JOURNAL OF VIROLOGY, Aug. 1996, p. 5430–5436 0022-538X/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 70, No. 8
Increased Pathogenicity in a Pseudorecombinant Bipartite Geminivirus Correlates with Intermolecular Recombination YU-MING HOU
AND
ROBERT L. GILBERTSON*
Department of Plant Pathology, University of California, Davis, California 95616 Received 30 November 1995/Accepted 9 May 1996
Most whitefly-transmitted geminiviruses possess bipartite DNA genomes, and this feature may facilitate viral evolution through pseudorecombination and/or recombination. To test this hypothesis, the DNA-A and DNA-B components of the geminiviruses bean dwarf mosaic virus (BDMV) and tomato mottle virus (ToMoV) were exchanged, and the resultant pseudorecombinants were serially passaged through plants. Both pseudorecombinants were infectious in Nicotiana benthamiana but induced attenuated symptoms and had reduced DNA-B levels. Serial passage experiments revealed that the BDMV DNA-A plus ToMoV DNA-B pseudorecombinant could not be maintained beyond three passages. In contrast, the ToMoV DNA-A plus BDMV DNA-B pseudorecombinant was maintained during serial passage through N. benthamiana and Phaseolus vulgaris and, after three to five passages, became highly pathogenic. Furthermore, the increased pathogenicity of this pseudorecombinant was consistently associated with an increased level of DNA-B, which eventuated in equivalent levels of both components. Sequence analysis of the DNA-B component of the more pathogenic pseudorecombinant revealed that intermolecular recombination had taken place in which most of the BDMV DNA-B common region was replaced with the ToMoV DNA-A common region. This recombinant DNA-B component, which contained the ToMoV origin of replication, was the predominant DNA-B component associated with the more pathogenic pseudorecombinant. These results provide the first demonstration of recombination between distinct bipartite geminiviruses and establish that the bipartite genome can facilitate viral evolution through pseudorecombination and intermolecular recombination. However, these pseudorecombinants were less pathogenic and had reduced DNA-B levels compared with the parental viruses. In this study, these pseudorecombinant geminiviruses were passaged through plants to determine their stability. We report here that serial passage of one of the pseudorecombinants resulted in the emergence of a highly pathogenic geminivirus with a recombinant genome.
Geminiviruses are a group of plant viruses that possess circular single-stranded (ss) DNA genomes encapsidated in small (18 by 30 nm) twinned icosahedral virions (reviewed in reference 38). In nature, geminiviruses are vectored by various leafhopper species or by whiteflies (Bemisia tabaci Genn.). Most of the whitefly-transmitted geminiviruses have divided genomes, composed of two ;2.6-kb ssDNAs designated DNA-A and DNA-B. The two DNA components share no sequence similarity except for an ;200-nucleotide (nt) noncoding region referred to as the common region (CR), which contains the viral replication origin (10, 20, 30). Essential viral functions are partitioned between the two components, with replication and encapsidation functions encoded by DNA-A and movement functions encoded by DNA-B (reviewed in reference 38). Geminiviruses are one of the rapidly emerging groups of plant viruses (4), which can be attributed to various factors, including increased insect vector populations and/or rapid viral evolution. However, while it has been speculated that geminiviruses have the capacity to evolve rapidly in response to changes in their environment (e.g., alterations in cropping systems and/or population dynamics of insect vectors), there have been few studies documenting geminivirus evolution. We have hypothesized that the bipartite genome might facilitate the evolution of whitefly-transmitted geminiviruses through pseudorecombination and recombination (14), mechanisms well established for evolution of multipartite RNA viruses (reviewed in references 18 and 33). Evidence supporting this hypothesis was provided by the finding that infectious pseudorecombinants could be made by exchanging the DNA components of two bipartite geminiviruses, bean dwarf mosaic (BDMV) and tomato mottle (ToMoV) (14).
MATERIALS AND METHODS Virus clones. Full-length infectious clones of BDMV (DNA-A in pBDA1 and DNA-B in pBDB1) and ToMoV (DNA-A in pTFA-1 and DNA-B in pTFB-1) have been previously described (12, 14). Inoculation of Nicotiana benthamiana with cloned viral DNAs and virus transmission. N. benthamiana plants at the five-to-seven leaf stage were inoculated with various combinations of DNA-A and DNA-B monomers excised from their respective recombinant plasmids (;5 mg/component) by rub inoculating celitedusted leaves with a pestle (14). In some cases, a protoplast bioassay was used to prepare inocula for rub inoculation (27, 28). In this assay, the cloned DNA-A and DNA-B monomers were transfected into N. tabacum protoplasts, and the protoplasts were maintained for 5 days in the dark at room temperature. Approximately 2 3 106 cells were homogenized in ice-cold 0.1 M potassium phosphate buffer (pH 8.0) in a tissue grinder, and the resulting suspension was rub inoculated onto N. benthamiana leaves. Virus sap transmission to N. benthamiana, Phaseolus vulgaris cv. ’Topcrop’ and Lycopersicon esculentum cv. ’Florida MH-1’ plants was carried out by grinding young, systemically infected leaves in ice-cold 0.1 M potassium phosphate buffer (pH 8.0) with a mortar and pestle. Sap was rubbed onto celite-dusted leaves of N. benthamiana (five- to seven-leaf stage), P. vulgaris (one-half to three-quarters expanded primary leaves), and L. esculentum (one- to two-true-leaf stage) with a pestle. Plants were maintained in a controlled environment chamber (250 mmol m22 s21 photosynthetically active radiation, 16 h/day, 308C). Fourteen to twentyone days postinoculation, plants were visually assessed for symptom development. Detection of geminivirus DNA by Southern blot hybridization and PCR. DNA manipulations were performed according to standard protocols (2, 21). For Southern blot hybridization analysis, 0.25 g of leaf tissue (generally taken from young, systemically infected leaves) was ground in 1.4 ml STE buffer (50 mM Tris pH 8.0, 100 mM NaCl, 10 mM EDTA) with 10% sodium dodecyl sulfate (SDS) and extracted with an equal volume of phenol-chloroform, and total nucleic acids were precipitated with ethanol. Total nucleic acids were recovered by centrifugation (10,000 3 g for 10 min) and resuspended in TE buffer. Approximately 1
* Corresponding author. Phone: 916-752-3163. Fax: 916-752-5674. E-mail:
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the dideoxynucleotide chain termination method with Sequenase (United States Biochemical). DNA sequences were assembled and analyzed with the software of the Genetics Computer Group, University of Wisconsin-Madison (5). Relationships among DNA sequences were determined by using the BESTFIT, GAP, and PILEUP programs.
RESULTS
FIG. 1. (A through D) Symptoms in N. benthamiana. (A) Mild-symptom phenotype for the first passage of the TA1BB pseudorecombinant (plants infected with the cloned DNA components). (B) Severe-symptom phenotype for the fifth passage of the TA1BB pseudorecombinant. (C) Epinasty and leaf crumpling caused by BDMV. (D) Epinasty caused by ToMoV. (E) Severesymptom phenotype for the 10th passage of the TA1BB pseudorecombinant in P. vulgaris.
mg of total nucleic acids was fractionated in a 1% agarose gel in Tris-acetateEDTA buffer and transferred to Hybond N1 nylon membranes (Amersham). Blots were hybridized with either a DNA-A-specific probe, which included PCRamplified BDMV and ToMoV DNA-A fragments (nt 475 to 1978), or a DNAB-specific probe, which included PCR-amplified BDMV and ToMoV DNA-B fragments (nt 450 to 2040). DNA probes were labeled with [a-32P]dATP by random priming according to the manufacturer’s instructions (Ambion). DNA extraction, PCR conditions, and degenerate primers for whitefly-transmitted geminiviruses have been previously described (15, 32). To amplify DNA-A and DNA-B fragments, primer PAL1v1978 paired with PAR1c475, and PBL1v2040 paired with PBV1c1304 (59-TTATAGATCTTCAACCGATATAA TC-39) or PBV1c703 (59-CTCATGTATGCGTTGGGCT-39) were used, respectively. PCR-amplified DNA fragments were cloned using the TA Cloning System (Invitrogen). DNA sequencing and sequence analysis. DNA sequencing was done by using
Passage of a pseudorecombinant geminivirus through plants leads to the appearance of a more pathogenic virus. The infectious pseudorecombinants made between BDMV and ToMoV are referred to hereafter as BA1TB (BDMV DNA-A 1 ToMoV DNA-B) and TA1BB (ToMoV DNA-A 1 BDMV DNA-B). Both pseudorecombinants induced attenuated symptoms in systemically infected N. benthamiana compared to those induced by the parental viruses. The stability of the pseudorecombinants was investigated using serial passage through plants. In these experiments, mechanical transmission was used because vector transmission cannot be conducted due to concerns related to viral containment. Because BDMV and ToMoV are sap transmissible (13, 14, 22), the capacity of the pseudorecombinants to infect N. benthamiana, P. vulgaris, and L. esculentum was determined by using extracts prepared from infected N. benthamiana. Based on symptom development and/or PCR analyses, BA1TB was transmitted only to N. benthamiana, and infected plants showed very mild symptoms or were symptomless. In contrast, TA1BB was transmitted to N. benthamiana and P. vulgaris, and infected plants showed mild but obvious symptoms that included leaf epinasty and crumpling (Fig. 1A). In all cases, the pseudorecombinants were transmitted at low frequencies (data not shown). These results established suitable hosts for the serial passage experiments and that, for sap transmission, TA1BB has a wider host range than BA1TB. Sap transmission was used to serially passage TA1BB through N. benthamiana and P. vulgaris. The inoculum for the initial transmission was prepared by using systemically infected leaves from an N. benthamiana plant infected with the cloned DNA components, and this plant was considered the first passage. In initial passages through N. benthamiana, TA1BB consistently induced mild symptoms that were indistinguishable from those observed in plants infected with the cloned DNA components. In initial passages through P. vulgaris, TA1BB induced leaf mottling and distortion, but not the degree of leaf distortion, crumpling, and stunting associated with BDMV infection. However, beginning with the fifth passage, symptoms induced in N. benthamiana and P. vulgaris by TA1BB became noticeably more severe, and the frequency of sap transmission increased from 17 to 33% in initial passages to nearly 100% (data not shown). In N. benthamiana, these symptoms included epinasty, chlorotic flecks, and severe distortion in systemically infected leaves and stunted and distorted plant growth (Fig. 1B). These symptoms were similar in severity but not identical in appearance to those induced by BDMV and were more severe than those induced by ToMoV (compare Fig. 1B, C, and D). In P. vulgaris, the severe symptoms included striking mosaic in older leaves and distortion and epinasty in newly emerged leaves (Fig. 1E), with the latter symptoms appearing to be similar to those induced by BDMV. The severe-symptom phenotype persisted in both hosts through five additional passages (Table 1). In another experiment, an extract prepared from transfected protoplasts was used to infect N. benthamiana to provide the initial inoculum for serial passage of TA1BB through P. vulgaris. Protoplast transfection provides a more efficient inoculation procedure than rub inoculation of monomeric DNAs (27). The results of this experiment were consistent with those observed in the first P. vulgaris experiment,
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TABLE 1. Disease symptom phenotypes, levels of viral DNA components, and CR sequences of the DNA-B component determined for the ToMoV DNA-A1BDMV DNA-B pseudorecombinant during serial passage through P. vulgaris and N. benthamiana Level of a : Expt
Host
1
P. vulgaris
2
N. benthamiana
3
P. vulgaris
Passage
1 3 5 8 10 1 3 5 8 10 1 3 5 10
DNA-B CRb
Symptoms
Mild Mild Severe Severe Severe Mild Mild Severe Severe Severe Mild Severe Severe Severe
DNA-A
DNA-B
Medium Medium High High High Medium Medium High High High Medium High High High
Low Low High High High Low Low High High High Low High High High
BDMV DNA-B BDMV DNA-B ToMoV DNA-A NDc ToMoV DNA-A BDMV DNA-B ND ToMoV DNA-A ToMoV DNA-A ToMoV DNA-A BDMV DNA-B ToMoV DNA-A ToMoV DNA-A ND
a
Levels of the DNA components were estimated based on the relative amount of PCR-amplified fragments. The DNA-B CR sequence was determined from a PCR-amplified fragment. c ND, not determined. b
except that the severe symptoms appeared after three passages. Taken together, the results of these experiments suggest that, during serial passage through plants, a change occurred in the TA1BB pseudorecombinant that resulted in an increase in pathogenicity and infectivity (frequency of sap transmission). Similar experiments were conducted in which BA1TB was serially passaged through N. benthamiana. Inoculum was prepared from N. benthamiana infected with the cloned DNA components (one experiment) or extracts prepared from transfected protoplasts (two experiments). In all three experiments, BA1TB was not maintained beyond three passages, and infected plants showed very mild symptoms or were symptomless (data not shown). The severe-symptom phenotype of TA1BB is associated with an increased level of DNA-B. During the serial passage of TA1BB, viral DNA components in newly emerged leaves of infected plants ;14 days after inoculation were detected by PCR and/or Southern blot hybridization analyses. In plants infected with cloned DNA components or extracts prepared from transfected protoplasts, DNA-B levels were reduced compared to those of DNA-A (Fig. 2 and 3 and Table 1). In early serial passages in which TA1BB induced mild symptoms, DNA-B levels remained low (Fig. 3 and Table 1). However, beginning with the passage in which the severe-symptom phenotype was observed, increased levels of DNA-B, which were equivalent to those of DNA-A, were detected in infected plants by PCR and Southern blot hybridization analyses (Fig. 2 and 3 and Table 1). Southern blot hybridization analysis also revealed slightly elevated DNA-A levels in plants with the severe-symptom phenotype (Fig. 3). The elevated levels of DNA-A and DNA-B were maintained throughout subsequent serial passages (Fig. 3 and Table 1). An intermolecular recombination event is associated with the increased pathogenicity of TA1BB. Because DNA-B levels had increased in plants with the severe-symptom phenotype, it was reasoned that the change in TA1BB may have occurred in the DNA-B component and, more specifically, in the CR, which contains the origin of replication. Therefore, the TA1BB DNA-B CR sequence was determined from selected infected plants during the course of the three serial passage experiments (Table 1). An ;1.3-kb DNA-B fragment containing the CR was amplified by PCR with primers PBL1v2040, a
degenerate primer that anneals at the 59 end of the BC1 gene (32), and PBV1c703, which anneals at the 59 end of the BV1 gene of BDMV and ToMoV. The PCR-amplified fragments were cloned, and the CR sequence was determined by using primers designed based on the ToMoV/BDMV CR sequence (59-GGCATTTTTGTAATAAGA-39), the BDMV hypervariable region (15) sequence (59-TGTTATTGGTGCACTGGTACTT39), and the ToMoV CR sequence (59-GAATGAGCAAGTTT GAGAAG-39). Analysis of CR sequences of the TA1BB DNA-B components from plants showing the severe-symptom phenotype revealed that, in all plants examined, most of the BDMV DNA-B CR had been replaced with the ToMoV DNA-A CR (Fig. 4 and Table 1). In contrast, the BDMV DNA-B CR sequence was found in plants with the mild-symptom phenotype. In the first P. vulgaris experiment, the CR sequences were determined for eight DNA-B clones obtained from a severe-symptom phenotype plant from the 10th pas-
FIG. 2. Ethidium bromide-stained agarose gel of DNA-A (A) and DNA-B (B) fragments amplified by PCR from N. benthamiana infected with the TA1BB pseudorecombinant from the first and fifth passages.
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FIG. 3. Southern blot hybridization analysis of total nucleic acids extracted from P. vulgaris infected with the TA1BB pseudorecombinant from the first, third, fifth, eighth, and tenth passages and from noninoculated (N) and BDMVinfected (BD) plants (the lower levels of DNA-A and DNA-B in the BDMVinfected leaves are due to the older age of the plant). Blots were probed with specific probes for DNA-A (A) or DNA-B (B). Positions of open circular dsDNA (oc), supercoiled dsDNA (sc), and ssDNA (ss) are indicated.
sage, and all were the ToMoV DNA-A CR. These results establish that, during passage through plants, the TA1BB DNA-B component had lost the BDMV DNA-B CR and acquired the ToMoV DNA-A CR and that the recombinant DNA-B component had become predominant in plants with the severe-symptom phenotype. The CR and flanking sequences of the recombinant DNA-B components examined in all three experiments were identical,
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indicating that in each case a similar recombination event had occurred. It was not possible to identify the precise nucleotides at which the recombination occurred, because the 59 and 39 ends of the BDMV and ToMoV CRs are identical (14) (Fig. 5). However, the recombination sites were mapped within 18and 24-nt sequences at the 59 and 39 ends of the recombinant DNA-B CR, respectively (Fig. 4). Furthermore, the 59 end of the recombinant DNA-B CR retained the first nucleotide of the BDMV DNA-B CR (T), and lacked the first two nucleotides (GG) of the ToMoV DNA-A CR, indicating that the recombination occurred after the start of the BDMV DNA-B CR (Fig. 4). The 39 end of the recombinant DNA-B CR lacked the last two nucleotides (GC) of the ToMoV DNA-A CR sequence and retained the last 20 nucleotides of the BDMV DNA-B CR, indicating that the recombination occurred before the end of the BDMV DNA-B CR (Fig. 4). DISCUSSION To test the hypothesis that the bipartite genome of whiteflytransmitted geminiviruses can facilitate viral evolution, two infectious pseudorecombinants (BA1TB and TA1BB) were serially passaged through plants. Both pseudorecombinants were phenotypically different from the parental viruses (BDMV and ToMoV) in that they induced attenuated disease symptoms and had reduced DNA-B levels. On the basis of these properties, it was reasoned that the pseudorecombinants were less viable than the parental viruses and that the appearance of variants with enhanced viability could be induced under suitable selection pressure. Indeed, serial passage of the TA1BB pseudorecombinant through P. vulgaris and N.
FIG. 4. Alignment of common region and flanking sequences for the ToMoV DNA-A component, the BDMV DNA-B component, and the DNA-B component of the TA1BB pseudorecombinant from the 1st (1B), 3rd (3B), 5th (5B), and 10th (10B) passages through P. vulgaris. Uppercase and lowercase letters represent CR and flanking sequences, respectively, and the ToMoV CR sequence is in boldface type. Asterisks indicate nucleotide differences between the ToMoV and BDMV sequences. The loop sequence in the potential stem-loop structure in the CR is underlined, and the recombinational hotspot within the loop is shown with arrowheads. Sequences containing the recombination junctions of the recombinant TA1BB DNA-B component (5th and 10th passages) are shown in italics and by brackets.
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FIG. 5. CR sequence alignments for the BDMV DNA-A and ToMoV DNA-B pseudorecombinant (A) and the ToMoV DNA-A and BDMV DNA-B pseudorecombinant (B). The BDMV and ToMoV CRs are 187 and 180 nt, respectively. Direct and indirect repeats are shown by thick and thin brackets, respectively, and the potential stem-loop structure is indicated with the stem sequence being underlined and the loop sequence shown in italics. The divergent region identified between the CR sequences is shown in boldface type.
benthamiana led to the appearance of a recombinant virus with equivalent levels of both DNA components and increased pathogenicity. The strong correlation found between DNA-B level and pathogenicity of the TA1BB pseudorecombinant extends upon findings of studies with the geminiviruses African cassava mosaic virus (ACMV) (24) and tomato golden mosaic virus (30). Furthermore, the increase in DNA-B level in the recombinant TA1BB virus also was associated with an overall increase in viability. Taken together with the fact that the DNA-B level of the nonviable BA1TB pseudorecombinant did not increase during serial passage, these results suggest that there is a tendency for a viable bipartite geminivirus to maintain similar levels of both DNA components. This is consistent with previous observations of similar levels of DNA-A and DNA-B in plants infected with naturally occurring bipartite geminiviruses (7, 17). Additionally, the results of this study establish that the BDMV movement proteins, which are encoded by the DNA-B component (25), are capable of mediating the efficient systemic spread of the ToMoV DNA-A component. Although additional genotypic changes may have occurred in the recombinant TA1BB virus, the replacement of most of the BDMV DNA-B CR with the ToMoV DNA-A CR was probably responsible for the increase in DNA-B level, because the CR contains the viral origin of replication. It was previously hypothesized that the low level of DNA-B in the TA1BB pseudorecombinant was due to the inefficient interaction of the ToMoV replication-associated (rep) protein with the BDMV DNA-B CR (14). It has now been established that the CR contains virus-specific elements (i.e., high-affinity binding sites) required for the efficient binding of the rep protein (9, 11), the only geminivirus-encoded protein absolutely essential for replication (6). Thus, the recombinant DNA-B component
contained the ToMoV rep protein-binding sites, allowing for more efficient replication mediated by the ToMoV rep protein and providing a selective advantage for this component. Our results also indicate that additional elements within the CR are required for efficient replication, because the high-affinity binding sites (direct repeats) and indirect repeats are nearly identical in the CRs of the pseudorecombinants (Fig. 5) (1, 8, 10). To identify these elements, alignments of the BA1TB and TA1BB CR sequences were generated with the GAP program of the University of Wisconsin Genetics Computer Group (5) (Fig. 5). In both alignments, a highly divergent region was identified between the high-affinity binding sites and the potential stem-loop structure (Fig. 5). This region may contain an additional specificity determinant(s) important for efficient replication. Further experiments are needed to determine the underlying basis for inefficient DNA-B replication in the BDMV/ToMoV pseudorecombinants. The recombination in TA1BB is the first example of intermolecular recombination between two bipartite geminiviruses. This clearly reflects the close relationship between BDMV and ToMoV and their capacity to form infectious pseudorecombinants (8, 14). Intermolecular recombination between DNA components of individual bipartite geminiviruses has been previously reported for ACMV (7, 31, 36) and bean golden mosaic virus (BGMV) (3). In these studies, a recombinational hotspot was identified within the conserved nonanucleotide sequence (TAATATTAC) in the loop structure of the CR (3, 7, 31). This nonanucleotide sequence has been shown to contain the nicking site (TACC) for initiating ssDNA replication via a rolling circle mechanism (16, 19, 34, 37). Although the precise location of the ToMoV DNA-A/BDMV DNA-B recombination could not be identified, the 39 junction of the recombinant DNA-B was mapped to a region that contained this recombinational hotspot (Fig. 4). This suggests that the ToMoV DNA-
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A/BDMV DNA-B recombination may have occurred by a mechanism similar to that of ACMV and BGMV and that recombination occurs during ssDNA replication. In this scenario, the ToMoV replication complex (including at least the ToMoV rep protein and a host DNA polymerase) binds to the CR of the BDMV DNA-B double-stranded replicative form and initiates ssDNA replication at the nicking site. Before completing the replication of the BDMV DNA-B ssDNA, the replication complex switches templates to the ToMoV DNA-A component at the identical 18-nt sequence located at the 59 end of the CR and completes ssDNA replication. This would result in the replacement of most of the BDMV DNA-B CR with the ToMoV DNA-A CR. Alternatively, the CR replacement may have occurred via replication-independent recombination between the ToMoV DNA-A and BDMV DNA-B double-stranded replicative forms, with the identical 59 and 39 CR sequences serving as crossover points. Several lines of evidence established differences between the BA1TB and TA1BB pseudorecombinants. The TA1BB pseudorecombinant was considerably more pathogenic, had a broader host range, and underwent intermolecular recombination. These results establish that pseudorecombination between BDMV and ToMoV is an asymmetric phenomenon that involves more than the simple reciprocal exchange of DNA components. This asymmetry cannot be attributed solely to host factors, because N. benthamiana is a common host for BDMV and ToMoV. Asymmetry also was reported for pseudorecombinants made between type II BGMV strains from Guatemala (BGMV-GA) and the Dominican Republic (BGMV-DR), in which wild-type symptoms were observed in P. vulgaris infected with BGMV-GA DNA-A and BGMV-DR DNA-B, whereas mild symptoms were observed in plants infected with the reciprocal combination (8). In contrast, pseudorecombinants made between tomato golden mosaic geminivirus or ACMV strains did not show attenuated or intermediate phenotypes (23, 40). Taken together, these results suggest that pseudorecombination is a complex phenomenon that involves interactions among virus- and host-encoded factors as well as viral DNA components. Consistent with the asymmetry found for the BDMV/ToMoV pseudorecombinants was the failure of more viable BA1TB variants to appear under the selection pressure of serial passage. The inability of BA1TB to be maintained during passage or to have increased DNA-B levels suggests that intermolecular recombination did not occur. The CR alignments revealed no differences in the putative recombination junctions for the BA1TB or TA1BB pseudorecombinants, indicating a similar potential for recombination. Moreover, on the basis of results of protoplast assays, the BDMV and ToMoV rep proteins are similar in their ability to replicate the ToMoV DNA-B and BDMV DNA-B components, respectively (17). This suggests that the failure to observe recombination in BA1TB was not due to a deficiency in ToMoV DNA-B replication mediated by the BDMV rep protein. It is possible that a critical number of cells need to be infected in order to reach a threshold DNA level necessary to achieve recombination and that this threshold was not reached for the BA1TB pseudorecombinant. In summary, the results of this study suggest that there is selection pressure for bipartite geminiviruses to maintain similar levels of both DNA components and support the hypothesis that pseudorecombination and recombination are mechanisms for geminiviral evolution in nature. This type of evolution may explain why bipartite geminiviruses are rapidly emerging, particularly in areas where mixed infections of related geminiviruses are found, such as in tomatoes in Mexico
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(4, 27) and India (26) and in squash in California (29). The hybrid DNA-A component of pepper huasteco geminivirus (39) and hybrid genome of beet curly top geminivirus (35) may have arisen by a similar mechanism. ACKNOWLEDGMENTS We thank Virginia M. Ursin and Steve D. Daubert for critical reading of the manuscript, Jeff Hall for assistance in preparation of figures, and Epaminondas J. Paplomatas and Douglas P. Maxwell for helpful discussions and encouragement. This work was supported in part by fellowships from the College of Agricultural and Environmental Sciences, University of CaliforniaDavis to Y.-M.H. and by grants from Petoseed and Asgrow Seed companies and the United States Department of Agriculture National Research Initiative Competitive Grants Program (9301256) to R.L.G. REFERENCES 1. Arguello-Astorga, G. R., R. G. Guevara-Gonzalez, L. R. Herrera-Estrella, and R. F. Rivera-Bustamante. 1994. Geminivirus replication origins have a group-specific organization of iterative elements: a model for replication. Virology 203:90–100. 2. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1994. Current protocols in molecular biology. John Wiley & Sons, Inc., New York. 3. Azzam, O., J. Frazer, D. de la Rosa, J. S. Beaver, P. Ahlquist, and D. P. Maxwell. 1994. Whitefly transmission and efficient ssDNA accumulation of bean golden mosaic geminivirus require functional coat protein. Virology 204:289–296. 4. Brown, J. K., and J. Bird. 1992. Whitefly-transmitted geminiviruses and associated disorders in the Americas and the Caribbean Basin. Plant Dis. 76:220–225. 5. Devereux, J., P. Haeberli, and G. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387–395. 6. Elmer, J. S., L. Brand, G. Sunter, W. E. Gardiner, D. M. Bisaro, and S. G. Rogers. 1988. Genetic analysis of the tomato golden mosaic virus II. The product of the AL1 coding sequence is required for replication. Nucleic Acids Res. 16:7043–7060. 7. Etessami, P., J. Watts, and J. Stanley. 1989. Size reversion of African cassava mosaic virus coat protein gene deletion mutants during infection of Nicotiana benthamiana. J. Gen. Virol. 70:277–289. 8. Faria, J. C., R. L. Gilbertson, S. F. Hanson, F. J. Morales, P. Ahlquist, A. O. Loniello, and D. P. Maxwell. 1994. Bean golden mosaic geminivirus type II isolates from the Dominican Republic and Guatemala: nucleotide sequences, infectious pseudorecombinants, and phylogenetic relationships. Phytopathology 84:321–329. 9. Fontes, E. P. B., P. A. Eagle, P. S. Sipe, V. A. Luckow, and L. HanleyBowdoin. 1994. Interaction between a geminivirus replication protein and origin DNA is essential for viral replication. J. Biol. Chem. 269:8459–8465. 10. Fontes, E. P. B., H. J. Gladfelter, R. L. Schaffer, I. T. D. Petty, and L. Hanley-Bowdoin. 1994. Geminivirus replication origins have a modular organization. Plant Cell 6:405–416. 11. Fontes, E. P. B., V. A. Luckow, and L. Hanley-Bowdoin. 1992. A geminivirus replication protein is a sequence-specific DNA binding protein. Plant Cell 4:597–608. 12. Gilbertson, R. L., J. C. Faria, S. F. Hanson, F. J. Morales, P. G. Ahlquist, D. P. Maxwell, and D. R. Russell. 1991. Cloning of the complete DNA genomes of four bean-infecting geminiviruses and determining their infectivity by electric discharge particle acceleration. Phytopathology 81:980–985. 13. Gilbertson, R. L., S. H. Hidayat, R. T. Martinez, S. A. Leong, J. C. Faria, F. J. Morales, and D. P. Maxwell. 1991. Differentiation of bean-infecting geminiviruses by nucleic acid hybridization probes and aspects of bean golden mosaic in Brazil. Plant Dis. 75:336–342. 14. Gilbertson, R. L., S. H. Hidayat, E. J. Paplomatas, M. R. Rojas, Y.-M. Hou, and D. P. Maxwell. 1993. Pseudorecombination between infectious cloned DNA components of tomato mottle and bean dwarf mosaic geminiviruses. J. Gen. Virol. 74:23–31. 15. Gilbertson, R. L., M. R. Rojas, D. R. Russell, and D. P. Maxwell. 1991. Use of the asymmetric polymerase chain reaction and DNA sequencing to determine genetic variability of bean golden mosaic geminivirus in the Dominican Republic. J. Gen. Virol. 72:2843–2848. 16. Heyraud, F., V. Matzeit, M. Kammann, S. Schaefer, J. Schell, and B. Gronenborn. 1993. Identification of the initiation sequence for viral-strand DNA synthesis of wheat dwarf virus. EMBO J. 12:4445–4452. 17. Hou, Y.-M., and R. L. Gilbertson. Unpublished data. 18. Lai, M. M. C. 1992. RNA recombination in animal and plant viruses. Microbiol. Rev. 56:61–79. 19. Laufs, J., W. Traut, F. Heyraud, V. Matzeit, S. G. Rogers, J. Schell, and B. Gronenborn. 1995. In vitro cleavage and joining at the viral origin of repli-
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