1 Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Istituto di Virologia Vegetale del CNR, Sezione di Bari, via Amendola 165/A, 70126 Bari, Italy
2 Istituto Agronomico Mediterraneo, Valenzano, Bari, Italy
Correspondence
Pasquale Saldarelli
csvvps04{at}area.ba.cnr.it
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ748510AJ748536 and AJ606338AJ606356.
Details of the GLRaV-3 isolates used are available as supplementary material in JGV Online.
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INTRODUCTION |
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Only one GLRaV-3 isolate (GenBank accession no. AF037268) has been sequenced completely (Ling et al., 1998, 2000
). Very limited sequence information is available for other isolates with reference to the RdRp domain (GenBank accession no. AY424407; Fajardo et al., 2001
), the heat-shock protein 70 homologue (HSP-70) (Saldarelli et al., 1998
) and the p20 protein (Habili et al., 1995
). By contrast, a great deal of sequence information exists on the variability and population structure of other members of the family Closteroviridae, mainly for Citrus tristeza virus (CTV), which occurs as a population of diverse isolates with different biological and epidemiological properties (Rubio et al., 1999
, 2001
; Kong et al., 2000
).
An attractive reason for the study of closterovirus variability is the size and complexity of the genome, which uses combined strategies of polyprotein processing and subgenomic RNAs for its expression and evolution (Karasev, 2000). Furthermore, GLRaV-3 and CTV infect vegetatively propagated hosts that persist in the field for a long time and are potentially subjected to repeated infections through insects and as a result of cultural practices (e.g. top grafting). The potential impact of virus variability in such long-lived hosts is not understood. Finally, from such an investigation, useful information could be derived for the development of more efficient, nucleic acid-based diagnostic tools and to provide a better choice of possible transgenes for inducing resistance in grapevines.
In the present paper, the population structure and genetic variability of 45 GLRaV-3 isolates from 14 countries were investigated by single-stranded conformation polymorphism (SSCP) and sequence analysis of three different genomic regions: those encoding the RNA-dependent RNA polymerase (RdRp), HSP-70 and the coat protein (CP).
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METHODS |
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Total RNA extraction, RT-PCR and cDNA cloning.
Total RNA was extracted from cortical scrapings of dormant cuttings that were collected from five different branches of each vine in December 2001. The extraction method was according to Foissac et al. (2000), by absorption of total RNA on to silica particles after guanidine-buffer treatment of plant tissues. The primer pairs P3U (5'-CGCTCATGGTGAAAGCAGACG-3') and P3D (5'-CTTAGAACAAAAATATGGAGCAG-3'), LC1 (5'-CGCTAGGGCTGTGGAAGTATT-3') and LC2 (5'-GTTGTCCCGGGTACCAGATAT-3'), and CP3U (5'-ATGGCATTTGAACTGAAATTAGGGC-3') and CP3D (5'-CGGCGCCCATAACCTTCTTACA-3') were designed based on the nucleotide sequence of isolate GLRaV-3 NY1 (GenBank accession no. AF037268) to amplify fragments of 653, 546 and 484 bp from the RdRp domain and the HSP-70 and CP genes, respectively. RT-PCR was carried out as described previously (Turturo et al., 2000
). Approximately 500 ng total RNA was denatured with 500 ng random primers (Roche) at 95 °C for 5 min and reverse-transcribed by incubation at 42 °C for 1 h in a reaction mixture (50 µl final volume) containing 1x Moloney murine leukemia virus (M-MLV) buffer (Invitrogen Life Technologies), 4·8 mM dithiothreitol, 200 µM each dNTP and 200 U M-MLV (Invitrogen Life Technologies). After cDNA synthesis, the reverse transcriptase was inactivated at 70 °C for 10 min. An aliquot (5 µl) of cDNA product was subjected to PCR in a 50 µl reaction mixture containing 1x PCR buffer (Promega), 1·5 mM MgCl2, 200 µM each dNTP, 200 nM each primer and 0·125 U Taq DNA polymerase (Promega). Cycling conditions comprised an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, annealing at 52 °C for 30 s for primers P3U/P3D and LC1/LC2 and at 57 °C for 20 s for primers CP3U/CP3D, followed by elongation at 72 °C for 1 min for primers P3U/P3D, for 50 s for primers LC1/LC2 and for 5 s for primers CP3U/CP3D. Final extension in all cases was at 72 °C for 7 min. Amplified products were analysed by 5 % PAGE and stained with silver nitrate (Beidler et al., 1982
). Amplicons of selected isolates were ligated into the pGEM-T Easy vector (Promega) following the manufacturer's instructions and cloned into Escherichia coli DH5
(Sambrook et al., 1989
). Ten clones of each isolate were selected randomly, subjected to further PCR and the amplified DNA was analysed by SSCP analysis as described below.
SSCP and sequence analysis.
SSCP analysis of amplified PCR products was done following the protocol described by Palacio & Duran-Vila (1999) with minor modifications. A 5 µl aliquot of PCR product was combined with 15 µl denaturing solution [95 % deionized formamide, 20 mM EDTA (pH 8·0), 0·05 % xylene cyanol and 0·05 % bromophenol blue], boiled for 10 min and chilled on ice. Denatured amplicons were separated by non-denaturing 10 % (RdRp and HSP-70) or 12 % (CP) PAGE at 200 V at 4 °C for 4 h. SSCP profiles were visualized by silver staining (Beidler et al., 1982
). Selected plasmids were purified by the boiling method (Sambrook et al., 1989
) and sequenced automatically (MWG). Multiple nucleotide alignments were made by using CLUSTAL X (Thompson et al., 1997
) version 1.8, a Windows interface for the multiple sequence-alignment software CLUSTAL V. Numbers of synonymous and non-synonymous nucleotide substitutions were calculated by using the DIVERGE software in the Genetics Computer Group (GCG) package (Devereux et al., 1984
), imposing the PamiloBianchiLi method (Pamilo & Bianchi, 1993
; Li, 1993
). The mean similarity between two aligned sequences was calculated and plotted by using the GCG PLOTSIMILARITY program (Devereux et al., 1984
). MEGA version 2.1 (Kumar et al., 2001
) was used to calculate nucleotide distances, using the method of Jukes & Cantor (1969)
for correction of superimposed substitutions. Phylogenetic analysis was done with the same software by using the neighbour-joining method (Saitou & Nei, 1987
) with 1000 bootstrap replicates to assess the robustness of the nodes. Genetic diversity (mean nucleotide distance between two randomly selected sequence variants) within and between isolates was estimated by the method of Lynch & Crease (1990)
. Statistical analysis of genetic diversity was performed by a Wilcoxon non-parametric test (Sokal & Rohlf, 1993
) using inter- and intra-isolate diversity values. Recombination events and identification of putative recombination junctions were detected by PHYLPRO version 0.8 (Weiller, 1998
).
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RESULTS |
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Evidence for recombination events
Low values of within-isolate genetic diversity were observed for the majority of the isolates in the three genomic regions (Table 1). Exceptions were found in some isolates (C6 in RdRp; AUSG5, MT48 and SY2 in HSP-70; and GR30, IL1, SS5 and SS15 in CP) whose patterns were composed of more than four bands, all showing a within-isolate genetic diversity higher than between-isolate diversity (data not shown), especially in the HSP-70 and CP genes. Moreover, phylogenetic analysis carried out to dissect relationships among clones originating from the latter isolates showed incoherent topologies (Fig. 3
a, b and c). For example, in the HSP-70 gene, some clones of isolate AUSG5 had smaller genetic distances from clones of other isolates (i.e. clones AUSG5-6 and SY2.4) than from clones originating from the parent isolate (Fig. 3b
). Moreover, some isolates (i.e. AUSG5) had two highly diverging variants showing high genetic distance values in two (HSP-70 and CP) of the three genomic regions analysed (Fig. 3a, b and c
). A similar population structure, previously described for CTV (Rubio et al., 2001
), could originate from mixed infection of two diverging viral variants that subsequently underwent recombination. A careful analysis of the phylogenetic trees relative to the three genomic regions showed an incongruent topology of clones AUSG5-2 and IL1-1 in the HSP-70 and CP genes, respectively (Fig. 3b and c
), as they did not cluster clearly with the other variants. Therefore, a systematic search for possible recombination events was performed by using PHYLPRO (Weiller, 1998
), a program that estimates the coherence of phylogenetic relationships in a set of aligned sequences. Putative recombination events are displayed in the form of a peak pointing downwards in a graph, which allows allocation of the point of recombination to a precise region. Based on this, evidence for recombination was identified in the HSP-70 and CP regions (Fig. 4
), but not in RdRp (data not shown). A new PHYLPRO analysis, done by excluding sequences with low phylogenetic correlation and introducing single sequences, identified ancestors of recombinant variants (data not shown), ultimately confirmed by analysis with the software PLOTSIMILARITY (Devereux et al., 1984
). This analysis suggested that the HSP-70 gene of AUSG5-2 originated from a recombination event between an AUSG5-3-like and an AUSG5-6-like variant around nt 370 (Fig. 5
a). A similar analysis suggested that the CP gene of IL1-1 originated from a recombination event between an IL1-2-like and an NIG3-like variant around nt 195 (Fig. 5b
). To determine whether these observed recombinant molecules originated from the in vitro template switching activity of reverse transcriptase (Negroni & Buc, 2001
) or from the Taq polymerase (Bradley & Hillis, 1997
), a negative control consisting of a mixture of total RNA of two different isolates whose SSCP patterns and sequences were known was included in the RT-PCR assay. DNA amplified from the mixture was cloned and 10 obtained clones were analysed by SSCP. Results using the primers LC1/LC2 (Fig. 6
a) and CP3U/CP3D (Fig. 6b
) for the HSP-70 and CP regions, respectively, showed that most of the clones had the same SSCP pattern and sequence as the two parental isolates. Sequencing of clones showing a non-parental pattern (i.e. clone 7 in Fig. 6
) did not reveal any in vitro recombination events.
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DISCUSSION |
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ACKNOWLEDGEMENTS |
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Received 23 June 2004;
accepted 2 October 2004.
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