Department of Molecular and Cell Biology, University of Cape Town, Private Bag, Cape Town 7000, South Africa1
Author for correspondence: Ed Rybicki. Fax +27 21 689 7573. e-mail ed{at}molbiol.uct.ac.za
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Abstract |
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Introduction |
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Whereas the generation of a recombinant linear replicon requires only a single recombination breakpoint, at least two breakpoints are required during the production of recombinant circular replicons. A single intermolecular homologous recombination event between two geminivirus replicative form (RF) DNAs will yield a circular dimeric form (CDF) containing two (+)-strand oris. Release of a recombinant unit length genome from the CDF could occur through one of two alternative mechanisms: (1) recombinational release involving a second intramolecular homologous recombination event, or (2) replicational release which occurs when replication of the CDF initiates at one its (+)-strand oris and terminates at the other. Whereas unit length recombinant genomes generated through recombinational release will have two breakpoints at random positions, those generated through replicational release will always have a breakpoint at their (+)-strand oris (Stenger et al., 1991 ).
Both replicational and recombinational release of unit length infectious genomes is believed to occur from geminivirus genome copies tandemly cloned on a Agrobacterium Ti plasmid during agroinoculation (Heyraud et al., 1993a ; Stenger et al., 1991
). There are a number of reasons why this feature of agroinoculation makes it an ideal tool for analysing geminivirus recombination: (1) the tandemly cloned geminivirus genome copies within an agroinfectious construct are in effect a linear representation of a CDF with the interface between the two genomes in the construct resembling a CDF cross-over site; (2) it is possible to individually simulate all different parts of a CDF using agroinfectious clones by changing the order and amount of viral genomic material that the clones contain; (3) only a very small proportion of the genomes released from heterodimeric agroinfectious clones (agroinfectious clones containing the genomes of two distinct virus genotypes) through recombination will be parental. Chimaeric genomes that are released should therefore face no or very little immediate competition from parental genomes, even when one or both parental genotypes are much fitter than all possible recombinant genotypes.
We have recently described the sequencing and biological characterization of the MSV isolates MSV-Kom and MSV-Set (Schnippenkoetter et al., 2001 ). MSV-Kom is highly virulent in maize and is an example of the MSV-A strain that is responsible for maize streak disease (Martin et al., 2001
). MSV-Set is considerably milder in maize than MSV-Kom and shares only
79% sequence identity with MSV-A isolates. In this paper we describe the use of a series of heterodimeric MSV-Kom/Set agroinfectious constructs to simulate a CDF that would result following an initial crossing-over event between the movement protein gene (MP) and long intergenic region (LIR) of MSV-Kom and MSV-Set. Through sequence analysis of recombinant RF DNAs cloned from agroinoculated plants, we identify the positions of recombination breakpoints and the compositions of mixed recombinant populations. We also analyse the biological properties of a group of the MSV-Kom/Set recombinants and speculate that there is most likely a strong selection pressure to minimize the amount of exogenous sequence within chimaeric genomes.
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Methods |
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Agroinoculation and symptom quantification.
The sweetcorn cultivar Jubilee (Starke Ayres Nursery, Cape Town, South Africa) was used during all agroinoculation experiments. Three-day-old seedlings were agroinoculated and maintained as has been described previously (Martin et al., 1999 ). The infection rates (IR), degrees of stunting (S) and chlorotic leaf streaking that were elicited by different agroinfectious virus constructs were quantified according to the methods described by Martin et al. (1999)
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Detection, cloning and sequencing of recombinant RF-DNAs.
MSV RF-DNAs were isolated from symptomatic plants as described by Palmer et al. (1998) . The composition of RF-DNA populations was analysed by Southern hybridization using the DIG kit (Boehringer Mannheim). RF-DNAs were digested with RsaI and SacI and restriction fragments were separated by 1% agarose gel electrophoresis before blotting them onto a nitrocellulose membrane. DIG-labelled MSV-Kom and MSV-Set genomes were individually used to probe blots to assess the representation of MSV-Kom and MSV-Set sequences within the RF-DNA populations.
RF-DNAs were randomly cloned into the BamHI site of pUC18. Clones containing MSV genomic DNA were identified by colony hybridization using DIG-labelled MSV-Kom and MSV-Set genomic DNA. Cloned recombinant genomes were isolated and restriction mapped using ApaI, BamHI, EcoRI, SalI and XhoI to both establish their identity and to determine the locations of any major recombinant regions.
Clones containing full-length MSV genomes were partially sequenced using an ALF Express automated sequencer (Pharmacia) to determine the positions of recombination breakpoints. Genetics Computer Group software (V7.1; GCG, Wisconsin, USA) was used for all DNA sequence manipulations and analyses.
Leafhopper transmission.
Viruses were transmitted by the leafhopper Cicadulina mbila to the maize genotypes sweetcorn, popcorn (Starke Ayres Nursery), Witplat, Vaalhartz Wit (VH Wit), Vaalharts Geel (VH Geel; Summer Grains Centre, Potchestroom, South Africa), PAN 6549, PAN 6552, PAN 6191, PAN 6363, PAN 6099, PAN 6195 and PAN 6364 (Pannar Seed Co., Greytown, South Africa), the wheat genotypes SST 66, SST 44, Marquis, Dias and Agent, and the barley genotypes Clipper, Adam Tas, Chokka and Festiquay (Elsenberg Agricultural Development Institute, Elsenberg, South Africa). Non-viruliferous leafhoppers were allowed to feed for between 48 and 96 h on agroinoculated maize plants showing streak symptoms. Viruses were transmitted to 2-week-old uninfected maize, wheat and barley seedlings. Viruliferous leafhoppers were allowed to feed on the plants for 5 to 10 days. All transmissions of each virus into particular host genotypes were repeated four times. Disease symptoms were rated on a 5 point rating scale (0=no streaks and no stunting, 4=80100% of leaf area chlorotic with severe stunting).
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Results and Discussion |
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RsaI and SacI restriction fragment length polymorphisms and Southern blot analysis were used to examine virus populations occurring in symptomatic plants agroinoculated with KosA, SekA and SekB. KosA agroinoculated plants contained predominantly MSV-Set-like RF DNAs and SekA agroinoculated plants contained predominantly MSV-Kom-like RF DNAs (Fig. 2). Whereas minor populations of MSV-Kom and MSV-Set-like RF-DNAs were also detectable in KosA and SekA infected plants, respectively, only MSV-Kom-like RF DNAs were detectable in SekB infected plants (Fig. 2
).
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Nine unique chimeric RF-DNAs were identified (Fig. 3): SekA.1, SekA.2, SekA.3, SekA.4 and SekA.5 from a SekA infected plant, SekB.1 from a SekB infected plant, and KosA.1, KosA.2 and KosA.3 from a KosA infected plant. Interestingly, clones identical to SekA.1 were also isolated from SekB and SekC infected plants, clones identical to SekA.2 were isolated from a SekB infected plant and a clone identical to KosA.1 was isolated from a KosB infected plant.
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Assuming that homologous recombination between MSV-Kom and MSV-Set genomes requires that they share at least two identical nucleotides at the cross-over site, there are a total of 334 sites at which these genomes can recombine. Whereas all 334 sites were available for recombinational release of unit length genomes from the SekA and KosA heterodimers, 114 sites could be used in the SekB heterodimer and 31 in the SekC and KosB heterodimers. Only six of these sites, all of which were within 200 nucleotides of the forced recombination breakpoint, were involved in the recombinational generation of all the genomes that we isolated from heterodimer inoculated plants (Fig. 4).
While it might appear that this genomic region surrounding the forced recombination breakpoint constitutes a recombination hot spot, it is also possible that selection in planta has favoured the survival of recombinants that most closely resemble wt MSV-Kom or MSV-Set genomes. The appearance of apparent recombination hot spots under conditions of selection that are otherwise absent when selection is removed has been observed in recombination studies of the coronavirus Mouse hepatitis virus (Banner & Lai, 1991 ). Our results indicate that selection has influenced the pattern of recombination that we observed. MSV-Kom is more pathogenic in maize than MSV-Set (Schnippenkoetter et al., 2001
). Accordingly, whenever agroinfectious heterodimers contained a full MSV-Kom genome (SekA, SekB, SekC and KosA), predominantly MSV-Kom-like genomes dominated the populations of clones that were recovered from agroinoculated plants. It is conceivable therefore that recombinants with more equitable portions of MSV-Kom and MSV-Set sequence may have been produced in heterodimer inoculated plants but that they were not detected because they were outcompeted by the more viable mostly MSV-Kom or MSV-Set-like recombinants that we did detect in these plants.
Only three of the nine unique chimaeras that we identified (SekA.3, SekA.4 and SekA.5) had recombination breakpoints in close proximity to the (+)-strand ori and were, therefore, potentially the direct products of replicational release from heterodimeric agroinfectious constructs (SekA in this case). All of the other chimaeras had apparently arisen through either direct recombinational release from heterodimeric agroinfectious constructs or homologous recombination between genomes that had already been replicationally released from these constructs.
It is, however, uncertain whether SekA.3, SekA.4 and SekA.5 could have originated through the conventionally accepted replicational release mechanism proposed by Stenger et al. (1991) . Whereas replicational release according to this model would be expected to yield chimaeras with recombination breakpoints precisely at the (+)-strand ori, (see the theoretical sequence in Fig. 5
), the breakpoints in SekA.3, SekA.4 and SekA.5 were between 11 and 15 nucleotides downstream of the ori. In all three clones, the breakpoint region included the nucleotide sequence TTACC a sequence which is identical to that surrounding the (+)-strand ori. Interestingly, SekA.4 and SekA.5 respectively contain 13 and 11 nucleotide sequence insertions at their recombination breakpoints. In both cases the insertions are bounded by TTACC residues (Fig. 5
).
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SekA.3, the recombinant with a breakpoint near the ori but without any nucleotide insertions, is either the direct product of recombinational release from the SekA heterodimer or the modified progeny of a replicationally released genome that contained a hairpin stem insertion. Whereas there is evidence that during the generation of SekA.4 and SekA.5 replication had been initiated from the expected position in the MSV-Set TAATATTAC (+)-strand ori sequence, if SekA.3 was the direct product of replicational release from SekA it would imply that replication had to have been initiated from the TTAC sequence within the 3' stem of the MSV-Set ori hairpin. It is, however, possible that SekA.3 is the deletion product of a recombinant resembling SekA.4 or SekA.5. Six and nine nucleotide direct repeats surrounding the nucleotide insertions in SekA.4 and SekA.5, respectively (Fig. 5) may have facilitated homologous recombinational deletion of these insertions. In the case of SekA.4, homologous recombination between the directly repeated sequences would result in the generation of SekA.3.
Biological characterization of recombinant genomes
To test the viability of the predominantly MSV-Kom-like recombinants which contained over 100 nucleotides of MSV-Set sequence, we constructed agroinfectious clones of SekA.1, SekA.2, SekA.3, SekA.4, SekA.5 and KosA.3 and examined the symptoms that they produced in agroinoculated sweetcorn plants (Fig. 6).
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It is probable that differences in pathogenicity between SekA.1, SekA.2, SekA.3, SekA.4 and SekA.5 are due entirely to differences in the sequences that they contain in the immediate vicinity of the (+)-strand ori. SekA.1 contains an ori hairpin sequence identical to that of MSV-Kom, SekA.2 contains a hairpin sequence identical to that of MSV-Set, and SekA.3, SekA.4 and SekA.5 contain hybrid MSV-Kom/Set hairpin sequences. It has been demonstrated using WDV that Rep oligomers bind with low affinity to the (+)-strand ori hairpin (Castellano et al., 1999 ). It is therefore feasible that the SekA.2, SekA.3, SekA.4 and SekA.5 are less pathogenic than SekA.1 because MSV-Kom Rep molecules (which all of these recombinants express) interact suboptimally with MSV-Set and hybrid MSV-Kom/Set ori sequences.
In addition to having potentially altered MSV-Kom Rep binding sequences, the SekA.3, SekA.4 and SekA.5 hybrid ori sequences would produce altered hairpin structures that are substantially less stable than those predicted for MSV-Kom and MSV-Set. It is perhaps not surprising therefore that these recombinants were less pathogenic than SekA.1 and SekA.2. It is, however, interesting that both SekA.4 and SekA.5 have nucleotide insertions within their hybrid ori sequences and that both are significantly more pathogenic than SekA.3, which contains no nucleotide insertions. While the most stable secondary structures predicted for the SekA.4 and SekA.5 ori sequences are more stable than that predicted for the SekA.3 sequence (data not shown), further experiments will be required to establish exactly how these small differences in sequence translate into such large differences in pathogenicity.
Our results may indicate why no naturally occurring mastrevirus recombinants with breakpoints within the (+)-strand ori hairpin have been discovered. Mastrevirus hairpin stem sequences are far less conserved than those of begomoviruses and curtoviruses. If mastreviruses sharing less than 85% nucleotide sequence identity recombine, our sequence data analysis suggests that genomes that are replicationally released from CDFs will tend to have nucleotide mismatches within the stem sequences of their (+)-strand ori hairpins. In a study involving the begomovirus Tomato golden mosaic virus, mutations that introduced nucleotide mismatches into the stem sequences of (+)-strand ori hairpins adversely effected (+)-strand replication (Orozco & Hanley-Bowdoin, 1996
). Although we have shown here that recombinants with hybrid hairpin stems are viable, we have also demonstrated that they are severely defective relative to their parental viruses and would most likely never survive for long enough in nature to be detected.
We investigated the impact of recombination on the biological characteristics of SekA.1 and KosA.3 in greater depth by leafhopper transmitting these viruses from agroinoculated highly symptomatic sweetcorn plants to a range of barley, wheat and differentially MSV-resistant maize genotypes. Because both chimaeras contained predominantly MSV-Kom sequence we were interested in determining whether the small portions of MSV-Set LIR (SekA.1) or MP (KosA.3) sequence they contained had altered their fitness relative to MSV-Kom in these hosts. The chimaeras did not induce any symptoms in the barley or wheat genotypes that were immune to symptomatic infection by MSV-Kom (Festiquay, Agent and SST-44; Fig. 7). Both of the chimaeras did, however, produce streak symptoms that were noticeably more severe than those produced by MSV-Kom in the barley genotype Chokka and the wheat genotype Dias (Fig. 7
).
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It has been speculated that recombination between geminiviruses has been a major contributing factor behind the recent emergence of a number of devastating crop diseases worldwide (Padidam et al., 1999 ). The long-term survival and establishment of a recombinant virus genotype in nature will depend strongly on it having a selective advantage over the overwhelming populations of its parental genotypes. We have shown here that a small subset of all possible chimaeras that could result following an initial crossing-over event between the MP gene and LIR of two different MSV strains is viable with biological characteristics that are distinct from those of the parental strains. Although none of the MSV-Kom/Set recombinants that we have dealt with here was more pathogenic than both MSV-Kom and MSV-Set in any of the host species and genotypes we examined them in, our results help explain the patterns of natural mastrevirus recombination noted previously. Our failure to detect recombinants containing more than
200 nucleotides of exogenous sequence fits well with sequence evidence that interstrain MSV recombinants generally contain fewer than
150 nucleotides of exogenous sequence (Martin et al., 2001
). The results presented here on the relative pathogenicities of recombinants with breakpoints at and around the (+)-strand ori provide an explanation for why this region is less of a recombination hot spot in mastreviruses than it apparently is in begomoviruses. We are currently using many of the MSV-Kom/Set recombinants described here together with analogous laboratory-constructed chimaeras to determine the positions and affinities of the replication specificity determinants within the LIRs of different MSV strains.
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Acknowledgments |
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References |
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Received 9 July 2001;
accepted 21 August 2001.