1 Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Av. PH Rolfs s/n, 36571.000 Viçosa-MG, Brazil
2 Departamento de Fitopatologia/BIOAGRO, Universidade Federal de Viçosa, Av. PH Rolfs s/n, 36571.000 Viçosa-MG, Brazil
Correspondence
Elizabeth Fontes
bbfontes{at}ufv.br
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ABSTRACT |
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INTRODUCTION |
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The genome of the bipartite begomoviruses is split between two genomic components, designated DNA-A and DNA-B (Lazarowitz, 1992). Both components are organized into divergent transcription units separated by an intergenic region (IR) of about 200 bp, which contains the replication origin and two divergent promoters (Hanley-Bowdoin et al., 1999
). DNA-A has the potential to code for five gene products (AV1, AC1, AC2, AC3, AC4) and DNA-B encodes two gene products (BV1 and BC1). Genetic and biochemical studies of viral genes and proteins have provided insights into their function. The AV1 gene product or CP represents the coat protein (Kallender et al., 1988
), necessary for insect transmission (Azzam et al., 1994
). The AC1 protein or Rep (replication-associated protein) is a sequence-specific DNA-binding protein essential for replication of both DNA-A and DNA-B genomic components (Elmer et al., 1988
; Fontes et al., 1992
, 1994a
, b
). The AC2 gene product or TrAP is a trans-acting factor needed for the expression of both AV1 (coat protein) and BV1 genes (Sunter & Bisaro, 1992
). The AC3 protein enhances the accumulation of viral DNA (Sunter et al., 1990
) and is also designated REn (replication enhancer protein). The BV1 protein, also named nuclear shuttle protein (NSP), facilitates movement of the virus from the nucleus to the cytoplasm, whereas the BC1 protein functions as a classic viral movement protein (MP) as it promotes an increase in the size exclusion limit of the plasmodesmata (Sanderfoot & Lazarowitz, 1996
).
Begomoviruses are considered one of the largest and most successful groups of plant viruses that infect a wide range of crops, particularly in tropical and subtropical regions. They are responsible for numerous diseases of economically imporant crops, such as cassava, cotton, bean, pepper and tomato (Simone et al., 1990; Brown & Bird, 1992
; Polston & Anderson, 1997
; Moriones & Navas-Castillo, 2000
). Recently, diseases caused by begomoviruses have become an even greater threat to Brazilian agriculture (Ambrozevicius et al., 2002
; Ribeiro et al., 2002
) due to the introduction of a new biotype of the whitefly, Bemisia tabaci, that colonizes tomato plants with high efficiency (Ribeiro et al., 1998
) and to the high recombinogenic properties of the virus (Padidam et al., 1999
). Interspecies recombination events have probably contributed significantly to the diversity of begomoviruses and their emergence as economically important pathogens (Deng et al., 1997
; Zhou et al., 1997
; Fondong et al., 2000
).
In this investigation, we report the identification and characterization of new species and strains of tomato-infecting geminivirus that belong to the genus Begomovirus, provisionally designated Tomato crinkle leaf yellows virus (TCrLYV) and Tomato chlorotic mottle virus, isolate MG-Bt1 (ToCMV-[MG-Bt1]). Remarkably, the DNA-A of ToCMV-[MG-Bt1] retains a high degree of sequence conservation with other species from the western hemisphere but differs significantly in its biological properties. The recombinant nature of this DNA-A variant of ToCMV is discussed.
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METHODS |
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The DNA-A of TCrLYV was cloned by a PCR-based method. Fragments of the viral genomes were PCR-amplified using total DNA from infected tomato leaves and a set of begomovirus DNA-A-specific degenerate primers, as previously described (Rojas et al., 1993). The amplified 1·1 kb TCrLYV DNA-A fragment was cloned into pUC118 and sequenced. Sequence analysis of the cloned fragment confirmed that it had originated from a DNA-A and allowed the design of overlapping DNA-A-specific primers that were used to generate full-length ClaI clones of the genomic component. Attempts to clone the full-length ClaI A component were unsuccessful, using a diversity of PCR-amplification cloning strategies. Nevertheless, cloning of overlapping fragments of TCrLYV DNA-A allowed the sequencing of the entire TCrLYV DNA-A.
Sequence analysis.
Phylogenetic analysis was done on matrices of aligned sequences using the neighbour-joining and bootstrap (1000 replications) options of DNAMAN 3.0 software. The sources and GenBank accession numbers of geminivirus DNA-A and DNA-B sequences used are provided in the supplementary table (available at http://vir.sgmjournals.org).
Construction of infectious DNA clones.
Infectivity assays were performed with plasmids containing partial tandem repeats of the genomic components of the viruses under investigation. To construct a partial repeat of ToCMV-[MG-Bt1], the full-length SacI insert of pUFV290 (pToCMV-A) was transferred to the unique SacI site of pUFV310, which contained a 1·4 kb SacIXhoI fragment of pToCMV-A inserted into the SacI and SalI sites of pUC118. The resulting clone contains 1·45 copies of ToCMV-[MG-Bt1] DNA-A, flanked by identical sequences of the AC1 gene and was designated pToCMVA-1·45 (pUFV398). Tandemly arranged partial repeats of TCrLYV DNA-B were constructed by moving the full-length EcoRI insert of pUFV248 (pTCrLYV-B) into the unique EcoRI site of pUFV267, which contained a 900 bp EcoRIBamHI fragment of pTCrLYV-B. The resulting clone, pUFV393 (also designated pTCrLYVB-1.34), harbours 1·34 copies of TCrLYV DNA-B and has a duplicated origin of replication.
Plant inoculation and viral DNA detection.
Nicotiana benthamiana plants were inoculated with plasmids containing partial tandem repeats of viral DNA components by biolistic delivery as previously described (Schaffer et al., 1995). Between 1421 days post-inoculation, both inoculated and apical leaves were harvested. Total nucleic acid was extracted as described (Fontes et al., 1994a
) and viral DNA was detected by a PCR-based assay using DNA-A- or DNA-B-begomovirus specific primers (Rojas et al., 1993
) and/or Southern blot analysis using radiolabelled DNA-A or DNA-B probes.
Replication assays of viral DNA.
Callus cultures were initiated from the pith of tomato plants as previously described (Cascardo et al., 2000; Alvim et al., 2001
). Cell culture lines were generated by transferring 2 g of friable calli to 25 ml of medium [MS salts supplemented with 3 % (w/v) sucrose, 0·0001 % (w/v) thiamin.HCl, 0·01 % (w/v) inositol, 0·2 µg 2,4-dichlorophenoxyacetic acid ml-1, 1·32 mM KH2PO4]. The cell culture was established after four subcultures in liquid medium prior to replication assays. Protoplasts preparation, transfection conditions and DNA extraction were according to Fontes et al. (1994a
, b)
. Total DNA was isolated from protoplasts 48 h post-transfection, digested overnight with SacI and DpnI (ToCMV-[MG-Bt1]) or EcoRI and DpnI (TGMV DNA-A, TGMV DNA-B, TCrLYV DNA-B), separated on a 1 % (w/v) agarose gel, blotted and hybridized with 32P-labelled DNA-A and DNA-B specific fragments as described (Sambrook et al., 1989
).
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RESULTS |
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The complete nucleotide sequences of clones pUFV290 (ToCMV-[MG-Bt1] DNA-A), pUFV70+pUFV245+pUFV246 (TCrLYV DNA-A) and pUFV248 (TCrLYV DNA-B) were determined. The genome organization of their viral components was similar to other western hemisphere begomoviruses. The intergenic region separating the divergent transcription units on both components harbours the conserved 30 bp stemloop structure found in all geminiviruses and the conserved TAATATTAC nonanucleotide sequence (Fig. 1) that contains the nicking site for initiation of virion-sense DNA replication (Laufs et al., 1995
; Stanley, 1995
). The DNA-A of the viruses encodes one virion-sense (AV1) and four complementary-sense (AC1, AC2, AC3, AC4) ORFs and TCrLYV DNA-B encodes one virion-sense (BV1) and one complementary-sense (BC1) ORF (Table 1
). ToCMV-[MG-Bt1] DNA-A encodes an additional ORF, designated AC5, with the potential to encode a protein of 250 amino acid residues.
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The ToCMV-[MG-Bt1] DNA-A is most closely related to ToCMV-[BA-Se1] (92 % sequence identity) and may be considered a strain of this virus. In addition to exhibiting a high degree of sequence conservation, the two share near-identical intergenic regions (94 % identity) and identical putative AC1 binding motifs (TGTGAATGGTG) upstream of the TATA box (Fig. 1). Based on sequence conservation of their Rep (AC1) proteins (93 % identity at the amino acid level) and origin of replication, one may predict efficient AC1-mediated trans-replication of the heterologous DNA-B genomic component from these closely related isolates.
Phylogenetic analysis and recombination events among recently identified tomato-infecting begomoviruses from Brazil
Phylogenetic analyses based on nucleotide sequence conservation of the Geminiviridae family have demonstrated that members of the Begomovirus genus form clusters according to their geographical origin with distinct branches for viruses from the Americas, Asia and Africa (Padidam et al., 1999). The inclusion of recently characterized begomoviruses from Brazil in sequence comparison analyses reinforces a geographical origin-based relatedness, as they are clustered together under sub-branches of the viruses from the Americas (Fig. 2
). A similar relationship holds when the deduced amino acid sequences of AV1, AC1, AC2 and AC3 served as the basis for comparison (data not shown). In contrast, based on AC4 phylogeny, ToCMV-[MG-Bt1] segregates more closely with clusters of the monopartite begomoviruses TYLCV and ToLCV from Africa and Asia (Fig. 3
). This observation may be relevant as the C4 ORF from the monopartite begomoviruses and the AC4 ORF of bipartite begomoviruses appear not to be functional analogues (Jupin et al., 1994
; Pooma & Petty, 1996
; Rigden et al., 1994
).
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A second recombination event of particular interest might have occurred between ToRMV-[Ub1] and ToCMV-[BA-Se1], resulting in ToCMV-[MG-Bt1], which retains ToCMV-[BA-Se1]-like sequences corresponding to the AC1 coding region and intergenic region. This statistically significant recombination event might have enabled the recombinant ToCMV-[MG-Bt1] DNA-A to recruit the DNA-B of ToCMV-[BA-Se1] and therefore, it may be considered a DNA-A variant of the bipartite ToCMV species. In fact, sequencing of a 300 bp DNA-B fragment, which was PCR-amplified from the DNA samples used to clone ToCMV-[MG-Bt1] DNA-A, revealed that it was identical to the corresponding region of ToCMV-[BA-Se1] DNA-B (not shown). The apparent recombinant nature of ToCMV-[MG-Bt1] was further confirmed by using its proposed recombined genome segments, such as the ToRMV-[Ub1]-like sequence (positions 1001601 on the ToCMV-[MG-Bt1] genome), as the basis for phylogenetic analysis (Fig. 4B). This caused a variation in the relative position of ToCMV-[MG-Bt1] shown in Fig. 2
, as it segregated more closely with ToRMV-[Ub1].
Infectivity of ToCMV-[MG-Bt1] DNA-A in N. benthamiana
The biolistic method was used to inoculate N. benthamiana and tomato plants with ToCMV-[MG-Bt1] DNA-A or TCrLYV DNA-B alone. A mock-inoculated control was included in each experiment. DNA-A components of ToCMV-[MG-Bt1] and TCrLYV exhibited similar genomic organization (Table 1) and were closely related to bipartite begomoviruses (Fig. 2
) that require both components for systemic infection. In fact, N. benthamiana inoculated with TCrLYV DNA-B alone was not systemically infected and did not accumulate viral DNA (Table 2
; Fig. 5
A, lanes 1316). Likewise, N. benthamiana inoculated with either infectious cloned TGMV DNA-A or TGMV DNA-B alone did not develop a systemic infection and viral DNA was not detected in upper leaves (Table 2
; Fig. 5A
, lanes 912). In contrast, inoculation of ToCMV-[MG-Bt1] DNA-A alone induced a systemic infection in N. benthamiana. The results of these analyses for three independent experiments are presented in Table 2
. ToCMV-[MG-Bt1] DNA-A induced stunting, leaf crumpling and mottling in 90 % of inoculated plants on average. In all ToCMV-[MG-Bt1] DNA-A-inoculated plants DNA-A accumulation was detected in inoculated (Fig. 5A
, lane 5) and newly emerging leaves (lane 7). The absence of a contaminating DNA-B in inoculated plants was confirmed by the failure to detect viral DNA-B in infected tissues by PCR analysis (Fig. 5A
, lanes 6 and 8) and Southern blotting (Fig. 6
B, lane 4). Taken together, these results indicate that ToCMV-[MG-Bt1] DNA-A is able to systemically infect N. benthamiana in the absence of a cognate DNA-B. This result was also obtained when plasmid DNA-A was delivered by mechanical inoculation, albeit to a much lesser extent (2/24 inoculated plants), demonstrating that ToCMV-[MG-Bt1] DNA-A infection was not an artifact of the biolistic method of inoculation. Inclusion of a cognate DNA-B in the inoculation assays (Fig. 6
, lane 5) caused a slight increase in ToCMV dsDNA-A accumulation (Fig. 6A
, compare lanes 4 and 5), although it did not alter the symptomatology of ToCMV DNA-A infection. In contrast, ToCMV-[MG-Bt1] DNA-A was not infectious in tomato, as judged by the lack of symptom development and inability to detect viral DNA in emerging leaves (not shown).
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Replication assays in tomato protoplasts demonstrated that ToCMV-[MG-Bt1] DNA-A trans-replicated the TCrLYV DNA B (Fig. 7, lane 7) but not that of TGMV (lane 8). The compatibility between heterologous DNA-A and DNA-B was correlated with sequence conservation between their replication module (AC1 protein and origin of replication). In fact, the replication module of ToCMV-[MG-Bt1] is more closely related to that of TCrLYV (AC1, 82 % identity at amino acid level; Ori, 85 % nucleotide sequence identity) than to that of TGMV (AC1, 71 % identity; Ori, 34 % identity). Consistent with the replication data, both DNA components were detected in inoculated and upper leaves of ToCMV-[MG-Bt1] DNA-A+TCrLYV DNA-B-infected plants (Fig. 5B
, lanes 58). The accumulation of an apparently replication compatible DNA-B form in N. benthamiana did not alter the symptomatology of ToCMV-[MG-Bt1] DNA-A-mediated disease, nor did it increase the DNA-A level. No symptoms were observed in tomato plants inoculated with the ToCMV-[MG-Bt1] DNA-A+TCrLYV DNA-B combination. Nevertheless, a symptomless infection was detected in 25 % of inoculated tomato plants, as both DNA-A and DNA-B were detected in newly emerged leaves (not shown).
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DISCUSSION |
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The recent characterization of a new bipartite begomovirus from the eastern hemisphere, named Sri Lankan cassava mosaic virus (SLCMV), demonstrated that its DNA-A exhibits features of a monopartite geminivirus (Saunders et al., 2002). In contrast to SLCMV DNA-A, which encodes an analogue of the V2 ORF in monopartite begomoviruses that is involved in virus movement, the coding capacity of ToCMV-[MG-Bt1] DNA-A does not include a V2 homologue gene. It includes, however, an atypical complementary-sense AC5 ORF with the potential to encode a protein of 250 amino acid residues. This putative AC5 ORF has been found on emergent begomovirus genomes and at least for Watermelon chlorotic stunt virus (WmCSV), has been proved not to be essential, as disruption of its coding capacity caused no effect in the virus-mediated infection process (Kheyr-Pour et al., 2000
). It remains possible, however, that AC5 from ToCMV-[MG-Bt1] and its WmCSV counterpart are genetically distinct, since they do not retain significant sequence conservation (32 % identity at amino acid level). Genetic analysis of the AC5 ORF from ToCMV-[MG-Bt1] DNA-A will allow us to address this possibility.
An alternative explanation for the capacity of ToCMV-[MG-Bt1] DNA-A to move systemically and develop symptoms in N. benthamiana may be related to its AC4 protein, which is most closely related to the C4-deduced protein from eastern hemisphere monopartite begomoviruses (Fig. 3). While the function of the AC4 ORF from bipartite begomoviruses remains elusive (Pooma & Petty, 1996
), the C4 gene product from monopartite begomoviruses, such as TLCV and TYLCV, has been proven to be an important determinant of symptom severity and to affect the systemic movement of the virus (Jupin et al., 1994
; Rigden et al., 1994
). The sequence relationship between the AC4 ORF of ToCMV-[MG-Bt1] and its counterpart of monopartite begomoviruses may imply similar functions.
ToCMV-[MG-Bt1] DNA-A possesses a hybrid genome on which the replication compatible module (AC1 and origin of replication sequences) was probably donated by ToCMV-[BA-Se1] and the remaining sequences appear to have originated from ToRMV-[Ub1] (Fig. 4). This modular organization of ToCMV-[MG-Bt1] DNA-A may accommodate the argument that the recombinant regions can act as composite interacting modules to confer distinct biological properties to the virus progeny. In this case, one candidate for participating with AC4 in the architecture of functional complexes involved in replacing DNA-B-encoded movement functions would be the coat protein. In fact, the coat protein from monopartite begomoviruses participates actively in the systemic movement of the virus (Ridgen et al., 1993
; Wartig et al., 1997
). Likewise, in non-adapted hosts, bipartite begomovirus-mediated systemic infection requires a functional coat protein (Pooma et al., 1996
). Although we do not yet know the effective contribution, if any, of interacting recombinant modules to the pathological properties of ToCMV-[MG-Bt1] DNA-A, such a model remains an attractive hypothesis that may explain the unique mechanism of ToCMV-[MG-Bt1] DNA-A-mediated disease in a permissive host. Genetic analysis of ToCMV-[MG-Bt1] recombinant DNA-A is currently in progress to investigate whether the predicted AC4 ORF performs similar function to the closely related C4 ORF from monopartite begomoviruses and to determine the contribution of the coat protein and/or AC5 ORF to virus pathogenesis.
We have also characterized a distinct tomato-infecting geminivirus, TCrLYV, that may be considered a new species of the Begomovirus genus, on the basis of DNA-A sequence identity (up to 84 % identity with ToCMV-[BA-Se1] DNA-A) and difference in phenotype when compared to the most closely related virus, ToCMV-[BA-Se1]. Nevertheless, TCrLYV DNA-B was efficiently trans-replicated by ToCMV DNA-A, isolate MG, indicating that TCrLYV does not fulfil all requirements for its classification as distinct virus, according to the current criteria that dictate geminivirus taxonomy. In fact, DNA-A-mediated trans-replication of heterogenomic DNA-B components is generally limited to isolate/strains of a particular virus. Several exceptions in the literature indicate, however, that this criterion can not be considered as absolute for the taxonomic classification of distinct species of Begomovirus (Gilbertson et al., 1993; Frischmuth et al., 1997
).
Replication assays in tomato protoplasts indicated that compatibility between heterologous DNA-A and DNA-B was correlated with sequence conservation of the replication module (AC1 and replication origin) of the viruses under investigation, as would be predicted from previous studies of AC1 specificity for replication origin recognition (Fontes et al., 1992, 1994a
, b
; Lazarowitz et al., 1992
; Jupin et al., 1995
; Chatterji et al., 2000
). In fact, TCrLYV DNA-B was trans-replicated by ToCMV-[MG-Bt1] DNA-A, but not by TGMV DNA-A in tomato protoplasts. Nevertheless, inoculation of N. benthamiana plants with TGMV DNA-A+TCrLYV DNA-B induced mild localized symptoms. We consider it unlikely that intermolecular recombination had occurred to drive AC1-mediated replication of the heterogenomic DNA-B, because sequencing of PCR-amplified fragments from total DNA of symptomatic leaves indicated that the DNA-B progeny kept its original replication origin sequence. In contrast to tomato protoplasts, the permissive nature of the N. benthamiana host may allow high levels of viral gene expression, which may compensate for a defective interaction between AC1 and origin of replication to support low levels of DNA-B replication. In fact, replication assays of a TGMV-derived replicon in tobacco protoplasts have demonstrated that AC1 is able to overcome a mutation in its high-affinity binding site if AC3 is provided in trans (Fontes et al., 1994b
). Therefore, the sequence-specific requirement for AC1-mediated trans-replication may vary to different extents depending on the efficiency of viral gene expression and hostvirus interactions.
In summary, we have identified and characterized two distinct tomato-infecting geminiviruses and presented evidence that a DNA-A variant of ToCMV exhibits unique pathological properties, despite its similar genomic organization to the DNA-A of western hemisphere bipartite geminiviruses. Our results provided further support for the significant role of recombination in begomovirus evolution, diversity and emergence as economically important pathogens. Until recently, TGMV was the only geminivirus known to be associated with tomato plants grown in Brazilian territory and was never considered a serious threat to the Brazilian agriculture. However, geminivirus-associated epidemics are currently threatening tomato production in Brazil, with the proliferation of new virus species. The Brazilian tomato-infecting geminiviruses described here and by others carry recombinant regions on their genome that are likely to be responsible for the more aggressive nature and enhanced fitness of the recombinant progenies.
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ACKNOWLEDGEMENTS |
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Received 16 August 2002;
accepted 18 November 2002.