John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK1
Author for correspondence: Simon Covey. Fax +44 1603 450045. e-mail simon.covey{at}bbsrc.ac.uk
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Abstract |
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Introduction |
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We have been exploring the potential to develop CaMV vectors capable of expressing relatively large reporter genes like that for glucuronidase (GUS). Vectors based upon interdependent complementation of bipartite defective CaMV replicons have not yet been developed, even though this type of complementation offers promise (Gronenborn, 1987 ; Hirochika & Hayashi, 1991
). CaMV mutants defective in movement can also be complemented by wild-type virus during systemic infection (Thomas et al., 1993
). We have determined whether this type of complementation can be exploited to express CaMV-based GUS gene vectors. Our experiments have shown that transient expression of the GUS gene from a replicating vector in the presence of helper virus is possible and that this can be distinguished from non-replicative expression of the reporter gene.
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Methods |
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Vector constructs.
CaMV replacement vector pBjigus1 was derived from pCa24, a clone of the CaMV Cabb B-JI genome inserted into pAT153 at the unique SalI site. The GUS gene was obtained from plasmid pJJ3431, kindly provided by Jonathan D. G. Jones (Jones et al., 1992 ). A GUS fragment was isolated following digestion of pJJ3431 with XhoI and XbaI and this was ligated into pGEM11 (Promega) generating pGemgus. A linker (Table 1
; oligos 1 and 2) with appropriate ends was inserted near the 5' end of the GUS gene following digestion of pGemgus with XhoI and NcoI, producing clone pGemlgus. Similarly, a second linker (Table 1
; oligos 3 and 4) was inserted into the 3' end of the GUS gene in pGemlgus after digestion with XbaI and NsiI to make clone pGemlgusl. The GUS gene was then isolated as an XhoINsiI fragment and cloned into the appropriate fragment of pCa24 digested with XhoI and partially digested with NsiI. The GUS gene was thus inserted between nucleotides 1644 (XhoI) and 3556 (NsiI) of the CaMV Cabb B-JI genome.
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Construct pPbs2 was produced from the CaMV Cabb B-JI genome by removal of the tRNA primer-binding site (pbs) at position 1, which was then relocated at the end of gene VII at position 333. The starting clone was pG1sbji, the Cabb B-JI genome inserted into pGEM1 at the SalI site. From this, an MluIXhoI fragment was isolated and cloned into pGEM7, to give pG7M-X. Mutagenesis of an A in pG7M-X to a C residue using oligos 9 and 10 (Table 1), thus creating a StuI site at position 22, was achieved with the QuikChange mutagenesis system (Stratagene). Similarly, an A to C change at position 322 created an EcoRV site, using oligos 11 and 12 (Table 1
). The resulting clone was digested with DraII and the large fragment re-ligated to give clone pG7m-xM2dra-. Mutagenesis with oligos 13 and 14 (Table 1
) created an NdeI site at 8011 to give pG7m-xM2dra-M1. This was digested with NdeI and StuI and a further linker (Table 1
; oligos 15 and 16) was inserted following removal of the pbs. This clone was then digested with EcoRV and a new pbs was inserted (Table 1
; oligos 17 and 18) to give pG7m-xM2dra-M3. Following digestion with MluI and XhoI, the fragment was inserted into pG1sbji to give pPbs2. The construct with the mutated pbs elements was combined with pBjigus1 by isolating an NsiI fragment containing the GUS gene and replacing the equivalent fragment in pPbs2 to produce pPbs2gus1.
Inoculations and detection of GUS activity.
Appropriate cloned DNAs were prepared and mixed as necessary. Co-inoculations contained 1 µg of each DNA; single inoculations contained 1 µg DNA. Inoculations were in 10 µl TE (10 mM TrisHCl, pH 7·6, 1 mM EDTA) with 0·03 g/ml Celite abrasive. The second true leaf of turnip seedlings was inoculated when the leaves were at least 2 cm long. GUS activity was detected after 7 days by using X-glucuronide exactly as described previously (Al-Kaff et al., 1998 ). Chlorophyll was removed by treatment of leaves with 100% ethanol.
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Results |
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GUS expression from replicating and non-replicating gene vectors
The above experiments suggested that a replication-defective CaMV construct containing a GUS gene could show expression following manual inoculation of leaves with vector DNA. The relatively small size of the background blue foci following inoculation of pBjigus1 DNA alone showed that measurable GUS activity was limited to one or a few cells, with little or no local movement of blue product to adjacent cells. As a positive control for localized GUS activity, we inoculated leaves with DNA of a simple 35SGUSoctopine synthase terminator construct (Fig. 1). Blue spots were observed after 1 week and were generally similar in size and distribution to those seen following single pBjigus1 inoculations (Fig. 3h
). No difference in the pattern of GUS expression was observed when DNAs were inoculated as linearized or supercoiled forms (data not shown).
We next wanted to show that the often larger zones of GUS activity located in the centres of local lesions following co-inoculation with wild-type virus had resulted from vector replication complemented by wild-type helper virus. The wild-type CaMV genome was modified by moving the pbs origin of reverse transcription from its normal location at the beginning of gene VII to the end of gene VII, generating construct pPbs2 (Fig. 1b). Inoculation of plants with pPbs2 resulted in no symptoms, from which we concluded that it was not infectious. The same mutation was then introduced into pBjigus1 and the construct (pPbs2gus1) was co-inoculated with wild-type virus. In this case, no large or small blue spots were observed in local lesions centres, but background blue spots were found following chlorophyll removal (Table 2
). This shows that the lesion-associated blue spots result from expression of replicated pBjigus1 following complementation by wild-type helper virus. We have found that local lesion-associated foci of GUS activity varied in size between large and small, with the latter being indistinguishable in size from the background non-lesion-associated blue spots. Thus, the size of blue foci was not used as a primary measure of replicative competence of a construct but rather its frequency of association with lesion centres.
Evidence that a CaMV splice acceptor produces a molecule with a cis-acting function
We were intrigued by the non-replicative expression of pBjigus1, since the GUS gene was internal in an equivalent position to CaMV gene III. Expression of the CaMV genome is complex and involves linked translation of internal genes mediated by the gene VI protein P6, a translational transactivator (Fütterer & Hohn, 1991 ; De Tapia et al., 1993
; Hohn & Fütterer, 1997
). Splicing is also an essential part of the CaMV replication cycle (Kiss-László et al., 1995
), although its specific involvement in expression of particular CaMV genes is not understood. A spliced RNA molecule has been detected in plants in which a splice donor in the 35S RNA leader (Kiss-László et al., 1995
) was fused to an acceptor site at 1508, located within gene II. Since mutagenesis of the 1508 splice acceptor abolishes CaMV infectivity, it has been suggested that the leader-to-1508 spliced molecule is a possible subgenomic mRNA for the essential CaMV gene III. To apply our transient expression system to this problem, we made a construct in which the GUS gene replaced the complete gene IIgene V sequence in such a way as to remove the splice acceptor at 1508, in construct pBjigus2 (Fig. 1b
). Since the gene III protein, P3, is produced by the wild-type helper virus to complement the missing protein in the vector, this should negate the requirement for the 1508 splice acceptor in pBjigus2. Leaves were co-inoculated with pBjigus2 and wild-type helper virus, with the expectation of observing lesion-associated GUS activity spots. However, in several experiments, we never observed lesion-associated GUS activity following co-inoculation with these constructs but found only background, non-lesion-associated, small blue spots (Fig. 3i
; Table 2
), from which we conclude that GUS expression had occurred in the absence of vector replication.
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Discussion |
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The observations of expression of the GUS gene as an internal open reading frame of the CaMV vector, even in the absence of helper virus, suggests strongly that RNA processing or expression of other viral genes had occurred. Translation of internal open reading frames can occur through the activity of the CaMV P6 translational transactivator protein (Fütterer & Hohn, 1991 ). However, it is not clear which CaMV genes are expressed by this process during virus replication in vivo. This is complicated further by the discovery of multiple splice donor and acceptor sequences in the 5' third of the genomic 35S RNA transcript. Moreover, a splice donor within the non-essential gene II at position 1508 is apparently essential for virus replication (Kiss-László et al., 1995
). Thus, expression of CaMV gene II might be expected to occur by P6-mediated translational transactivation, with gene III expression resulting from a splice between a donor site in the 35S RNA leader sequence at position 7909 and the essential acceptor in gene II at 1508 (see Fig. 4
). In construct pBjigus1, the GUS gene is located in a position equivalent to gene III, with its possible mode of expression through a spliced mRNA (Fig. 4b
). In construct pBjigus2, the GUS gene was inserted in the gene II position in such a way as to eliminate the splice acceptor at 1508 (Fig. 4c
). Transgene expression in this case could have occurred by P6 transactivation, although we cannot exclude the possibility that some other splice produced the necessary mRNA. The unexpected consequence of deleting the 1508 splice acceptor was that it rendered the vector incapable of replication, judged by the absence of lesion-associated GUS activity when co-inoculated with helper virus. The pBjigus2 construct lacked gene III or an equivalent gene, so the requirement for a splice to the missing acceptor should have been obviated. This suggests that the primary role of the 1508 splice acceptor is not for expression of gene III but that it is required for some other essential virus function required in replication. Alternatively, the region deleted in this construct might play a role in some other process, although it is difficult to speculate what this might be, especially since gene II is non-essential except for the splice acceptor. Our CaMV vectorhelper system, which allows us to distinguish replication-associated gene expression from non-replicative gene expression, should provide a new approach to investigate this and other virus mechanisms.
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Acknowledgments |
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References |
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Received 13 July 2000;
accepted 28 September 2000.
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