eggplant tRNA ligase of different monomeric linear
ELVd (?) RNAs opened at different sites. (A) ELVd folded in the predicted
minimum free-energy conformation. Arrows indicate positions in which the
different linear ELVd substrates subjected to ligation were opened. (B) ELVd
circularization assay with recombinant eggplant tRNA ligase. Reaction prod-
ucts were separated by denaturing PAGE and ELVd (?) RNAs revealed by
Northern blot hybridization. Lanes 1 to 6, controls with no protein added.
Lanes 7 to 12, eggplant tRNA ligase added. Substrate RNAs were opened be-
tween positions A333 and G1 (lanes 1 and 7), A263 and G264 (lanes 2 and 8),
U245 and U246 (lanes 3 and 9), U176 and C177 (lanes 4 and 10), A103 and
A104 (lanes 5 and 11), and U89 and U90 (lanes 6 and 12). The positions of
FIG 4 Circularization by the recombinant eggplant tRNA ligase of self-cleav-
age (?) and (?) monomeric linear ASBVd (lanes 1 and 2), PLMVd (lanes 3
and 4), CChMVd (lanes 5 and 6), and ELVd (lanes 7 and 8) RNAs. Reaction
products obtained in the presence of [?-32P]ATP were separated by denatur-
ing PAGE, and the gel was autoradiographed. The positions of monomeric
and an asterisk, respectively.
Chloroplastic tRNA Ligase Circularizes Avsunviroidae
August 2012 Volume 86 Number 15jvi.asm.org 8273
RNA transcription during viroid replication through a rolling-
circle mechanism produces oligomeric viroid strands (4). In
members of the family Pospiviroidae, only the (?) oligomeric
RNAs are processed to monomers (3). This processing has been
proposed to occur through a conserved mechanism in which the
upper CCR strand and flanking inverted repeats) of two contigu-
ous units in the oligomeric RNA intermediate interact (kissing
loops) and promote the adoption of a palindromic quasi-double-
stranded structure that would be the substrate of a host type III
RNase (20). The resulting monomeric linear viroid intermediate
with 5=-phosphomonoester and 3=-hydroxyl termini (21) would
be ultimately circularized by the host DNA ligase 1 redirected to
act as an RNA ligase (Nohales et al., submitted).
In members of the family Avsunviroidae, oligomeric viroid
RNAs of both (?) and (?) polarities self-cleave to monomers
we have shown that the chloroplastic isoform of eggplant tRNA
ligase most likely mediates circularization of both (?) and (?)
monomeric linear RNAs during replication of ELVd. This pro-
posal is supported by several observations. First, a recombinant
the monomeric linear ELVd (?) RNA, resulting from the ham-
merhead ribozyme self-cleavage of a head-to-tail dimeric tran-
script (mimicking the replication intermediates in vivo), but not
the same 5=-hydroxyl and 2=,3=-cyclic phosphodiester terminal
groups (Fig. 3). Second, the recombinant eggplant tRNA ligase
efficiently catalyzes in vitro circularization of the ELVd (?) mo-
nomeric RNA replication intermediate resulting from self-cleav-
age (Fig. 4). And third, the ratio of monomeric circular to total
monomeric ELVd (?) RNA decreased when dimeric (?) ELVd
transcripts were transiently expressed in N. benthamiana plants
preinoculated with a VIGS vector to induce silencing of endoge-
nous tRNA ligase, in contrast to the situation observed with
CCCVd, a member of the family Pospiviroidae, with a different
processing pathway (Fig. 5).
ated by the chloroplastic tRNA ligase of this green alga. The spec-
ificity reported here regarding the ligation site is also in accor-
dance with the circularization of ELVd RNA in C. reinhardtii
chloroplasts demanding a quasi-double-stranded structure in the
middle of the molecule wherein the ligation site maps at a short
loop motif (33).
The recombinant eggplant tRNA ligase also mediates the effi-
cient circularization in vitro of the (?) and (?) self-cleavage mo-
three components of the family Avsunviroidae (Fig. 4), thus sup-
porting the involvement in replication of the tRNA ligase homo-
logues from their corresponding hosts. In the case of PLMVd, the
vitro in the presence of Mg2?through a reaction which is not the
reverse of the cleavage by the hammerhead ribozyme, because a
2=,5=-phosphodiester bond is produced (7). The monomeric lin-
ear ELVd (?) RNA resulting from self-cleavage also shows the
same behavior (D. Molina-Serrano, R. Flores, and J.-A. Daròs,
unpublished results). However, despite the detection of some cir-
cular PLMVd RNA forms locked through a 2=,5=-phosphodiester
linkage in infected peach tissue (6), it is unlikely that this is the
main circularization mechanism during replication in the family.
First, and similar to what occurs with the hammerhead ribozyme
reverse reaction, the efficiency of this reaction is very low com-
pared to that of the tRNA ligase-mediated circularization. And
second, reverse transcription analysis of the monomeric circular
ASBVd and ELVd (?) RNAs isolated from infected tissues indi-
likely the involvement of a recently described chloroplastic RNA
ligase activity analogous to the bacterial and archaeal 2=-5= RNA
phosphodiester, 2=-phosphomonoester junction (13). However,
the 2=-phosphomonoester group seems to be absent in mature
ASBVd and ELVd circular RNAs accumulating in infected tissues
(15, 34). In contrast, the presence of a 2=-phosphomonoester
group was previously reported at the ligation site of two viroid-
FIG 5 Effect of tRNA ligase silencing on circularization of ELVd (A) and
CCCVd (B) monomeric RNAs. Dimeric (?) ELVd and CCCVd transcripts
to silence the GFP transgene or the endogenous tRNA ligase. RNAs were pu-
rified at different days from the agroinfiltrated areas of three different plants
phosphorimetry. The plots represent the average ratio of monomeric circular
to total monomeric (circular plus linear (?) ELVd (A) and CCCVd (B) RNAs
tRNA ligase-silenced plants (red symbols). Error bars indicate the standard
deviations of the triplicate measurements.
Nohales et al.
jvi.asm.org Journal of Virology
tion by an RNA ligase (28). RT-PCR amplification of circular
PLMVd and CChMVd RNAs accumulating in infected tissues
less likely, from a 2=,5=-phosphodiester linkage. Removal of the
the activity of a 2=-phosphotransferase, similar to what occurs
during eukaryotic tRNA maturation (1, 46). Interestingly, A.
thaliana and rice 2=-phosphotransferases also contain amino-ter-
minal transit peptides and have been shown to target the chloro-
plast during transient expression (14).
acterized for their restricted host range, since they only infect the
plants in which they were initially discovered and a few phyloge-
roplastic isoform of eggplant tRNA ligase efficiently mediates in
vitro circularization of the genuine monomeric RNAs of both po-
larities of all Avsunviroidae implies that the remarkable host spec-
This work was supported by the Ministerio de Ciencia e Innovación
(MICINN) from Spain through grants BIO2008-01986, BIO2011-26741,
and BFU2008-03154. M. A. Nohales and D. Molina-Serrano were the
cación y Ciencia.
We thank Verónica Aragonés for excellent technical assistance. We
thank Aaron J. Plys (Department of Biological Sciences, University of
Cyprus, Nicosia) for critical reading of the manuscript.
1. Abelson J, Trotta CR, Li H. 1998. tRNA splicing. J. Biol. Chem. 273:
2. Ambrós S, Hernández C, Desvignes JC, Flores R. 1998. Genomic struc-
ture of three phenotypically different isolates of peach latent mosaic vi-
roid: implications of the existence of constraints limiting the heterogene-
ity of viroid quasispecies. J. Virol. 72:7397–7406.
3. Branch AD, Benenfeld BJ, Robertson HD. 1988. Evidence for a single
rolling circle in the replication of potato spindle tuber viroid. Proc. Natl.
Acad. Sci. U. S. A. 85:9128–9132.
4. Branch AD, Robertson HD. 1984. A replication cycle for viroids and
other small infectious RNAs. Science 223:450–455.
5. Canny MD, Jucker FM, Pardi A. 2007. Efficient ligation of the Schisto-
soma hammerhead ribozyme. Biochemistry 46:3826–3834.
6. Côté F, Lévesque D, Perreault JP. 2001. Natural 2=,5=-phosphodiester
bonds found at the ligation sites of peach latent mosaic viroid. J. Virol.
7. Côté F, Perreault JP. 1997. Peach latent mosaic viroid is locked by a
2=,5=-phosphodiester bond produced by in vitro self-ligation. J. Mol. Biol.
and facilitates its hammerhead-mediated self-cleavage. EMBO J. 21:749–
9. Daròs JA, Marcos JF, 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.
10. De la Peña M, Gago S, Flores R. 2003. Peripheral regions of natural
hammerhead ribozymes greatly increase their self-cleavage activity.
EMBO J. 22:5561–5570.
11. de la Peña M, Navarro B, Flores R. 1999. Mapping the molecular
determinant of pathogenicity in a hammerhead viroid: a tetraloop within
the in vivo branched RNA conformation. Proc. Natl. Acad. Sci. U. S. A.
12. Ding B. 2009. The biology of viroid-host interactions. Annu. Rev. Phyto-
13. Englert M, Beier H. 2005. Plant tRNA ligases are multifunctional en-
zymes that have diverged in sequence and substrate specificity from RNA
ligases of other phylogenetic origins. Nucleic Acids Res. 33:388–399.
14. Englert M, et al. 2007. Plant pre-tRNA splicing enzymes are targeted to
multiple cellular compartments. Biochimie 89:1351–1365.
15. Fadda Z, Daròs JA, Fagoaga C, Flores R, Duran-Vila N. 2003. Eggplant
latent viroid, the candidate type species for a new genus within the family
Avsunviroidae (hammerhead viroids). J. Virol. 77:6528–6532.
16. Feldstein PA, Hu Y, Owens RA. 1998. Precisely full length, circularizable,
complementary RNA: an infectious form of potato spindle tuber viroid.
Proc. Natl. Acad. Sci. U. S. A. 95:6560–6565.
17. Flores R, Daròs JA, Hernández C. 2000. The Avsunviroidae family:
viroids containing hammerhead ribozymes. Adv. Virus Res. 55:271–323.
18. Flores R, Hernández C, Martínez de Alba AE, Daròs JA, Di Serio F. 2005.
19. Flores R, Owens RA. 2008. Viroids, p 332–342. In Mahy BWJ, Van
Regenmortel MHV (ed), Encyclopedia of virology. Elsevier, Oxford,
20. Gas ME, Hernández C, Flores R, Daròs JA. 2007. Processing of nuclear
viroids in vivo: an interplay between RNA conformations. PLoS Pathog.
21. Gas ME, Molina-Serrano D, Hernández C, Flores R, Daròs JA. 2008.
Monomeric linear RNA of citrus exocortis viroid resulting from process-
ing in vivo has 5=-phosphomonoester and 3=-hydroxyl termini: implica-
tions for the RNase and RNA ligase involved in replication. J. Virol. 82:
22. Gómez G, Pallás V. 2010. Noncoding RNA mediated traffic of foreign
mRNA into chloroplasts reveals a novel signaling mechanism in plants.
PLoS One 5:e12269. doi:10.1371/journal.pone.0012269.
23. Hernández C, Flores R. 1992. Plus and minus RNAs of peach latent
mosaic viroid self-cleave in vitro via hammerhead structures. Proc. Natl.
Acad. Sci. U. S. A. 89:3711–3715.
24. Hertel KJ, Herschlag D, Uhlenbeck OC. 1994. A kinetic and thermody-
namic framework for the hammerhead ribozyme reaction. Biochemistry
25. Hutchins CJ, et al. 1985. Comparison of multimeric plus and minus
forms of viroids and virusoids. Plant Mol. Biol. 4:293–304.
26. Hutchins CJ, Rathjen PD, Forster AC, Symons RH. 1986. Self-cleavage
of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic
Acids Res. 14:3627–3640.
27. Khvorova A, Lescoute A, Westhof E, Jayasena SD. 2003. Sequence
elements outside the hammerhead ribozyme catalytic core enable intra-
cellular activity. Nat. Struct. Biol. 10:708–712.
28. Kiberstis PA, Haseloff J, Zimmern D. 1985. 2= phosphomonoester, 3=-5=
phosphodiester bond at a unique site in a circular viral RNA. EMBO J.
29. Konarska M, Filipowicz W, Domdey H, Gross HJ. 1981. Formation of a
2=-phosphomonoester, 3=,5=-phosphodiester linkage by a novel RNA li-
gase in wheat germ. Nature 293:112–116.
30. Liu Y, Schiff M, Marathe R, Dinesh-Kumar SP. 2002. Tobacco Rar1,
EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance
to tobacco mosaic virus. Plant J. 30:415–429.
31. Makino S, Sawasaki T, Endo Y, Takai K. 2005. Purification and sequence
determination of an RNA ligase from wheat embryos. Nucleic Acids
Symp. Ser. 2005:319–320.
32. Marcos JF, Flores R. 1993. The 5= end generated in the in vitro self-
occurring linear viroid molecules. J. Gen. Virol. 74:907–910.
33. Martínez F, Marqués J, Salvador ML, Daròs JA. 2009. Mutational
analysis of eggplant latent viroid RNA processing in Chlamydomonas re-
inhardtii chloroplast. J. Gen. Virol. 90:3057–3065.
34. Molina-Serrano D, Marqués J, Nohales MA, Flores R, Daròs JA. 2012.
2–5= RNA ligase. RNA Biol. 9:326–333.
35. Navarro B, Flores R. 1997. Chrysanthemum chlorotic mottle viroid:
unusual structural properties of a subgroup of self-cleaving viroids with
hammerhead ribozymes. Proc. Natl. Acad. Sci. U. S. A. 94:11262–11267.
36. Navarro JA, Daròs JA, Flores R. 1999. Complexes containing both po-
larity strands of avocado sunblotch viroid: identification in chloroplasts
and characterization. Virology 253:77–85.
37. Navarro JA, Vera A, Flores R. 2000. A chloroplastic RNA polymerase
Chloroplastic tRNA Ligase Circularizes Avsunviroidae
August 2012 Volume 86 Number 15jvi.asm.org 8275
resistant to tagetitoxin is involved in replication of avocado sunblotch
viroid. Virology 268:218–225.
38. Nelson JA, Shepotinovskaya I, Uhlenbeck OC. 2005. Hammerheads
derived from sTRSV show enhanced cleavage and ligation rate constants.
39. Pick L, Furneaux H, Hurwitz J. 1986. Purification of wheat germ RNA
ligase. II. Mechanism of action of wheat germ RNA ligase. J. Biol. Chem.
40. Prody GA, Bakos JT, Buzayan JM, Schneider IR, Bruening G. 1986. Autolytic
41. Rodio ME, et al. 2007. A viroid RNA with a specific structural motif
inhibits chloroplast development. Plant Cell 19:3610–3626.
42. Ruiz MT, Voinnet O, Baulcombe DC. 1998. Initiation and maintenance
of virus-induced gene silencing. Plant Cell 10:937–946.
43. Schürer H, Lang K, Schuster J, Mörl M. 2002. A universal method to
44. Schwartz RC, Greer CL, Gegenheimer P, Abelson J. 1983. Enzymatic
mechanism of an RNA ligase from wheat germ. J. Biol. Chem. 258:8374–
45. Tsagris EM, Martínez de Alba AE, Gozmanova M, Kalantidis K. 2008.
Viroids. Cell. Microbiol. 10:2168–2179.
46. Wang LK, Shuman S. 2005. Structure-function analysis of yeast tRNA
ligase. RNA 11:966–975.
Nohales et al.
jvi.asm.orgJournal of Virology