1 Cornell Research Foundation, Cornell University, 20 Thornwood Drive, Ithaca, NY 14850, USA
2 Queensland Agricultural Biotechnology Institute, DPI Queensland, 80 Ann Street, Brisbane, Queensland 4001, Australia
Correspondence
Kathleen Hefferon
klh22{at}cornell.edu
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ABSTRACT |
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INTRODUCTION |
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BeYDV contains four conserved open reading frames (ORFs); these are oriented bidirectionally between large and small intergenic regions known as LIR and SIR, respectively (Liu et al., 1998). The virion-sense strand, containing ORFs V1 and V2, encodes both the movement (MP) and coat protein (CP) genes (Liu et al., 1998
). The Rep gene, which is encoded by two overlapping ORFs C1 and C2, and contains an 86 nt intron, resides on the complementary-sense strand and is expressed in the opposite orientation from the virion-sense genes. BeYDV complementary-sense gene expression is regulated by a splicing event in which RepA (C1) is expressed from the unspliced transcript, and Rep (C1 : C2) is expressed from the spliced transcript. The LIR is composed of an origin of replication as well as the cis-acting elements required for regulating both virion-sense and complementary-sense transcription (Kammann et al., 1991
). The SIR contains elements necessary for transcriptional termination in addition to a host-derived primer, which is considered to play a role in complementary-sense strand DNA synthesis (Donson et al., 1984
; Hayes et al., 1988a
; Liu et al., 1998
).
Virus replication is believed to take place via a rolling-circle mechanism in which a complementary-sense DNA strand is synthesized by host replication machinery from the primer located within the SIR to produce a double-stranded (ds) replication intermediate (Saunders et al., 1991; Stenger et al., 1991
). Multiple ss circular copies of the virion-sense strand are produced by the introduction of a nick at a hairpin structure located within the LIR, followed by DNA synthesis and strand-displacement. Finally, the nascent DNA strand is ligated to produce a ss circular molecule. Both nicking and ligating events have been shown to be carried out by Rep, encoded by ORFs C1 and C2. RepA is thought to have an autoregulatory effect on Rep gene expression by binding to a Rep binding site located within the LIR and inhibiting complementary-sense gene expression (Palmer & Rybicki, 1998
). In addition, RepA is believed to play a role in transactivating virion-sense expression. Several conserved motifs have been identified within the Rep genes of other mastreviruses, including those required for oligomerization, DNA binding and cleavage (Palmer & Rybicki, 1998
). An LXCXE consensus motif for RepA binding to the retinoblastoma-related (RBR) protein has also been identified; the interaction of RepA with RBR enables the virus to retain the environment of the cell in S-phase, thus generating conditions suitable for supporting viral DNA replication (Liu et al., 1999a
).
The employment of geminivirus replicating vectors, or replicons, as putative gene amplification systems has been explored by a number of groups (Hayes et al., 1988b, 1989
; Timmermans et al., 1994
; Needham et al., 1998
; Woolston et al., 1998
). Timmermans et al. (1992)
have shown that a geminivirus vector was capable of replication when the Rep gene was provided in trans from a second vector containing the complete virus genome. In the present study, we examined in more detail the expression of various Rep gene products and investigated their capacity to support replication in trans of a minimal Rep and RepA-deficient reporter cassette containing the cis-acting sequences required for BeYDV replication. We placed Rep and RepA under independent promoter control and analysed their respective effects on replication. In an attempt to maximize the expression levels of our minimal BeYDV construct, we investigated the role of the CP in regulating these processes. Finally, we analysed the effect of the constitutively expressed Rep gene products on BeYDV infection.
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METHODS |
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Construction of plasmids.
Plasmids pSKBYD1.4 and pintron were kindly provided by J. Stanley (Liu et al., 1998
). A schematic diagram of the constructs made is shown in Fig. 1
. For the in trans studies, Rep, RepA and
intron ORFs were PCR-amplified from pSKBYD1.4 and p
intron, and subcloned into pIBT210 (Haq et al., 1995
) to generate p35SRep, p35SRepA and p35S
intron (Fig. 1b
). A fourth construct, p35S
BRep, was generated by removal of the 727 bp BamHI fragment of p35SRep and religating the template. p35SCP was constructed by PCR-amplifying the CP ORF and inserting it into the plasmid pIBT210.
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To generate the HA-tagged Rep constructs, the coding regions of Rep, intron and RepA were amplified by PCR, tagged at either the N or C terminus with a double haemmagglutinin (HA) epitope and inserted into a plant expression vector containing the CaMV 35S promoter and nopaline synthase terminator (Kovtun et al., 2000
). RepA and Rep RBR-binding mutants were constructed using the primer RBRLXCXQ (5'-GCAGATCCTGGTGGCAGCGGAGATCTGATTCTGTGAAGGG) by site-directed mutagenesis using the Bio-Rad mutagenesis kit.
Immunoblot analysis.
Approximately 50 µg total protein extracted from NT-1 cells were ground in sample loading buffer (100 mM Tris-HCl, pH 6·8, 1 % SDS, 20 % glycerol). Each sample was boiled for 10 min and loaded onto a gradient gel. Protein was electrotransferred onto nitrocellulose membranes. Blots were incubated overnight with anti-HA antibody at a concentration of 0·1 µg ml-1 and proteins were visualized using an enhanced chemiluminescence kit (Amersham).
GUS assay.
NT-1 cells cobombarded with pBYGUS as the reporter plasmid were analysed for GUS activity using the protocol of Jefferson (1987). Briefly, 1 g NT-1 cells was crushed using a micropestle and resuspended in GUS extraction buffer (50 nM NaPO4, pH 7·0, 0·1 % SDS, 10 mM Na2EDTA and 10 mM 2-mercaptoethanol). Total protein concentrations were determined by Bradford assay (Bio-Rad) using BSA as a standard. GUS activities were determined by fluorometric assay using 1 mM 4-methylumbelliferyl
-D-glucuronide (MUG) as substrate and 4-methylumbelliferone (MU) as standard. Assays were monitored by fluorometry (DYNEX technologies Fluorolite 1000). Data are presented as µmol min-1 (mg total protein)-1.
Southern blot analysis.
For Southern blot analysis, 2 µg of each plasmid DNA was cobombarded into a thick slurry of NT-1 cells which had been slowly pipetted onto Petri dishes containing NT-1 media plus 8 g agar l-1 (Sigma). Plates were then incubated at 28 °C for up to 8 days, depending on the experiment performed. DNA was extracted from cells using the procedure according to Wilke (1996). Ten µg total DNA of each sample were digested with HindIII and loaded onto a 1 % agarose gel. DNA was transferred onto nitrocellulose by capillary action (Sambrook et al., 1989
). A 1 kbp DNA fragment encoding GUS was 32P-labelled by random priming according to the conditions recommended by the manufacturer (Life Technologies) and used as a probe for hybridization in 25 mM Tris/HCl, pH 7·2, 1 mM EDTA and 7 % SDS at 65 °C. Filters were washed in 40 mM Tris/HCl, pH 7·2, 1 mM EDTA and 5 % SDS at 65 °C and signal detected by the STORM Optical Scanner (Molecular Dynamics) system. Signals were quantified using IMAGEQUANT software (Molecular Dynamics).
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RESULTS |
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Examination of Rep RBR-binding mutant
Since RepA possesses an RBR-binding domain (LXCXE) which is believed to be involved in cell cycle control, we were interested in determining the effect of this domain on our expression system. To this end, we constructed N-terminally HA-tagged Rep and RepA as well as the mutants 35SHARepARBRLXCXQ and 35SHAintronRBRLXCXQ (Fig. 3
a). The LXCXQ mutation was selected for these experiments because constructs containing this mutation had been previously shown by Liu et al. (1999a)
, using the two-hybrid yeast system, to have a greatly reduced ability to bind to maize RBR. These constructs were cobombarded into NT-1 cells along with the pBYGUS expression cassette. We compared the expression profiles of each mutant to wild-type N-terminally HA-tagged Rep and
intron by Western blot analysis. We found no difference in the level of expression of Rep gene products between wild-type and RBR-binding mutant constructs (Fig. 3b
, compare lane 1 with 3 and 4 with 6). In both cases, the level of RepA expression exceeded that of Rep. To determine whether the reduced ability of RepA to bind to RBR plays a role in replication, we cobombarded NT-1 cells with the pBYGUS expression cassette and either p35SRepARBRLXCXQ and p35S
intron, or p35SRepA and p35S
intronRBRLXCXQ. Despite the consistency among levels of gene products throughout these constructs, the levels of corresponding replication products from tissue cobombarded with p35S
intron and p35SRepARBRLXCXQ were significantly reduced (Fig. 3c
, compare lane 1 with 2). On the other hand, cells cobombarded with constructs p35RepA and p35S
intronRBRLXCXQ resulted in an accumulation of replication products comparable to that of wild-type p35SRepA and p35S
intron (Fig. 3c
, compare lane 1 with 3), suggesting that the drop in replication efficiency is more likely to be due to the decreased ability of RepA to bind to RBR rather than a general mutation which reduced replication efficiency in general.
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DISCUSSION |
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Replication was supported and reporter gene expression enhanced when RepA and Rep, comprising both C1 and C1 : C2 gene products, and intron, comprising the C1 : C2 gene product, were either expressed from the context of the native Rep promoter or placed under 35S promoter control. Attempts to increase reporter gene expression by simultaneously providing Rep in cis as well as in trans resulted in a decrease in the relative level of gene expression, although the replication efficiency increased more significantly. It was interesting to note in this case that the levels of GUS activity did not increase proportionally with the increase in DNA accumulation. Gooding et al. (1999)
demonstrated that wheat dwarf virus virion-sense gene expression may be tissue-dependent, as it became compromised in suspension cell cultures compared with embryogenic tissues. The authors suggested that the lack of corresponding increase in gene expression was due to an association of the viral DNA with a subset of minichromosomes that renders the virus in a replicationally committed state that promotes DNA synthesis rather than transcription. Such a phenomenon would provide a similar explanation of the results presented in this study. It is not clear whether changes in gene expression brought about by independent expression of Rep and RepA would be masked by changes in the replication efficiency. A more refined approach using non-replicating constructs to further examine the roles of Rep and RepA in regulating gene expression from complementary and virion-sense promoters is currently under way.
With the exception of BeYDV and TYDV, all geminiviruses that infect dicots possess a single continuous Rep gene; however, in monocots, splicing of Rep ORFs C1 and C2 is a general feature (Schalk et al., 1989). Gene expression has been demonstrated to be enhanced by the presence of an intron, most likely by affecting mRNA stability (McCullough et al., 1991
). Some monocot genes containing introns are prevented from being spliced in dicots. Digitaria streak virus (DSV), for example, exhibits inefficient splicing in wheat and maize; however its splicing is non-functional in tobacco (Mullineaux et al., 1990
). It has been considered that a component of the splicing machinery that predominates in monocots is expressed in dicots in limiting amounts or in a temporal or tissue-specific manner (Mullineaux et al., 1990
). Previously, examination of replication using a virus containing an intronless version of the Rep gene of BeYDV resulted in several-fold higher levels of replication products than the spliced version (Liu et al., 1998
). While such results were not observed in the present study, this discrepancy may be due to a difference in cell preparation and DNA delivery technology (virus-infected protoplasts were used in previous studies, as opposed to biolistic delivery into intact cells used in the current study). In addition, we did not observe significant differences in expression of C-terminally tagged versions of Rep derived from 35SRep or 35S
intron constructs, which could also explain the similarities in replication efficiency and GUS activity (Fig. 2a
). The results of our Western blot analysis of N-terminally tagged Rep gene products expressed in NT-1 cells implies that RepA accumulated to greater levels than Rep. Using a yeast expression system, Liu et al. (1999a)
, recorded that expression of BeYDV Rep and Rep A yielded bands on a Western blot of similar intensity; perhaps the splicing efficiency varies between these two systems. Since intron splicing may be host-specific and is either inactive or works less efficiently in other plant hosts, it may be wise to use the spliced form for more widespread application of this vector system.
It is interesting to note that whereas providing Rep and RepA from construct 35SRep or Rep alone from construct p35intron conferred comparable increases in replication efficiency, suggesting a more minor role for RepA in this assay, we found that supplementing Rep with RepA both individually under 35S promoter control enhanced replication. Perhaps this discrepancy in our results can be accounted for by the artificial alteration in the ratio of Rep to RepA, brought about by the coexpression of these two gene products under independent constitutive promoter control. The fact that a decrease in replication was observed when 35SRepA and pBYcisGUS (containing Rep in cis and under native reporter control) were cobombarded into NT-1 cells would strengthen this argument.
It was previously demonstrated that RepA of BeYDV contains an RBR-binding domain and binds to a plant homologue of the cell cycle regulator retinoblastoma protein (Liu et al., 1999a). RepA uses this property to reprogram the host and create a replication-competent environment (Gutierrez, 2000a
, b
). By altering this LXCXE motif, we showed that while the level of expression of the mutant Rep gene products remained similar to that of wild-type Rep gene products, replication efficiency was severely diminished, most likely due to the reduced ability of RepA to bind to RBR. Liu et al. (1999a)
conducted a series of experiments with Rep mutants in which altering the strength of RBR binding, as determined by the two-hybrid yeast system, did not appear to significantly affect virus replication in systemically infected Phaseolus vulgaris tissue. In the present report, we have demonstrated that the introduction of a mutation to the LXCXE consensus motif of RepA resulted in a marked reduction in replication efficiency of our BeYDV replicon system. Since our experiments were performed on NT-1 cells, which were plated out for synchronous growth, it is possible that the efficiency of RBR binding to RepA is more pronounced under these conditions than when an entire plant is infected.
In a number of geminiviruses, the absence of CP has been shown to result in the abolition of virus infectivity as well as a diminishment of ssDNA levels (Azzam et al., 1994; Qin et al., 1998
; Woolston et al., 1998
; Liu et al., 1998
). When a non-specific ssDNA binding protein was used as a substitute for tomato leaf curl virus CP, ssDNA accumulated to wild-type levels (Padidam et al., 1996
, 1999
). Liu et al. (1997b
, 1999b
, 2001)
have indicated that the CP of maize streak virus, another mastrevirus, binds to ssDNA to facilitate both nuclear trafficking of viral DNA as well as cell-to-cell movement. It is possible that early on in infection, the CP is available to interact with but not abundant enough to fully encapsidate viral DNA until later on in the infection process. Consequently, the CP may regulate ssDNA accumulation by sequestering it away from the replication pool (Qin et al., 1998
). In this report, expression of the CP gene resulted in a marked increase in ssDNA accumulation and a decrease in reporter gene expression. Since we have eliminated the native CP gene from our expression system, we have optimized reporter gene expression.
In this report, we have examined the effects of constitutively expressed Rep and RepA on BeYDV replication. Further work concerning their effects on reporter gene expression using replication-deficient constructs is currently under investigation.
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ACKNOWLEDGEMENTS |
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Received 9 July 2003;
accepted 13 August 2003.
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