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1 Institute of Infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Observatory 7925, South Africa
2 James Graham Brown Cancer Center, University of Louisville, 529 South Jackson Street, Louisville, KY 40202, USA
3 Department of Pharmacology and Toxicology, University of Louisville, 570 South Preston Street, Louisville, KY 40202, USA
4 14 Ashby Drive, Bungendore, NSW 2621, Australia
5 Department of Molecular and Cell Biology, University of Cape Town, Private Bag, Rondebosch, Cape Town 7701, South Africa
Correspondence
Edward P. Rybicki
Ed.Rybicki{at}uct.ac.za
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Supplementary figures are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, 1994a, b; Lazarowitz et al., 1992; Argüello-Astorga et al., 1994b; Jupin et al., 1995; Choi & Stenger, 1996; Gladfelter et al., 1997; Akbar Behjatnia et al., 1998; Sanz-Burgos & Gutierrez, 1998; Castellano et al., 1999; Chatterji et al., 1999, 2000). Sequence analysis of this genomic region has revealed iterative elements called iterons, the sequences of which are a major determinant of Rep-binding specificity for viruses in the related genera Begomovirus and Curtovirus (Fontes et al., 1992, 1994a, b; Argüello-Astorga et al., 1994a, b; Choi & Stenger, 1995, 1996; Chatterji et al., 2000; Ramos et al., 2003). Complementation of replication functions by begomo- and curtoviruses is restricted to individuals that share nearly identical iteron sequences (Stanley et al., 1985; Lazarowitz et al., 1992; Gilbertson et al., 1993; Fontes et al., 1994b; Hanley-Bowdoin et al., 1996, 2000; Gladfelter et al., 1997; Chatterji et al., 2000; Saunders et al., 2002; Ramos et al., 2003). In addition to the iterons, undefined sequences located between the iterons and the inverted-repeat sequence (stem–loop) of begomovirus genomes are potential secondary replication-specificity determinants (RSDs) (Fontes et al., 1994b; Hou & Gilbertson, 1996; Gladfelter et al., 1997). Whereas the correct spatial arrangement of begomovirus iterons, in relation both to one another and to the inverted repeat, is essential for efficient replication (Argüello-Astorga et al., 1994b; Gladfelter et al., 1997; Orozco et al., 1998; Chatterji et al., 2000), the precise arrangements of iterons and inverted-repeat sequences are less constrained in the curtoviruses (Choi & Stenger, 1996). Also, the inverted-repeat sequences of either curtoviruses or begomoviruses apparently contain no primary RSDs (Fontes et al., 1994b; Choi & Stenger, 1996).
Unlike the situation with begomoviruses, the mastrevirus V-ori inverted-repeat sequences are highly diverse and it has been hypothesized that both the inverted-repeat and rep-proximal iteron (RPI) sequences might constitute RSDs in this genus (Argüello-Astorga et al., 1994a, b). Evidence that both the iterons and the potential stem–loop structure created by the inverted repeat as a whole are required during mastrevirus replication has been obtained in mutational studies of the maize streak virus (MSV) long intergenic region (LIR) (Schneider et al., 1992). Conversely, a wheat dwarf mastrevirus (WDV) mutant with a deletion of the entire inverted-repeat sequence was able to initiate replication. There was, however, a defect in termination of replication, resulting in high-molecular-mass concatemeric forms of viral DNA (Kammann et al., 1991). Heyraud et al. (1993) subsequently found that the mutant virus was able to initiate replication from a second site (TACCC) resembling the nicking site in the inverted-repeat sequence. This second initiation site is apparently unique to WDV and it is likely that the intact inverted repeat is required for replication initiation and termination by all other geminiviruses.
Both Rep–LIR interactions and the core LIR sequences required for replication are well defined in the genome of the mastrevirus WDV (Suarez-Lopez et al., 1995; Suarez-Lopez & Gutierrez, 1997; Sanz-Burgos & Gutierrez, 1998; Castellano et al., 1999; Missich et al., 2000). The upper bounds of the minimal LIR sequence absolutely required to support WDV replication lie between 188 and 178 nt 5', and 25 and 28 nt 3', of the V-ori, with an extra approximately 70 nt 5' and 30 nt 3' of this region enabling efficient replication (Sanz-Burgos & Gutierrez, 1998). Electron-microscopic visualization of Rep–LIR binding and DNA footprinting revealed two high-affinity and one low-affinity Rep-binding sites within the LIR in the vicinity of the complementary-sense gene TATA box, the virion-sense gene TATA box and the inverted repeat, respectively (Sanz-Burgos & Gutierrez, 1998; Castellano et al., 1999). Although evidence of a Rep–inverted-repeat sequence (stem–loop) complex is strongly suggestive of Rep-binding signals required for replication (and hence RSDs) residing here, the role of the Rep–LIR complexes in replication is less certain, in that they may also be active in regulation of complementary- and/or virion-sense gene expression.
Whilst WDV and MSV are both grass-infecting mastreviruses, they are only very distantly related (approx. 48 % LIR nucleotide sequence identity); this is far lower than for any other intrageneric comparisons for curto- or begomoviruses (Rybicki, 1994). The WDV LIR is also 101 nt longer than that of MSV. A degree of caution is therefore needed when using these excellent WDV studies to infer either the minimal LIR sequences required for MSV replication or the locations of Rep binding in the MSV LIR.
In this study, we sought to define features of the MSV LIR that are functional during replication. First, we examined LIR deletion mutants by using transient replication assays to define the upper bounds of the minimal LIR sequence absolutely required for replication. trans-Replication assays were then used to demonstrate complementation of replication functions between viruses with divergent LIR and iteron sequences. Finally, we examined chimaeras of inefficiently trans-replicating viruses by using agroinoculation and competitive trans-replication assay experiments to localize the primary MSV RSD to a 67 nt region surrounding the RPI. Our results indicate that sequences in this region interact cooperatively with those in the inverted repeat to determine the efficiency of MSV replication.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Willment et al., 2002) and are referred to here as pMSV-Kom, pMSV-VM, pMSV-Tas, pMSV-VW, pMSV-Set and pPanSV-Kar, respectively.
pKEP176 (Palmer, 1997) is a pUC19-based construct containing the MSV-Kom V1 gene followed by the 
-glucuronidase (uidA) gene (a replacement of the V2 gene), and was used as an intermediate construct in the creation of plasmids pLIR1[–90,+148],LIR2[–90,+148] and pLIR1[–106,+148],LIR2[–106,+148] (described below).
pKomPstI [called pMSVPstI by Palmer & Rybicki (2001)] is a replication-incompetent tandem dimer of MSV-Kom. pKomRep [called pKEP132 by Palmer (1997)] is an expression cassette containing the MSV-Kom rep gene cloned between the rice actin 1 promoter and the nopaline synthase (nos) terminator (both from pCOR113; McElroy et al., 1991), for high-level expression of Rep in maize suspension cells.
pSekA.2, an agroinfectious, mostly MSV-Kom chimaera containing MSV-Set LIR sequence from 24 nt 5' of the V-ori to 148 nt 3' of the V-ori (nt –24 to +148), has been described previously (Schnippenkoetter et al., 2001b).
LIR deletion constructs.
Partially dimerized deletion mutants of the MSV-Kom LIR (Fig. 1) containing (i) wild-type (wt) 5' LIR (LIR-1) and truncated 3' LIR (LIR-2) sequences, (ii) wt LIR-2 and truncated LIR-1 sequences and (iii) truncated LIR-1 and LIR-2 sequences were constructed as detailed below and as shown in Supplementary Fig. S1 (available in JGV Online).
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] consists of pMSV-Kom with the virion-sense open reading frames replaced by the cauliflower mosaic virus (CaMV) 35S promoter and bar gene, and forms the backbone upon which LIR-1 deletion mutants were constructed (Supplementary Fig. S1).
pKori0-2 (Supplementary Fig. S1) is the backbone upon which LIR-2 deletion mutants were constructed. pKori0-2 was constructed by using the PCR primers LIR-Kom (5'-CTATCTAGACGACGACGGAGGTTGAG-3'; XbaI site underlined) and SIR-Kom (5'-CCGCTCGAGTAATTCATATAGATC-3'; XhoI site underlined) to amplify a 1529 nt fragment containing the MSV-Kom LIR (wt LIR-1), V1, V2 and short intergenic region (SIR) sequences. This fragment was then inserted into the SalI site of a vector already containing the TaqI fragment of the chloramphenicol acetyltransferase (cat) gene from pBSG8-15 (Georges Rapoport, Institut Pasteur, Paris, France) and the PstI–XbaI fragment containing the CaMV 35S promoter from pMF6 (Callis et al., 1987). Both the XhoI site (compatible with SalI) and the XbaI site (blunt-ligated with SalI) of the PCR fragment were lost during the cloning steps.
pKori0-1-based constructs, each of which contains a wt LIR-2 and a truncated LIR-1 sequence, were named according to the portion of the LIR-1 that they contained relative to the V-ori (Fig. 1
). The 3' LIR-1 truncations, which have a 5' XbaI site, were made by deleting sequences between the (i) DraI and BamHI sites and (ii) ApaI and BamHI sites (Fig. 2; Supplementary Fig. S1), inserting the fragments into pUC18 to create a 3' XbaI site and cloning the truncated LIR-1 sequence into the XbaI site of pKori0-1, to give pLIR1[–163,+54] and pLIR1[–163,+27], respectively. pLIR1[–163,+148] has no LIR-1 deletion.
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; Supplementary Fig. S1), inserting the fragments into pUC18 to create flanking XbaI sites and cloning the truncated LIR-2 sequence into the XbaI site of pKori0-2, to give pLIR2[–106,+148] and pLIR2[–90,+148], respectively. pLIR2[–163,+148] has no LIR-2 deletion. Two mutants containing 5' deletions in both LIR-1 and LIR-2 were also constructed. To do this, XhoI–BamHI fragments containing XbaI–AccI and XbaI–TaqI LIR deletions were inserted into the XhoI/BamHI sites of pKEP176 immediately upstream of the V1 gene to yield pKoriA and pKoriB, respectively. These contain a 5'-truncated LIR1, V1 gene and uidA gene. SacI fragments of pLIR2[–106,+148] and pLIR2[–90,+148], containing the SIR, cat gene, CaMV 35S promoter and 5'-truncated LIR2 sequences (Supplementary Fig. S1) were inserted into the SacI sites of pKoriA and pKoriB 3' of the uidA sequence, to yield pLIR1[–90,+148],LIR2[–90,+148] and pLIR1[–106,+148],LIR2[–106,+148], respectively. These contain a 5'-truncated LIR1, V1 gene, uidA gene, SIR, cat gene, CaMV 35S promoter and a 5'-truncated LIR2, with the genes either side of the SIR in the same orientation, as shown in Supplementary Fig. S1 (available in JGV Online).
The complete LIR sequences of pLIR1[–163,+27], pLIR1[–163,+54], pLIR2[–90,+148], pLIR2[–106,+148], pLIR1[–90,+148],LIR2[–90,+148] and pLIR1[–106,+148],LIR2[–106,+148] were confirmed (by using an ABI Prism automated sequencer; Applied Biosystems) to have the correct sequence and orientation.
LIR chimaera constructs.
The 274 nt BamHI–SacI fragment of pMSV-Kom was used to replace the corresponding fragment of pMSV-Set to produce the chimaera pSSBK. An approximately 370 nt NsiI–ApaI fragment was exchanged between the viruses to obtain the mostly MSV-Kom chimaera pKNASNsiI and the mostly MSV-Set chimaera pSNAK. Whereas pSNAK contains a functional rep, pKNASNsiI is missing 10 nt between two closely situated NsiI sites in pMSV-Set and therefore has a frameshift mutation within rep. The complementary-sense strand side of the LIR, up to the rpeI element (Fenoll et al., 1988, 1990), and the first 185 nt of rep were exchanged between pMSV-Set and MSV-Kom. The 811 nt XhoI–RsrII fragments of pKNASNsiI and pMSV-Kom were exchanged between these constructs to produce the chimaeras pKNRSNsiI and pKRAS. Both chimaeras contain mostly MSV-Kom sequences, but whilst KNRSNsiI contains MSV-Set sequence between the NsiI and RsrII sites, pKRAS contains MSV-Set sequence between the RsrII and ApaI sites. pKNRSNsiI has the same 10 nt deletion in rep as does pKNASNsiI. The 2227 nt RsrII fragments of pSNAK and pMSV-Set were exchanged to produce the chimaeras pSNRK and pSRAK. The approximately 2400 nt SacI fragment of pMSV-Set was used to replace the corresponding SacI fragments of pSNRK and pSNAK to produce the chimaeras pSSRK and pSSAK, respectively.
Partially dimeric (1.1mer) agroinfectious clones of pKNRSNsiI, pKNASNsiI, pKRAS, pSSBK, pSNAK, pSNRK, pSRAK, pSSRK and pSSAK in the binary cloning vector pBI121 (Clontech) were constructed as described previously (Schnippenkoetter et al., 2001b). Although no attempt was made to obtain properly agroinfectious clones of pKNRSNsiI and pKNASNsiI (because both contained a 10 nt deletion in rep), these clones were partially dimerized for use during replication assays in black Mexican sweetcorn (BMS) cells.
A series of Rep-defective mutants of the LIR chimaeras was constructed for use in transient replication assays. The 10 nt rep NsiI fragment of pMSV-Set and the mostly MSV-Set chimaeras pSRAK, pSSRK, pSSAK and pSSBK was deleted to produce a set of self-replication-incompetent constructs called pSetNsiI, pSRAKNsiI, pSSRKNsiI, pSSAKNsiI and pSSBKNsiI. Construction of the self-replication-incompetent clones pKNRSNsiI and pKNASNsiI has been described above. A pMSV-Kom PstI frameshift mutant, called pKomPstI, was used as a self-replication-incompetent wt MSV-Kom LIR control. The XhoI–RsrII fragment of pKomPstI was used to replace the corresponding fragment of pKRAS to produce the self-replication-incompetent chimaera pKRASPstI. The PstI mutation in both pKomPstI and pKRASPstI results in translation of a protein containing the N-terminal 24 aa of Rep and additional altered amino acids due to the frameshift mutation.
Transient replication assays.
Replication proficiency of LIR deletion mutants and complementation of replication functions between viruses with divergent LIR and iteron sequences were assayed by a biolistic bombardment-mediated transient assay system as described by Shepherd et al. (2005). In each experiment, bombardments with DNA of individual plasmids or plasmid combinations were performed in duplicate and all experiments were repeated at least twice.
DNA was extracted from BMS (Pioneer Hi-Bred) suspension culture cells 3 or 4 days after bombardment, as described by Xie et al. (1995), and digested with DpnI to remove input plasmid DNA (i.e. methylated, bacteria-derived DNA). Electophoresis through 0.8 % 0.5x TBE/agarose gels was used to separate either various rolling-circle replication DNA intermediates or restriction-enzyme fragments following digestion. DNA was then capillary-transferred onto nylon membranes (Hybond-N+; Amersham Biosciences) and detected by using digoxigenin-labelled (Roche Diagnostics) pMSV-Kom- or CAT-specific probes, following standard protocols (Sambrook et al., 1989). Evidence of rolling-circle replication in bombarded cells was also examined by various replicative form (RF)-specific PCR assays. These involved different combinations of primers specific for the CaMV 35S promoter sequence (5'-CAACCACGTCTTCAAAGC-3') and two previously described degenerate primers (Willment et al., 2001): 215–234 and 1770–1792 (which bind at nucleotide co-ordinates 361–343 and 1746–1764, respectively; numbering starts from the last A of the conserved V-ori TAATATTAC sequence of MSV-Kom, in the 5'
3' direction of the primer). Degenerate-primer RF-specific PCR assays used were described by Shepherd et al. (2005).
Agroinoculation experiments.
Agroinfectious clones of the LIR chimaeras were used to transform Agrobacterium tumefaciens C58C1 (pMP90) (Koncz & Schell, 1986) by using the freeze–thaw method of Holsters et al. (1978). Agroinoculation of 3-day-old sweetcorn ‘Jubilee’ seedlings (Starke Ayres) and quantification of disease symptoms by image analysis were carried out as described previously (Martin et al., 1999). In each of three to six repeated agroinoculation experiments, the ten chimaeras, plus wt MSV-Kom and MSV-Set, were each used to infect 14 plants. In each experiment, the number of plants showing symptomatic infections with the various chimaeric and wt viruses was recorded after 29 days. Viral genomic DNA was isolated from the agroinfected plants, and Southern blot analysis or degenerate-primer PCR amplification and restriction fragment-length polymorphism (RFLP) analysis with CfoI were used to confirm the identity of agroinfectious clones, as described previously (Willment et al., 2001).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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).
Three days after bombardment, RF-DNA was detectable by both RF-specific PCR and Southern analyses in BMS cells bombarded with pLIR1[–163,+148] (two wt MSV-Kom LIRs) and pLIR1[–163,+54] (3'-truncated LIR-1 and wt LIR-2; Fig. 2), but not in those bombarded with pLIR1[–163,+27] (3'-truncated LIR-1 and wt LIR-2; Fig. 2). This indicates that the 3' boundary of the minimal LIR sequence required for replication lies between +27 and +54 nt 3' of the V-ori (defined as the last A of the conserved TAATATTAC sequence in the loop of the potential stem–loop structure, 163 nt 3' of the first MSV-Kom LIR nucleotide; Fig. 1).
Both Southern and RF-specific PCR analyses of the LIR-2 mutants co-bombarded with pMSV-Kom indicated that only the construct with two wt LIR sequences, pLIR2[–163,+148], was trans-replicated efficiently (Fig. 2). However, when the LIR-2 mutants were co-bombarded with pKomRep, both Southern and PCR analyses indicated the presence of small amounts of pLIR2[–90,+148] and pLIR2[–106,+148] (both containing a wt LIR-1 and 5'-truncated LIR-2) RF-DNA. No RF-DNA was detectable in BMS cells co-bombarded with pKori0-2 (containing only a wt LIR-1 and no LIR-2) and either pMSV-Kom or pKomRep, demonstrating that the absence of LIR-2 prevents replication completely.
We suspected that some replication may have occurred directly from the input DNA when pLIR2[–90,+148] and pLIR2[–106,+148] were co-bombarded with the Rep expression cassette, but that the released RF-DNAs were probably replication-incompetent. This hypothesis was supported by the fact that no RF-DNA was detected by either Southern or PCR analysis following co-bombardment of pLIR1[–90,+148],LIR2[–90,+148] and pLIR1[–106,+148],LIR2[–106,+148] with either pMSV-Kom or pKomRep. pLIR1[–90,+148],LIR2[–90,+148] and pLIR1[–106,+148],LIR2[–106,+148] respectively contain the same LIR-2 deletions as pLIR2[–90,+148] and pLIR2[–106,+148], but also contain these same deletions in their LIR-1. Therefore, whilst the region of nt –163 to –90 5' of the V-ori is probably not absolutely required for the termination of replicational release from the LIR-2 deletion mutants, the 5' boundary for the minimal LIR sequence required for efficient replication resides between nt –163 and –106 5' of the V-ori (Fig. 3).
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), in WDV, it ranges between –188 and –178 nt 5', and +25 and +28 nt 3', of the V-ori (Sanz-Burgos & Gutierrez, 1998). This indicates that, despite the viruses sharing only approximately 48 % LIR sequence identity and the WDV LIR being 101 nt larger than that of MSV, the 5' and 3' boundaries of the core LIR sequences required for replication have been conserved between the viruses. It also suggests that the spatial arrangements of other features determined within the WDV LIR may have similarly conserved analogues within the MSV LIR.
Identification of replication specificity between MSV-Kom and other viruses
The ability of different MSV and PanSV isolates to complement the replication functions of MSV-Kom was tested by co-bombarding BMS cells with the MSV-Kom rep-replacement construct pLIR2[–163,+148] (containing two wt LIRs, with rep replaced by the CaMV 35S promoter and cat gene; Supplementary Fig. S1) and pMSV-VM, pMSV-VW, pMSV-Tas, pMSV-Set or pPanSV-Kar. Respectively, the Reps of these wt cloned viruses share 98, 87, 88, 82 and 65 % amino acid sequence identity with that of MSV-Kom.
Southern and RF-specific PCR analyses determined that pLIR2[–163,+148] was trans-replicated by all of the wt MSV isolates and PanSV-Kar (Fig. 4; Supplementary Fig. S2, available in JGV Online). This result is somewhat surprising, as the Rep proteins of PanSV-Kar and MSV-Kom share only 65 % amino acid sequence identity. Although such leniency in trans-replication specificity has been observed between begomovirus Rep proteins and both defective satellite (Dry et al., 1997; Lin et al., 2003) and DNA- (Briddon & Stanley, 2006) molecules, in begomo- and curtoviruses, trans-replication has only been demonstrated between isolates sharing >80 % Rep amino acid sequence identity (Gilbertson et al., 1993; Frischmuth et al., 1997; Hill et al., 1998; Unseld et al., 2000; Brown et al., 2002; Saunders et al., 2002; Ramos et al., 2003). In some cases, begomovirus strains with Reps sharing >90 % amino acid sequence identity exhibit specificity for their cognate origins (Chatterji et al., 2000). Intriguingly, in triplicate experiments, we also observed consistently that the PanSV-Kar Rep provided better complementation of MSV-Kom replication functions than did the much more closely related MSV-Set Rep, which shares 82 % amino acid sequence identity with that of MSV-Kom. Despite its inability to trans-replicate MSV-Kom efficiently, the MSV-Set Rep is perfectly functional, as evidenced by MSV-Set being both agroinfectious (Schnippenkoetter et al., 2001a) and replication-proficient in BMS cells (Fig. 4a). It is also very unlikely that pLIR2[–163,+148] RF-DNAs detected in these trans-replication experiments arose via a recombinational mechanism (rather than through trans-replication), as BMS cells bombarded with the MSV-Kom rep-replacement construct alone yielded no evidence of RF-DNAs in either Southern or PCR analysis.
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; Rybicki, 1994; Padidam et al., 1995; Martin et al., 2001), the iterative sequence elements identified by Argüello-Astorga et al. (1994a, b) as potential RSDs are found within the LIRs of all currently sequenced mastreviruses. The sequences of these elements are, however, not conserved between MSV isolates such as MSV-Kom and MSV-Set (Schnippenkoetter et al., 2001a). In the PanSV and MSV isolates that we have analysed, there are also considerable differences in the spacing of the RPI relative to the rep TATA box and the V-ori (Fig. 3). The RPI is 103, 87 and 65 nt 5' of the V-ori and 39, 22 and 11 nt 5' of the rep TATA box in MSV-Set, MSV-Kom and PanSV-Kar, respectively. Only in the case of MSV-Set do these differences (potentially acting as secondary RSDs; Fontes et al., 1994a) potentially impact on the ability of its Rep to recognize and trans-replicate MSV-Kom.
An additional, if not more obvious, factor that may have contributed to PanSV-Kar trans-replicating MSV-Kom better than MSV-Set is that the latter virus has tracts of highly divergent sequence within its LIR. For example, the MSV-Kom LIR sequence around the RPI from –102 to –78 nt 5' of the V-ori resembles that of PanSV-Kar significantly more than that of MSV-Set (Fig. 3). Conversely, the inverted-repeat sequences of the MSV-Kom and MSV-Set potential stem–loops are much more similar to one another than they are to that of PanSV-Kar. This similarity extends to the MSV-Kom iteron sequences in the inverted repeat, which have a 4 nt overlap with those of MSV-Set, but none with those of PanSV-Kar. This suggested to us that the RSD of MSV-Kom, which is more compatible with that of PanSV-Kar than that of MSV-Set, probably resides within sequences at, or around, the RPI and not in the iteron sequences in the stem–loop.
Identification of MSV-Kom RSDs
Based on the replication-specificity results above, we constructed a series of MSV-Kom–MSV-Set chimaeras to identify the region(s) within the MSV-Kom LIR sequence responsible for its presumably inefficient interaction with the MSV-Set Rep. The infectivity of another MSV-Kom–MSV-Set LIR chimera, pSekA.2, has been described previously (Schnippenkoetter et al., 2001b). The infectivity of all of these LIR chimaeras was tested by agroinoculation of maize.
In all the plants agroinfected with the two predominantly MSV-Kom chimaeras, pSekA.2 and pKRAS, pKRAS consistently produced the most severe symptoms (Fig. 5). This chimaera, which has the 48 nt MSV-Kom inverted-repeat sequence replaced by that of MSV-Set (which differs at eight sites from the MSV-Kom sequence), infected a similar number of plants to, and produced only slightly milder symptoms in maize than, MSV-Kom (Fig. 5). However, the predominantly MSV-Set chimaera containing the MSV-Kom inverted-repeat sequence, pSRAK, was substantially less infectious and produced much milder symptoms in maize than did MSV-Set. pSRAK only induced symptoms on the second and third leaves of infected plants, with all symptomatic plants apparently recovering by the fourth-leaf stage. These results indicated that, whilst the inverted repeat does not contain a primary RSD that would prevent MSV-Kom from trans-replicating MSV-Set efficiently, the region might contain the primary RSD that prevented MSV-Set from trans-replicating MSV-Kom efficiently.
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), the resulting chimaera, pSSAK, was as infectious as, and produced only slightly milder symptoms in maize than, MSV-Set. This indicates that the attenuation in symptoms observed in pSSRK, rather than being due to replacement of a primary RSD, is probably caused by disruption of a sequence-specific interaction between an element in the MSV-Set inverted repeat and this 67 nt 5' region. The possibility of an interaction between the region –120 to –53 nt 5' of the V-ori and the inverted repeat is supported by the observation that pSSRK, the predominantly MSV-Set chimaera containing the MSV-Kom 67 nt 5' region and the MSV-Set inverted repeat, produces symptoms almost as mild as those produced by pSRAK (Fig. 5). Whilst the agroinfection experiments demonstrated the viability of various LIR chimaeras, they did not reliably indicate the location of the RSD that prevented the MSV-Set Rep from trans-replicating MSV-Kom efficiently. To assay more directly the capacity of Reps from MSV-Set and MSV-Kom to initiate and sustain replication from chimaeric LIR sequences, we developed a competitive transient trans-replication assay. Self-replication-incompetent PstI and NsiI mutants of the various chimaeric LIR sequences were co-bombarded with pMSV-Kom or pMSV-Set and evidence of their trans-replication was assayed by RFLP coupled with Southern and RF-specific PCR analyses. The rationale behind this approach was that, unless the LIR sequences of the replication-incompetent viruses contain the appropriate cis-acting replication-specificity elements, they should be out-competed for Rep binding by the wt LIR sequences of the replication-competent helper virus. Within the 4 day time period of the experiment, one would expect that only chimaeras with appropriate RSDs should have been trans-replicated to levels approaching that of the wt helper virus.
With PstI, the PCR products of the self-replication-incompetent genomes are digested into 712 and 596 nt fragments, whereas the wt genomes remain uncut (1306 nt for MSV-Kom and 1321 nt for MSV-Set; expected PCR–RFLP fragments for all wt and chimaeric virus genomes are shown in Table 1). With CfoI, the PCR product of wt MSV-Set is digested into 11, 25, 601 and 685 nt fragments and wt MSV-Kom into 2, 16, 37, 80, 319 and 853 nt fragments (in both cases, only the larger two fragments are resolvable on a 1.5 % agarose gel). Chimaeric genomes contain mixtures of MSV-Kom and MSV-Set and all have different restriction patterns from those of the wt viruses. For example, the predominantly MSV-Set chimaera SSAK, having gained four extra CfoI sites due to the portion of MSV-Kom that it contains, is digested into fragments of 2, 11, 16, 25, 80, 216, 357 and 589 nt (only the 216, 357 and 589 nt bands are visible in Fig. 6). Therefore, fragments that are seen in addition to the wt fragments are indicative of trans-replication of the self-replication-incompetent chimaeric genomes.
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). pKNRSNsiI and pKNASNsiI, which are also predominantly MSV-Kom chimaeras containing the MSV-Set 77 nt surrounding the RPI between –130 and –53 nt 5' of the V-ori, did compete successfully with pMSV-Set for the MSV-Set Rep. Conversely, whilst pSetNsiI and pSRAKNsiI were unable to compete with MSV-Kom for the MSV-Kom Rep, all predominantly MSV-Set chimaeras containing the MSV-Kom 67 nt surrounding the RPI between –120 and –53 nt 5' of the V-ori (pSSBKNsiI, pSSRKNsiI and pSSAKNsiI) did compete successfully with MSV-Kom for its Rep (Fig. 6). These results show clearly that the incompatible RSD impeding MSV-Set trans-replication of MSV-Kom resides within this 67 nt region of MSV-Kom. They also indicate that an analogous primary RSD resides within a 77 nt region containing the RPI, between –130 and –53 nt 5' of the MSV-Set V-ori. Whilst it is possible that the RPI is the RSD, it is also possible that any combination of the RPI, sequences surrounding the RPI, and the spatial arrangement of the RPI or other sequences in this region in relation to the inverted repeat, constitute the RSD.
Conclusions
In this study, we have identified two major differences between MSV and begomovirus virion-strand replication origins. First, like WDV (Sanz-Burgos & Gutierrez, 1998) but unlike begomoviruses (Orozco et al., 1998), MSV replication involves sequences 3' of the inverted repeat (Fig. 3). Despite WDV and MSV sharing lower nucleotide sequence identity than do begomoviruses, topocuviruses and curtoviruses to one another, the extent of the LIR sequence required for replication in these species is approximately the same and is possibly conserved in all mastreviruses.
The second and most striking difference between the MSV and begomovirus V-oris that we have identified in this study is that RSDs in MSV are far more flexible than they are in begomoviruses. Whilst complementation of replication functions is generally only possible between begomoviruses sharing 
80 % Rep amino acid sequence identity (Lazarowitz et al., 1992; Gilbertson et al., 1993; Frischmuth et al., 1997; Hill et al., 1998; Saunders et al., 2002; Ramos et al., 2003), we have demonstrated reasonably efficient trans-replication of mastreviruses with Reps sharing only 65 % amino acid sequence identity.
Despite these differences, we have established clearly that the cis-elements required for MSV replication show greater architectural similarity to those of the other geminivirus genera than was expected previously (Argüello-Astorga et al., 1994a, b). Whilst our discovery of a sequence-specific interaction between the RPI and inverted-repeat sequences of MSV is not entirely surprising, contrary to expectations, we have also shown that sequences surrounding the RPI, rather than the V-ori inverted repeat, constitute the primary RSD of MSV – a situation that is possibly the same for other mastreviruses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 30 August 2006;
accepted 20 February 2007.
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