1 CIRAD, UPR 75, Station de Neufchâteau, Sainte-Marie, F-97130 Capesterre Belle-Eau, Guadeloupe, French West Indies
2 CIRAD/UMR BGPI, TA 41/K, Campus International de Baillarguet, F-34398 Montpellier Cedex, France
3 UMR GD2P, INRA et Université Bordeaux 2, IBVM, Campus INRA de la Grande Ferrade, BP 81, F-33883 Villenave d'Ornon Cedex, France
Correspondence
Pierre-Yves Teycheney
teycheney{at}cirad.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AY729491AY729643 (RdRp sequences) and AY730729AY730758 (CP/3' NCR sequences).
Tables showing the names, genomic groups, origins and sequences generated from banana accessions are available as supplementary material in JGV Online.
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INTRODUCTION |
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Banana mild mosaic virus (BanMMV) is a recently characterized flexuous virus infecting bananas (Gambley & Thomas, 2001) and an unassigned member of the family Flexiviridae (Adams et al., 2004
). Banana (Musa spp.) is a widely cultivated monocotyledonous crop belonging to the family Musacae. Diploid, triploid or more rarely tetraploid cultivated banana varieties (cultivars) are parthenocarpic and often sterile, and are therefore mostly propagated vegetatively. They originate from two diploid Musa species, Musa acuminata Colla (AA genome) and Musa balbisiana Colla (BB genome) (Simmonds & Shepherd, 1955
; Stover & Simmonds, 1987
) and are classified in five main genomic groups designated AA, AAA, AAB, ABB and AAAA according to their genetic make-up. The 7352 nt, positive-sense, polyadenylated genomic RNA of BanMMV bears five open reading frames encoding a replication-associated protein with RdRp activity, three small proteins necessary for cell-to-cell movement within its host (TGBp1 to TGBp3) and a coat protein (CP). Vertical transmission by vegetative propagation is currently the only known means of propagation of this virus, since no biological vector has been identified nor has experimental mechanical inoculation on susceptible hosts been successful so far (Thomas et al., 1999
).
Here, the first analysis of the genetic variability of BanMMV genomic RNA is reported. The aim of the present study was to assess the molecular diversity of a virus not known to be transmitted from plant to plant in a vegetatively propagated crop. This was achieved through extensive cloning and sequencing of two distinct regions of the BanMMV genomic RNA.
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METHODS |
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Determination of BanMMV sequences.
Immunocapture of BanMMV particles was performed on banana leaf homogenates with purified anti-BanMMV IgGs (kindly provided by B. E. Lockhart, University of Minnesota, St Paul, USA) using the technique of Wetzel et al. (1992) with minor modifications (P.-Y. Teycheney and others, unpublished results).
For amplification of a portion of the ORF1 encoding the viral RdRp, RT-nested PCR with inosine-containing degenerated primers and Biotaq DNA polymerase (Eurobio) was performed on immunocaptured virus particles using the protocol, primers and conditions developed by Foissac et al. (2005). The two nested internal primers used target conserved motifs II and V near the active site of the polymerase (Fig. 1
) so that the amplified region (310 nt excluding the primers) contains conserved motifs III and IV of the RdRp (Koonin, 1991
; Koonin et al., 1991
). As such, this region is expected to correspond to one of the most conserved regions of the BanMMV proteins.
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Before the reverse transcription step, encapsidated RNAs were released from the immunocaptured particles by incubation at 95 °C for 2 min in 15 µl DEPC-treated water. First strand cDNAs were prepared using AMV reverse transcriptase (USB) according to the manufacturer's instructions. PCR primers were used at 10 pmol and PCR parameters were as follows: pre-incubation at 95 °C for 3 min; 35 cycles at 95 °C for 30 s, 56 °C for 1 min, 72 °C for 30 s; and a final elongation step at 72 °C for 10 min.
All PCR products were cloned into plasmid pGEM-T (Promega) and one to seven distinct cDNA clones per PCR product were used for sequencing. Sequencing was performed by Genome Express. All mutations were individually confirmed using original electrophoregrams.
To provide an evaluation of the mutations that might be introduced by the nested PCR method used to analyse the RdRp region, amplifications were performed on in vitro transcribed RNA targets and the PCR products obtained were cloned and sequenced as described above.
Nucleotide sequence analysis.
Multiple sequence alignments and initial phylogenetic reconstructions (neighbour-joining) were performed using the program CLUSTAL_X with randomized bootstrapping evaluation of branching validity (Thompson et al., 1997). Mean diversities, genetic distances (p-distances calculated on amino acid or nucleotide identity), NeiGojobori synonymous/non-synonymous substitution rates (Nei & Gojobori, 1986
) and Nei's Gst coefficient of differentiation (Nei, 1987
) were calculated using MEGA2 (Kumar et al., 2001
). Potential recombination events in the datasets were evaluated using GENECONV v.1.81 software, available at http://www.math.wustl.edu/
sawyer/geneconv/ (Sawyer, 1989
), following manual editing of the CLUSTAL_X alignments, when appropriate.
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RESULTS |
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All sequences were obtained from cDNAs amplified from immunocaptured virus particles, which ensures that only encapsidated sequences were analysed. A total of 154 sequences were obtained from the 68 banana accessions for the BanMMV RdRp region and 30 sequences were from 10 accessions for the BanMMV CP/3' NCR (Supplementary Tables S1 and S2 available in JGV Online). The absence of recombination events was verified in both datasets using the program GENECONV (Sawyer, 1989) (data not shown).
Within the RdRp dataset, all sequences are fully collinear, without indels, when compared to the reference BanMMV sequence (Gambley & Thomas, 2001). However, the coding capacity of three of the 154 sequences is affected by point mutations introducing in-frame stop codons. In the CP/3' NCR dataset, a similar situation was observed concerning the CP coding region: no indels are observed but mutations interrupting the reading frame are observed in two cDNA clones. The genomes containing such stop codon-introducing mutations are likely to produce non-functional proteins and, particularly in the case of the RdRp, to represent non-viable sequences unless they can be complemented by other sequence variants.
The 3' NCR presented a different situation (Fig. 2) with the simultaneous presence of highly conserved regions and three zones of indel polymorphism. Although such polymorphisms are frequent in the non-coding regions of RNA viruses, the situation reported here appears to indicate a high level of variability. In particular, the indel polymorphism region immediately following the stop codon varied in size from 1 to 20 nt, whereas that adjacent to the poly(A) tail varied in size from 2 to 4 nt.
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In order to evaluate the potential effects of the RT-nested PCR procedure used for analysis of the RdRp region on the sequence variability observed, control amplifications were performed on in vitro transcribed RNA targets. The RT-PCR products were cloned and 10 cDNA clones were sequenced. Sequence analysis revealed an overall mutation rate introduced by the RT-nested PCR procedure of 0·47 % (result not shown). Given the mean divergence between isolates (see below), this error rate can be estimated to contribute to a little less than 5 % of the total observed diversity. Pairwise divergence levels (nucleotide identity) were calculated for all sequences from the RdRp dataset. The genomic sequence from another member of the family Flexiviridae, Apple stem pitting virus (ASPV, genus Foveavirus; D21829), was added to the dataset in order to provide a comparative estimation of the divergence level against another virus species belonging to the same family. Pairwise values obtained between BanMMV sequences were 028·4 %, whereas those calculated between BanMMV sequences and ASPV were 34·740·8 %. The distribution of the pairwise percentages of divergence observed is shown in Fig. 3. There is a clear separation between the peaks corresponding to intra-BanMMV values (solid bars) and those corresponding to BanMMV/ASPV values (open bars). This pattern confirms that all the sequences obtained correspond to isolates of BanMMV. The highest level of divergence observed between BanMMV isolates (28·4 %) can be compared with the species discrimination criteria for the family Flexiviridae, which states that isolates represent distinct species when they share less than 72 % identical nucleotides between their complete CP or RdRp genes (Adams et al., 2004
).
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Overall, the ratio of non-synonymous to synonymous substitution rates (dN/dS), which gives a measure of the evolutionary constraints to variation (Kimura, 1980, 1983
), is in the same range for both BanMMV RdRp and CP complete datasets (Table 1
). This ratio is well below 0·1 in both cases, i.e. in the range of the lowest values reported for plant viruses (García-Arenal et al., 2001
; Chare & Holmes, 2004
). The dN/dS value calculated for the BanMMV CP dataset is, in particular, in the range of those calculated for CP sequences of other members of the family Flexiviridae (Chare & Holmes, 2004
). This result indicates that despite accumulating synonymous mutations to a high level, the genome of BanMMV seems to be under a level of evolutionary constraint similar to that of other plant viruses.
Very different situations were observed, however, between the various BanMMV sequences. This is illustrated in Fig. 4, which presents a plot of the pairwise percentage of divergence (identity) in the CP amino acid sequences against the pairwise percentage of divergence in the corresponding nucleotide sequences. While there is a general positive correlation between these two measures of divergence, variability in the association is strong and the correlation observed is poor (r2 of only 39 % for the best-fit linear regression), suggesting a complex relationship between synonymous and non-synonymous substitutions. For example, a similar divergence value of 18 % at the nucleotide level was observed for sequences showing 1·515·0 % amino acid divergence. Reciprocally, an amino acid sequence divergence value of 1·5 % was observed for pairs of isolates displaying 219 % nt divergence. Similar results were obtained when performing the same analysis on the RdRp dataset (data not shown).
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A significant increase in the dN/dS ratio was observed for the RdRp sequences from plants from which only closely related sequences were recovered, such as plant 44, which displayed a mean dN/dS value of 0·35 between the six sequences obtained (data not shown). This may reflect a relaxation of selection constraints, possibly due to trans-complementation effects between closely related variants present in the same host.
Inter-plant comparisons, which represent over 98 % of all pairwise comparisons, provided generally high divergence between sequences (Fig. 3). Nevertheless, a small proportion of comparisons (0·6 %) yielded low (below 2 %) divergence values. In two cases (sequences 1·1 and 40·6, sequences 8·4 and 22·3), identical sequences were even recovered from different plants. These results indicate that, in some instances, different plants share BanMMV sequences that are as closely related as those observed within individual plants.
Factors structuring the genetic diversity of BanMMV
Mean pairwise distance values obtained using subsets of the BanMMV RdRp dataset were further analysed in order to pinpoint possible factors that might influence the structure of the genetic diversity of BanMMV. Three structuring parameters were evaluated: individual host plant, host plant genomic group (AA, AAA, AAAA, AAB, ABB and TT) and geographical origin of host plant. For each parameter, relevant sequences of the RdRp dataset were pooled in subpopulations and the mean intra- and inter-subpopulation diversities were calculated, as well as the entire population diversity and Nei's Gst coefficient of differentiation between subpopulations (Nei, 1987). Sequences of the CP/3' NCR dataset were also used to estimate host plant influence on the genetic structure of the population. However, the CP/3' NCR dataset could not be used to estimate the effects of host plant genomic group or country of origin due to the low number of plants analysed. The results are shown in Table 2
. With each parameter analysed, mean inter-subpopulation diversity was found to be lower or within the range of intra-subpopulation diversity, indicating an overall low level of subpopulation differentiation. The fraction of the overall genetic diversity due to subpopulation differentiation was further evaluated using Nei's Gst parameter (Nei, 1987
). Relatively low values of 0·13 and 0·259 were calculated using the host plant genomic group and the host plant country of origin, respectively, as structuring parameters. This result indicates that the subpopulations defined by these two parameters show a low level of differentiation. However, in a few cases, phylogenetically supported clusters of plants originating from the same geographical area were observed (results not shown), resulting in reduced intra-subpopulation diversity values. Such a situation was encountered for isolates from Colombia (plants 87 and 88), Mayotte (plants 100 and 101) and Vietnam (plants 85, 86 and 89). In the majority of cases, however, sequences retrieved from plants with the same geographical origin did not cluster together, strongly suggesting that the diversity of BanMMV is poorly structured by the geographical origin of the host plants.
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Although not previously documented, the overall limited effect of host or geographical parameters on BanMMV populations could potentially result from plant-to-plant spread of BanMMV isolates within CIRAD's field collection. In order to obtain insights into a possible plant-to-plant transmission of BanMMV, the percentage of plants sharing RdRp sequence variants harbouring less than an arbitrary cut-off of 2 % nucleotide identity divergence were plotted against the distance separating these plants. This cut-off value was retained because it roughly corresponds to the variability observed within plants containing only closely related isolates and may, therefore, represent a conservative measure of the variability envelope of individual virus isolates. Fig. 5 shows the level of infection by identical or closely related variants as a function of the physical distance between plants sharing those variants (expressed as the number of intervening banana plants). A steep gradient was observed, indicating that closely located plants were more likely to share closely related variants than more distant ones.
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DISCUSSION |
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The results reported here show that the variability of BanMMV results mostly from the accumulation of synonymous mutations. This translates into a very high variability of the third nucleotide of the codons, with only 3 and 6 % of the third bases conserved in the RdRp and CP datasets, respectively. The third base variability contributes to 76 and 81 % of the total variability of the analysed regions of CP and RdRp genes, respectively. In contrast to other plant viruses in which conservation of the encoded proteins cannot fully account for the low overall molecular variability of the viral genome (García-Arenal et al., 2001, 2003
), the results reported here can be taken as an indication that little selection pressure is applied on BanMMV coding genomic sequences above that necessary for the conservation of the encoded proteins. This hypothesis is corroborated by observation of similar levels of amino acid conservation between pairs of isolates showing wide variations in their nucleotide sequences (Fig. 3
). Similarly, high variabilities and low dN/dS values have been reported for other members of the family Flexiviridae (Chare & Holmes, 2004
; Shi et al., 2004
), suggesting that this property might be widely shared by members of this family.
Despite the fact that the CP region exhibits a slightly reduced variability (particularly at the amino acid sequence level), the overall trends are similar in the two regions analysed. The relatively close dN/dS values observed (CP, 0·054; RdRp, 0·085) indicate that both regions are similarly constrained. These regions correspond to parts of their respective ORFs likely to be under stronger selection pressure than other regions of the ORFs. This is particularly true for the RdRp region, which is close to the viral polymerase catalytic domain and encompasses previously identified conserved motifs (Koonin, 1991). The results reported here are therefore likely to somewhat underestimate the whole genome variability of BanMMV.
The third functional region analysed in this study, the viral 3' NCR, is usually a highly conserved region of the genome of positive-sense RNA viruses since it frequently contains RNA motifs corresponding to the negative-strand RNA promoter necessary for virus replication. The present results indicate that although this region is more conserved than the RdRp and CP coding regions analysed, it still shows a high variability, including two blocks of extensive indel polymorphism. Gambley & Thomas (2001) postulated that contrary to the situation observed in most positive-sense RNA viruses, the BanMMV negative-strand promoter may be, in part, located further upstream than the 3' NCR, in the 3' end of the CP gene. However, the ACUAAA motif they identified as being possibly involved is not conserved between the isolates of the CP dataset (results not shown) and the region immediately around this putative motif is not significantly more conserved than the whole coding region analysed. It is, therefore, likely that the BanMMV negative-strand promoter is located in the 3' NCR and that the need to maintain a structure and function essential to virus replication accounts for the lower variability observed in that region, compared to the other regions analysed in this study. In this respect, it is worth noting that a 9 bp hairpin (in bold and underlined in Fig. 2
) is strongly conserved through co-variation at base-paired positions in almost all the 3' NCR sequences obtained.
When considering intra-plant diversity, two very contrasting situations were encountered: about half (43 %) of the plants analysed yielded only closely related sequence variants, with mean pairwise divergence values below 23 %, whereas other plants (57 %) contained two or more highly divergent isolates, resulting in much higher intra-plant diversity values (up to 21·8 % in the case of plant 30). Similar proportions of plants containing only closely related sequences (40 %) or divergent sequences (60 %) were observed when analysing the CP dataset, confirming that co-existence of widely diverging BanMMV isolates in a given Musa genotype is frequent.
The first situation probably reflects the heterogeneous nature of virus populations due to high error rates of viral RNA polymerases. Such a genetic structure comprising a major genotype or master sequence and a set of minor variants is well documented for bacterial, animal and plant viruses (Domingo & Holland, 1997; García-Arenal et al., 2001
). The second situation, in which highly divergent sequences are simultaneously detected in a single plant, is also frequently observed and might result from two non-exclusive factors: genetic divergence during long-term association of virus isolates with vegetatively propagated banana genotypes or plant-to-plant transfer of virus isolates. Genetic divergence could reflect either a lowering of selection pressures through complementation between co-infecting variants or, alternatively, an increase in positive selection created by the need to escape host defence mechanisms during persistent infections (Simmonds, 2004
; Tuplin et al., 2004
). Sequence-specific post-transcriptional gene silencing (Voinnet, 2005
; Waterhouse et al., 2001
), with its ability to counter-select all sequences close to the inducing molecule and, conversely, its inactivity against widely divergent variants, could potentially represent such a driving force in virus evolution. In this respect, it is noteworthy that analysis of the phylogenetic trees built from BanMMV RdRp and CP datasets (not shown) shows that the phylodynamic situation of BanMMV compares to that of other viruses causing persistent infections (Grenfell et al., 2004
).
The other possibility to explain the co-existence of widely divergent isolates in a single plant is plant-to-plant transfer of isolates, which has never been reported for BanMMV (Gambley & Thomas, 2001). However, two observations support the existence of horizontal spread of BanMMV isolates in the field. Firstly, identical RdRp sequences were retrieved from two independent pairs of plants. Secondly, the results presented in Fig. 5
are best explained if one postulates the existence of a steep gradient of dispersion of the virus between adjacent plants. These observations do not, however, provide any insight into the mechanism(s) underlying this plant-to-plant transfer, which could represent either an unsuspected consequence of cultural practices or an as yet undescribed vector-borne transmission. Further work is clearly needed to clarify this point.
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ACKNOWLEDGEMENTS |
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Received 22 May 2005;
accepted 7 July 2005.
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