Departamento de Mejora y Patología Vegetal, CEBAS-CSIC, Campus Universitario de Espinardo, PO Box 4195, 30071 Murcia, Spain1
Ministry of Agriculture and Food, Tirana, Albania2
Istituto Agronomico Mediterraneo, Valenzano, Bari, Italy3
Author for correspondence: Vicente Pallás. Fax +34 96 3877859. e-mail vpallas{at}ibmcp.upv.es
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Abstract |
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Introduction |
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Hop stunt viroid (HSVd) belongs to the Pospiviroidae family. It has been found in a wide range of hosts including hop, cucumber, grapevine, citrus, plum, peach, pear (Shikata, 1990 ) and, recently, apricot and almond (Astruc et al., 1996
; Cañizares et al., 1999
). The infection seems to be latent in some hosts such as grapevine (Shikata, 1990
; Polivka et al., 1996
) and apricot (Astruc et al., 1996
). In other cases, specific disorders such as hop stunt (Shikata, 1990
), dapple fruit disease of plum and peach (Sano et al., 1989
) and citrus cachexia (Diener et al., 1988
; Semancik et al., 1988
) have been associated with HSVd infection.
Historically, HSVd sequence isolates have been divided into three groups (i.e. plum-type, hop-type and citrus-type) on the basis of overall homology (Shikata, 1990 ). The characterization of ten new sequence variants from three different Prunus species and the subsequent phylogenetic analysis revealed the appearance of two new groups that very probably derived from recombination events (Kofalvi et al., 1997
). In addition, it was shown that the previous hop-type group itself is likely to be the result of a recombination between members of the plum-type and citrus-type groups.
Until now, characterization of the primary structure of HSVd isolates has been carried out using isolates from Spain, France, Italy, USA and Japan. In this work we present the characterization of 16 new sequence variants of HSVd obtained from four Mediterranean countries (Cyprus, Greece, Morocco and Turkey) from where no sequence data were available before this work. Molecular variability comparisons and phylogenetic analyses revealed that sequence variants belonging to the two minor recombinant subgroups are more frequent than previously thought and that there are stretches of sequences on the viroid molecule that are highly conserved, suggesting key roles in the viroid life-cycle. In addition we identified a hammerhead-like structure within the TR domain that is strictly conserved in all the sequence variants characterized so far and that can be considered as an evolutionary link between typical viroids and those having the ability to undergo self-cleavage via hammerhead ribozymes.
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Methods |
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RTPCR amplification, cloning and sequencing of viroid isolates.
RTPCR was performed as described (Astruc et al., 1996 ) by using avian myeloblastosis virus reverse transcriptase (Promega) for the RT and Pfu DNA polymerase (Stratagene) for PCR amplification. The oligonucleotides used were the antisense 26-mer VP-19 (5' dGCCCCGGGGCTCCTTTCTCAGGTAAG 3', complementary to HSVd residues 6085) and the sense 27-mer VP-20 (5' dCGCCCGGGGCAACTCTTCTCAGAATCC 3', residues 78102). Both primers lie in the strictly conserved central region of HSVd and contain the unique endonuclease restriction site SmaI (underlined). Following RTPCR, electrophoretic analysis confirmed the presence of a monomeric PCR product of the expected size. The PCR products were phenol-extracted, ethanol-precipitated and digested with the SmaI endonuclease. The resulting DNA fragments were cloned in the SmaI site of dephosphorylated pUC18 plasmid. Since this pair of primers covers almost the totality of the CCR, another pair of primers (VP-98 and VP-99) was designed to study the molecular variability of this part of the molecule. These primers (VP-98 5', dCTCCAGAGCACCGCGGCCCTC 3', complementary to residues 120140; and VP-99, 5' dCTGGGGAATTCTCGAGTTGCCGC 3', HSVd residues 123) flank the CCR of HSVd and contain EcoRI and SacII restriction sites, respectively (underlined). The PCR products were phenol-extracted, ethanol-precipitated and digested with EcoRI and SacII. The resulting DNA fragments were cloned in a previously digested Bluescript II KS+ plasmid.
For all isolates, cDNA clones were identified by restriction analysis. Selected clones were sequenced in both orientations by using universal primers with an automated DNA sequencer (ABI PRISM 337; Perkin-Elmer). The new sequence variants were named following the rules described previously (Kofalvi et al., 1997 )
Computer analysis of the sequences.
Multiple alignments of HSVd sequences were obtained using ClustalW (Thompson et al., 1994 ). The alignment was corrected manually to maximize sequence homology. Phylogenetic analyses were performed using the following programs of the PHYLIP 3.5c package (Felsenstein, 1993
). DNADIST was used to calculate genetic distances, NEIGHBOUR (UPGMA or neighbour-joining methods) to cluster the variants from the distance data, DRAWTREE to draw the resulting phylogenetic tree and SEQBOOT (100 repetitions) and CONSENSE to perform bootstrap analysis.
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Results and Discussion |
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Viroid apricot sources found to be homogeneous within the limits of the sparse sample group used include Maoui 1.2 from Morocco, Septik from Turkey, Canino from Cyprus and Kolioponlou and Pr. Porou from Greece. However the other sources were heterogeneous, with two or three different sequences for two or three cDNA clones sequenced. It is worth noting that the two cDNA clones sequenced from the source Pr. Porou from Greece were identical to HSVd.apr9 obtained from the Canino cultivar from Morocco (Table 1).
The closest HSVd sequence for most of the sequence variants from Morocco, with the exception of apr13, was HSVd.apr4, a sequence variant previously identified in Bulida apricots from Spain (Kofalvi et al., 1997 ). The variant from Turkey was found to be very similar to HSVd.h1, a sequence variant originating from Japan. All variants from Greece, except the one obtained from Gr7.1 and Gr7.2 cDNA clones, were found to be very similar to HSVd.g3, which came from a German grapevine. Finally, all the variants from Cyprus, except apr18 and apr21, which are very similar to HSVd.g3 and HSVd.apr1 respectively, were found to be very similar to HSVd.apr2, a sequence variant that, as stated above, was previously detected in apricot cultivars originating from Spain and Italy (Kofalvi et al., 1997
) and in plum from Japan.
The above results could be explained by the frequent plant exchange between different countries or, alternatively, by a parallel evolution of the viroid molecule, whose variability is restricted to certain polymorphic positions.
Several lines of evidence allowed us to conclude that we obtained a high degree of fidelity in the characterization of the new sequences variants. (i) Two different RTPCR reactions were carried out for each isolate. The first one was done by using VP-19 and VP-20 primers that were designed to the central part of the molecule (see Methods). With this pair of primers the sequence of 251 out of 300 nt of the viroid molecule was determined for each isolate. To obtain clones representing the central part and surrounding areas VP-98 and VP-99 were designed and a different RTPCR was carried out. The sequence of three clones for each isolate revealed that all of the mutations observed in these partial clones were coincident in all isolates with the mutations observed with the almost full-length clones obtained in the other RTPCR. (ii) We used a thermostable DNA polymerase endowed with proofreading activity to minimize the introduction of substitutions during PCR amplification. It has been determined that the error rate of Pfu DNA polymerase (<3x10-6 errors per bp per cycle) is ten times lower than the error rate of Taq polymerase (Bracho et al., 1998 ). (iii) All the polymorphic positions found are present in at least five out of the 16 new sequence variants characterized in this work. In addition they are identical to other polymorphic positions previously described for other Prunus hosts (Kofalvi et al., 1997
) and even for different non-Prunus hosts.
Phylogenetic analysis of the new HSVd variants
Alignment and phylogenetic analyses of the HSVd sequence variants characterized in this work were carried out together with the 38 HSVd sequences previously reported, giving a total of 54 HSVd sequences. Previously, HSVd variants were divided, according to overall sequence homologies (Sano et al., 1989 ) and phylogenetic analysis (Hsu et al., 1994
), into three major groups: citrus-, hop- and plum-type. After characterizing ten new sequence variants, Kofalvi et al. (1997)
redefined this classification into five groups, including the three previous ones and two new groups, having only two or three members, respectively. These two new groups could be considered as the results of recombination events between members of the plum- and citrus-type (now named P-C group) or between members of the plum- and hop-type or cit3 sequence variant (now named P-H/cit3 group) (Kofalvi et al., 1997
).
Phylogenetic analysis of all sequences, including the sequence variants characterized in this work, showed that five out of the seven variants from Cyprus are included in the recombinant group P-C, which previously contained the variants HSVd.apr2 and HSVd.apr5 (Fig. 1). In the other recombinant group (P-H/cit3) are the three new variants from Greece, HSVd.apr18 from Cyprus and HSVd.apr13 from Morocco (Fig. 1
). These results reflect the facts that recombination events are more frequent than previously expected on HSVd and that intraspecific recombination could be a general mechanism in the evolution of viroids (Candresse et al., 1997
). Finally, four out of the five variants from Morocco clustered into the plum-type group, confirming the homogeneous origin of the Moroccan isolates and showing their close relationship to the Spanish sequence variant isolated from the Bulida apricot, the most extended cultivar growth in the southeast of Spain having 81% HSVd infection (Cañizares et al., 1998
).
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The presence of the inverted repeat sequences within the C domain of typical viroids allows the formation of a cruciform structure alternative to the rod-like conformation (Fig. 3), similar to the alternative structure described in the PSTVd group (Liu & Symons, 1998
). Interestingly, the upper part of this cruciform structure (hairpin I) is strictly conserved in all the 54 sequence variants characterized, whereas in the lower part a reduced variability is allowed (Fig. 3
). The presence of a G residue (marked with an arrowhead in Fig. 3
) between U231 and C232 always led to the disappearance of U207 and A228 (marked with asterisks). This covariation might indicate the requirement of an unpaired residue in the proximal region of the lower stem of the cruciform structure that could be involved in a tertiary interaction with another residue on the loop located immediately downstream of the stem (U207A228 or C212extra G). Another important feature that is apparent from the cruciform representation is that the ends of both stemloops are strictly conserved and complementary (see shaded area in Fig. 3
), suggesting a tertiary structure that may have an important role in the viroid infection process.
Citrus viroids (CVds) have been classified into five groups of viroids variants (Durán-Vila et al., 1988 ). Group II contains HSVd-related variants including those inducing cachexia disease (CV-IIb, CV-IIc and CVd-903), no pathogenic symptoms (Cvd-IIa) and one inducing mild cachexia reactions (CVd-909). Within the variable domain of the HSVd sequence variants infecting citrus (CVds-II) it has been proposed that a cluster of six nucleotide changes regulates the induction of cachexia disease (Reanwarakorn & Semancik, 1998
). It is relevant that the cachexia-inducing sequence (CVd-IIb) is not present in any of the sequence variants obtained from any non-Citrus host. The most similar sequence variant to CVd-IIb is apr14, in which only two substitutions are needed to convert it to a cachexia-inducing sequence. For all the Moroccan and Greek sequence variants, three nucleotides would have to change to revert them to CV-IIb. In these two countries, Prunus and Citrus hosts are cultivated in close proximity, emphasizing the need to control the sanitary status of apricot even though HSVd is considered to be latent in this crop. Curiously, a detailed analysis of the lower part of this cruciform structure revealed that in all citrus sequence variants except those inducing cachexia disease (CVd-IIb, CVd-IIc and Ca-909) the upper stem (marked with a shaded arrow in Fig. 3
) was disturbed by the changes of C204G and G205A. This could explain why CVd-909, in spite of lacking all six nucleotide changes defined above in the cachexia-inducing sequence (in the V domain), incites mild cachexia reactions (Reanwarakorn & Semancik, 1999
). Thus, a rule could be drawn for all citrus sequences characterized so far: those sequence variants with the six nucleotide changes within the V domain and with a very stable stem in the lower part of the cruciform structure would induce a severe cachexia (e.g. CVd-IIb, CVd-IIc); those sequence variants having one of the two features would induce mild cachexia (e.g. Ca 993 and Ca 903, see Table 1
in Reanwarakorn & Semancik, 1999
) and those sequences having an unaltered stem (e.g. CVd-IIa and the rest of citrus sequences) would not induce cachexia disease.
On the basis of phylogenetic analysis of all known viroid sequences, the viroid-like satellite RNAs and the viroid-like domain of the Hepatitis delta virus RNA, it has been suggested that viroids with self-cleavage capability could be considered as an evolutionary link between typical viroids and satellite RNAs (Elena et al., 1991 ). If we assume the hypothesis of the early RNA world containing RNA (ribozyme) rather than protein catalysts, the present day RNA-catalysed reactions could then be considered as ancestral to similar reactions that have become protein-dependent. In this context, Diener (1996)
has suggested that viroids may have evolved from satellite RNAs while still free-living molecules, with both presumably acquiring a dependence on their host (viroids) or helper virus (satellite RNAs) after becoming intracellular entities. It is then reasonable to think that, in this scenario, typical viroids could have maintained relics of the self-cleaving structures. In Fig. 4
, we show a partial hammerhead structure formed in the TR domain of the HSVd molecule in which nine out of 13 absolutely conserved nucleotides of a typical hammerhead ribozyme (Hertel et al., 1992
) are present (boxed nucleotides in Fig. 4
). As a reference, the ribozyme of Peach latent mosaic viroid (PLMVd) (Hernández & Flores, 1992
) has been included in Fig. 4
. It is worth noting that a similar hammerhead-like structure with identical levels of similarity to the one observed in the plus-strand was also found in the negative-strand of the HSVd molecule (not shown). In addition, the presence of a high number of nucleotides that are conserved in a hammerhead ribozyme in a region of the negative polarity of a self-cleaving circular RNA associated with Rice yellow mottle virus has been considered as a vestige of an ancestral functional hammerhead (Collins et al., 1998
). By introducing three substitutions and one insertion between helix I and II in the HSVd pseudo-ribozyme, a canonical hammerhead ribozyme could be reverted. To the best of our knowledge this is the first time that a hammerhead-like structure has been described for a typical viroid and it could represent an evolutionary link between typical viroids and those having the ability to undergo self-cleavage via hammerhead ribozymes.
Interestingly, the motif covering the putative HSVd pseudo-hammerhead is strictly conserved among the 54 known sequence variants, indicating that this viroid region is not prone to sequence variability and suggesting a putative key role in the viroid life-cycle. It has been proposed that self-cleavage reactions will also be involved in the replication of the PSTVd group of viroids (Symons, 1997 ) and preliminary supporting evidence was recently provided for CCCVd (Liu & Symons, 1998
). Experiments are in progress to determine the in vivo viability of an HSVd mutant bearing the four changes required to acquire ribozyme activity.
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Acknowledgments |
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Footnotes |
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b Present address: Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, Avenida de los Naranjos s/n, 46022 Valencia, Spain
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Received 13 November 2000;
accepted 22 December 2000.