The Plant Cell, Vol. 4, 213-223, February 1992 O 1992 American Society of Plant Physiologists
Coordinate Regulation of Replication and Virion Sense
Gene Expression in Wheat Dwarf Virus
Julie M. 1. Hoferya9'Elise L. Dekker,b Helen V. Reynolds,' Crispin J. WoolstonIai2Brian S. CoxYb
a n d Philip M. Mullineauxa
a John
lnnes Institute, John lnnes Centre for Plant Science Research, Colney Lane, Norwich NR4 7UH, United Kingdom
Department of Plant Sciences, University of Oxford, Oxford 0x1 3RA, United Kingdom
We have investigated the relationship between viral DNA replication and virion sense gene expression in wheat dwarf
virus (WDV), a member of the geminivirus group, by testing a series of deletion mutants in transfected Triticum monococcum (einkorn) protoplasts. Mutants contained a transcription fusion of the chloramphenicol acetyltransferase coding
sequence to the virion sense promoter that replaced the viral coat protein coding sequence. The deletion analysis revealed that WDV replication and virion sense transcription can proceed independently and are controlled in part by
nonoverlapping elements i n the large intergenic region. These data and those from a C2 open reading frame (ORF)
frameshift mutant also showed that the product of the C2 ORF (C1-C2 protein) is independently involved in both DNA
replication and activation of the virion sense promoter. The amino acid sequences encoded by C2, which are highly consewed in the geminivirus group, show some homology to the DNA binding domain of the myb-related class of plant
transcription factors. The possible involvement of the host in controlling the function of the C142 protein and the implication of these data for the development of WDV-based gene vectors are discussed.
INTRODUCTION
The geminiviruses are plant DNA viruses and those that
infect monocotyledonous (monocot) hosts are particularly
good candidates for the study of interactions between transcription and DNA replication in plants (Accotto et al., 1989;
Mullineaux et al., 1990). Wheat dwarf virus (WDV) is a member of the subgroup of monocot-infectinggeminiviruses that
includes maize streak virus (Howell, 1984; Mullineaux et al.,
1984), Digitaria streak virus (DSV; Donson et al., 1987), and
chloris striate mosaic virus (Anderson et al., 1988). The genome of WDV is monopartite and consists of a circular 2.7-kb
molecule of single-stranded DNA, which replicates in infected
tissue as double-stranded DNA (dsDNA) (MacDowell et al.,
1985; Woolston et al., 1988). Sequence analysis reveals the
presence of four open reading frames (ORFs) (Vl, V2, C1,
and C2) common to all monocot-infecting geminiviruses,
and mutagenesis studies have been used to determine
their functions. V1 may be involved in viral spread and symptom development (Mullineaux et al., 1988; Boulton et al.,
1989; Lazarowitz et al., 1989), and V2 is the capsid protein
(Morris-Krsinich et al., 1985). Neither of these ORFs is required for dsDNA replication in protoplastsderived from Triticum monococcum (einkorn) cell suspension cultures
(Woolston et al., 1989; Matzeit et al., 1991); however, both
To whom correspondence should be addressed.
Current address: Department of Applied Biology, University of
Hull, Hull HU6 7RX, United Kingdom.
complementary sense ORFs, C1 and C2, are essential
(Schalk et al., 1989; Ugaki et al., 1991). The large intergenic
region (LIR; Davies et al., 1987) of the viral genome is located
between the 5'ends of ORFs V1 and C1. A highly conserved
nanonucleotide sequence within the LIR comprises part of
the head of a potential stem-loop structure and is thought to
be the origin of replication (ofi; MacDowell et al., 1985;
Revington et al., 1989). This motif resembles the ori of the
single-stranded DNA bacteriophage (pX174 (Shlomai and
Kornberg, 1980) and is almost identical to a site required for
the initiation of DNA synthesis in the human adenoviruses
(Rawlins et al., 1984).
WDV transcription proceeds bidirectionallyfrom within the
LIR (Dekker et al., 1991). A single, abundant, virion sense
transcript directs the synthesis of the V1 and V2 ORF products. As in DSV, two complementary sense transcripts, one
spliced and one unspliced, appear to direct the synthesis of
a 41-kD splice-mediatedfusion of the C1 and C2 ORFs (C1-C2
protein) and a 30-kD C1 product, respectively (Accotto et al.,
1989; Mullineaux et al., 1990; Dekker et al., 1991). Neither
product has yet been detected in vivo. Whereas the C1-C2
ORF product is essential for the replication of WDV dsDNA
in einkorn protoplasts(Shalk et al., 1989; Matzeit et al., 1991),
a role for the putative unspliced complementary sense transcript product, C1, has not yet been defined. The subgroup
of geminiviruses that infects dicotyledonous (dicot) plants encodes an AC1 (or AL1) ORF product that is equivalent to the
214
The Plant Cell
WDV C1-C2 protein (Hanley-Bowdoin et al., 1990; Etessami
et al., 1991). All other factors required for geminivirus replication and transcription are probably of cellular origin. This dependente on host proteins makes the geminiviruses an
excellent model system for investigating replicationand its relation to gene expression in plants (Accotto et al., 1989). In
addition, the participation of cellular factors in the regulation
of gene expression should be addressed if geminiviruses are
to be developed further as plant transformation vectors.
We used a protoplastsystem to investigatethe organization
of control elements in WDV, and our results revealed that independent sequences in the LIR are required for replication
and virion sense promoter activity. We have shown that the
Cl-C2 protein is also essential for both processes and can
contribute to each independently. We consider the possible
involvement of the host cell in regulating transcription and
replication and whether the C1-C2 protein might be a transcription factor.
pWDV2
7.2kb
RESULTS
Standard Constructs
We used three standard WDV constructs, shown in Figure l A ,
to study the relationshipbetween WDV virion sense promoter
activity and vira1 dsDNA replication. pWDV2CAT contains the
chloramphenicol acetyltransferase (CAT) coding sequence
(Alton and Vapnek, 1979) cloned between the BstEll (nucleotide 500) and Mlul (nucleotide 1246) sites of WDV, which
replaces the WDV V2 ORF in a transcription fusion to the
virion sense promoter (for details of construction, see Methods). Smaller, replicating WDVCAT molecules (a possible
replicon form is shown in Figure 16) are generated from
pWDV2CAT in transfected protoplasts, probably as a result of
replication (Woolston et al., 1989).
pWDV3CAT (Figure 1A) is replication defective due to
frameshift mutations in both C2 ORFs, which would result in
prematurely terminated translation of the C1-C2 ORF fusion
product. In every other respect, pWDV3CAT is identical to
pWDV2CAT.
A second replication defective construct, pWDV3.35SCATNOS (Figure lA), contains the same C2 ORF frameshift
mutations; however, the CAT coding sequence is under the
control of a cauliflower mosaic virus (CaMV) 35s promoter
and a nopaline synthase polyadenylation sequence. The
35SCATNOS fragment was inserted in an antisense orientation with respect to the WDV virion sense promoter to ensure
that the CaMV 35s promoter was positioned as far as possible from any regulatory sequences in the LIR.
Time-Course Experiments
Einkorn protoplasts transfected with pWDV2CAT and
pWDV3CAT were sampled at 24-hr intervals for 6 days after
Figure 1. pWDVCAT Vectors.
(A) pWDV2 is a 1.4-mer tandem repeat of WDV inserted into
pBluescript KS+ (Alting-Mees and Short, 1989). Vira1 ORFs VI, C1,
and C2 are marked. A polylinker has been introduced 113 bp 3'to the
BstEll site (nucleotide 500) of WDV, and a unique BamHl site has
been inserted 7 bp 5' to the BstEll site to facilitate the introduction
of reporter genes. UTT is a universal translation termination oligonucleotide (Mullineaux et al., 1988) inserted to prevent translation read
through from ORF VI. Deletion mutations removed sequences from
ORF V1 and the LIR (indicated by a doubleheaded arrow). The complementary sense TATA box is marked with an open triangle; the
virion sense TATA box is marked with afilled triangle. The CAT coding
sequence (Alton and Vapnek, 1979) was inserted into the EstEll and
Bglll sites of pWDV2 to generate pWDV2CAT. Ndel sites (') were
filled in and self-ligated to generate pWDV3CAT, a replicationdefective vector containing frameshift mutations in both C2 ORFs.
pWDV3.35SCATNOScontains the CaMV 35s promoter, the CAT coding sequence, and the NOS polyadenylation sequence inserted into
the Xhol site of the polylinker in an antisense orientation with respect
to the WDV virion sense promoter.
(B) Predicted form of a WDV2CAT replicon generated from
pWDV2CAT DNA in transfected einkorn protoplasts.
Replication and Gene Expression in WDV
inoculation. Figure 2 shows a DNA gel blot analysis (Southern,
1975) of DNA extracted from protoplasts inoculated with
pWDV2CAT (lanes b to g) that revealed persistent opencircular (oc), supercoiled (sc), and linear forms of plasmid
DNA and additional smaller DNA species, which were the sc
and oc forms of the WDV2CAT replicon (Figure 1B). This assignment of DNA forms was based on the analysis outlined
below and on previous experiments (Woolston et al., 1989).
The smaller DNA forms were not detectable in DNA extracted
from pWDVSCAT-transfected protoplasts (Figure 2, lanes h to
m). Digestion of total DNA with BamHI converted plasmid
and WDV replicon DNAs to linear forms of the predicted
sizes, 7.2 kb and 3.2 kb, respectively, and DNA gel blot analysis using a CAT-specific DNA probe showed the reporter
gene to be present in both DNA species (J.M.I. Hofer and P.M.
Mullineaux, unpublished data). Furthermore, the smaller
forms of DNA (oc and sc) were susceptible to digestion with
Day
Day
1 2 3 4 5 6
1 2 3 4 5 6
Input
DNA
oc*
wt oc
sc'
wt sc
I wt ss
a
l!
b c d e f g
:
.r
Ui
h i j k l m
n
Figure 2. Replication of WDVCAT Constructs in Einkorn Protoplasts.
An autoradiograph is shown of a DNA gel blot of DNA extracted from
transfected protoplasts that were separated on a 1% agarose gel and
probed using a full-length Ncol monomer of WDV-specific DNA. Lane
a contains 2 ng of input pWDV2CAT DNA. Lanes b to g were each
loaded with 5 ng of DNA extracted from pWDV2CAT-transfected protoplasts on days 1 to 6 after inoculation. Lanes h to m were each
loaded with 5 ng of DNA extracted from pWDV3CAT-transfected protoplasts on days 1 to 6 after inoculation. Mbol-sensitive (*), oc, and
sc forms of double-stranded replicon DNA were generated in
pWDV2CAT-transfected protoplasts but were absent in pWDVSCATtransfected protoplasts. Lane n contains DNA extracted from wildtype (wt) WDV-infected wheat seedlings. Single-stranded (ss) viral
DNA was not routinely detected in transfected protoplasts.
215
Mbol, whereas the plasmid DNA was not (J.M.I. Hofer and
P.M. Mullineaux, unpublished data). This suggested that the
WDV replicon molecules had an altered pattern of DNA methylation, which rendered them sensitive to this enzyme, and
were therefore likely to have been synthesized de novo
(Woolston et al., 1989). The replicative DNA forms observed
in protoplasts transfected with pWDV2CAT were identical to
those observed in wild-type WDV-transfected protoplasts
reported previously (Woolston et al., 1989).
These time course experiments confirmed that an intact C2
ORF was required for the generation of replicative WDV
dsDNA forms. They also demonstrated that the ability of an
inoculated plasmid to give rise to replicating WDV monomers
may be assayed by the presence or absence of the small
Mbol-sensitive dsDNA forms identified in Figure 2.
Half of each sample harvested for DNA extraction was retained and assayed for CAT activity as shown in Figure 3. In
our expression system, a transcriptionally fused CAT gene
was used to measure the activity of the WDV virion sense promoter. In protoplasts transfected with pWDV3.35SCATNOS
(Figure 3A), maximum CAT activity was observed 24 hr after
inoculation and was maintained throughout the experiment.
A similar CaMV 35S promoter activity profile has been
demonstrated in maize protoplasts after uptake of a plasmid
containing the CAT reporter gene (Maas et al., 1990). Transient gene expression in protoplasts is a well-established
technique (Fromm et al., 1985; Werr and Lorz, 1986; Prols et
al., 1988), and our results for construct pWDV3.35SCATNOS
were typical, showing that input plasmid DNA was capable
of driving the expression of the CAT reporter gene.
Virion sense promoter activity measured in protoplasts
transfected with pWDV2CAT showed a different profile (Figure 3B). Promoter activity was initially low, then increased,
concomitant with an increase in the number of molecules of
replicating dsDNA (Figure 2, lanes b to g). Similar promoter
activity profiles were obtained in protoplasts transfected with
a WDV dimer construct, pWDV1-59 (see Methods; data not
shown), and a pWDVCAT construct reported previously
(Matzeit et al., 1991).
In complete contrast to the results above, no CAT activity
was measured in protoplasts transfected with pWDVSCAT
(Figure 3C). This indicated that the virion sense promoter was
not active on input DNA in pWDVSCAT-transfected protoplasts and that either actively replicating DNA or the expression of the C2 ORF was necessary for activation of the
virion sense promoter.
Deletion Mutagenesis of the LIR
Figure 4 shows a series of deletion mutants derived from
pWDV2CAT that were constructed to determine the relationship between the virion sense promoter and DNA replication.
In these experiments, transfected protoplasts were harvested
and assayed 4 days after inoculation when levels of virion
sense promoter activity and viral replication were approaching a maximum (Figures 2 and 3).
216
The Plant Cell
% CAT
A
activity
70
90 70 90 90 100
3-CM
1-CM
CM
day
123456
%CAT
B
activity
10 30 ?o 70 90 100
3-CM
1-CM
CM
G
day 1 2 3 4 5 6
CM
day 1 2 3 4 5 6
Figure 3. CAT Activity in Einkorn Protoplasts Transfected with
WDVCAT Constructs.
(A) Protoplasts transfected with pWDV3.35SCATNOS. One hundred
percent is equivalent to 0.25 nmol of chloramphenicol converted to
3-CM per 106 protoplasts per hour.
(B) Protoplasts transfected with pWDV2CAT. One hundred percent is
equivalent to 0.30 nmol of chloramphenicol converted to 3-CM per
106 protoplasts per hour.
(C) Protoplasts transfected with pWDV3CAT. A1% background of CAT
activity was detected.
A preliminary analysis determined that deletion of the V1
ORF between nucleotides 299 and 500 affected neither
dsDNA replication nor virion sense gene expression (J.M.I.
Hofer, H.V. Reynolds, and P.M. Mullineaux, unpublished
data), which is consistent with results obtained for WDV V1
deletion mutants reported elsewhere (Laufs et al., 1990;
Ugaki et al., 1991). Deletions that removed sequences between nucleotides 2746 and 500 (pSSB430 and pABP514;
Figure 4A) resulted in a loss of CAT activity and a loss of the
mutants' ability to replicate. A deletion between nucleotides
2683 and 39 (pECA101; Figure 4A) resulted in the same
phenotype. Therefore, elements located between nucleotides
2683 and 299 were essential for both replication and virion
sense gene expression.
A deletion that removed sequences between nucleotides
2750 and 39 (pAPA38; Figure 4A) did not affect either virion
sense promoter activity or dsDNA replication deleteriously.
This small segment of the LIR was therefore not required for
either activity (Figure 48).
Another group of mutants that were deleted between
nucleotides 142 and 500 (pAVB365, pBSB347, and pALB288;
Figure 4A) gave rise to WDV replicons, yet CAT activity was
very low. This indicated that elements between nucleotides
142 and 500 in the LIR were dispensable for WDV dsDNA
replication and that dsDNA replication could proceed in the
absence of virion sense promoter activity (Figure 4B).
Protoplasts transfected with a mutant deleted between
nucleotides 2746 and 81 (pAPS84; Figure 4A) yielded a fourth
phenotype. A relatively high level of virion sense promoter activity was maintained, although no dsDNA replicons could be
detected by DNA gel blot analysis (Southern, 1975). This
demonstrated that virion sense promoter activity and dsDNA
replication in WDV were independent events, and this mutant
delineated a region between nucleotides 2746 and 81 that
was necessary for dsDNA replication (Figure 4B). A summary of the domains in the WDV LIR defined by this series
of deletion mutants shows that although replication and virion
sense promoter functions could be separated, some sequences were also shared (Figure 4B).
We investigated further the activity of the virion sense promoter in replication defective WDV constructs. The profile
of promoter activity of mutant pAPS84, which increased
with time in a similar manner to the profile of pWDV2CAT, is
shown in Figure 5. Introduction of a frameshift mutation in
the C2 ORF of pAPS84 to generate a double mutant,
pAPS84.WDV3CAT (Figure 4A), abolished CAT activity and
Transfected protoplasts were harvested on days 1 to 6 after inoculation and assayed for CAT activity. Typical autoradiographs of the
different acetylated forms of chloramphenicol (CM), 3-acetylchloramphenicol (3-CM), and 1-acetyl-chloramphenicol (1-CM) that
were separated by thin-layer chromatography are shown. Chloramphenicol acetylation was quantified by scanning densitometry and
expressed relative to the amount of 3-acetyl-chloramphenicol measured on day 6 (100%).
Replication and Gene Expression in WDV
LIR
217
t
CAT Acthrlty
Relative t o
WDV2CAT
Repllcatlon
< 2%
J
J
c 2%
J
< 2%
X
< 2%
X
66% (SE12%)
J
45% (SE11%)
< 2%
X
X
pWDV3CAT
< 2%
X
pAPS84.WDV3CAT
< 2%
X
< 2%
1 CQ
1
I
CAT
I
CAT
>
>
Figure 4. WDV Deletion Mutants.
(A) The WDV V2 ORF was replaced with the CAT coding sequence to generate transcriptionfusions to the virion sense promoter. Deletion mutations were made in the LIR, a noncoding region of the WDV genome extending between the V I and C1 ORFs. CAT activity was determined for
each deletion mutant from an extract prepared from transfected einkorn protoplasts harvested 4 days after inoculation. The values given are
averages obtained from at least eight individual transfection experiments. CAT activity measurements were included with the provision that a
positive result was obtained from pWDV2CAT-transfectedprotoplasts in the same experiment. One hundred percent is equivalent to 0.30 nmol
of chloramphenicol converted to 3-acetyl-chloramphenicol per 106protoplasts per hour (SE, standard error). Replication was scored according
to detection of WDV replicon DNA (refer to Figure 2).
(8)A summary representation of functional regions in the LIR where dotted rectangles delineate the regions required for dsDNA replicationand
cross-hatched rectangles delineate the regions required for virion sense gene expression defined in this study.
confirmed that the C2 ORF was required for virion sense promoter activity, independent of the ability to produce replicons.
DlSCUSSlON
pWDV2CATis a WDVvector designed to measure WDV virion
sense gene expression from a transcriptional fusion of the
virion sense promoter to the CAT reporter gene and was constructed such that tandemly repeated sequences enable the
release of replicative dsDNA monomers in einkorn protoplasts. The replication defective construct, pWDV3CAT,
showed that an intact C2 ORF, expressed as a splicemediated fusion to the C1 ORF (Schalk et al., 1989; Dekker
et al., 1991), is essential for WDV dsDNA replication in
einkorn protoplasts. Our deletion analysis enabled us to identify two regions within the LIR that are also required for
dsDNA replication (Figure 48). The first region, defined by
mutants PECA101 and pAPA38 (nucleotides 2689 to 2746),
contains both mapped complementary sense transcript start
sites and the predicted complementary sense TATA box
(nucleotide 2720; Dekker et al., 1991); therefore, failure of mutant PECA101 to replicate was probably due to disruption of
the expression of the C1C2 transcription unit. The second
essential region, delineated by mutants pAPA38, pAPS84,
pSSB430, and pAVB365 (nucleotides 34 to 142; Figure 4B),
is probably not directly involved in the expression of complementary sense ORF products because pAPA38 was competent in both replication and virion sense transcription, yet
pAPS84, with a slightly larger deletion extending away from
the complementary sense ORFs, failed to generate replicons.
The Plant Cell
218
1O0
90
80
.-a
70
.->
5 60
cd
I50
40
8
'
30
20
10
1 2 3 4 5 6
Days after inoculation
Figure 5. CAT Activity in Einkorn ProtoplastsTransfected with Deletion Mutant pAPS84.
Typical time c o m e s of CAT activity are shown for protoplasts transfected with mutant pAPS84 (dashed line) compared to protoplasts
transfected with pWDV2CAT (solid lhe) and pAPS84.WDV3CAT (dot-
ted line).One hundred percent represents0.30 nmol of chloramphenicol converted to 3-acetyl-cloramphenicol per 106 protoplasts per
hour.
The second region includes the conserved geminivirus
nanonucleotide sequence (nucleotides 77 to 85) and provides further evidence that this motif is part of the geminivirus core ori, either as a DNA nicking site (Rogers et al., 1986;
Saunders et al., 1991) or as a recognition site for the binding
of a replication complex (Huberman, 1987; Challberg and
Kelly, 1989).
Two regions of the WDV LIR are required for virion sense
promoter activity (Figure 46). The first region was delineated
by mutants PECA101 and pAPA38 and includes the complementary sense TATA box (Dekker et al., 1991). As previously described, the virion sense promoter is activated by the
C1-C2 protein and, therefore, is indirectly dependent on an
active, complementary sense promoter. The second domain
(nucleotides 81 to 299; Figure 46) contains the virion sense
transcription start sites and sequences 105 bp upstream, including the putative virion sense TATA box at nucleotide 225
(Dekker et al., 1991). Mutants pAPS84, pAV6365, and our preliminary V1 ORF deletions defined this region. Other parts of
the LIR may also contribute to virion sense promoter function
in the form of enhancer or activator sequences. The LIR of
maize streak virus contains GC-rich elements, homologous
to the Spl binding site of mammalian promoters, which activate virion sense transcription in maize protoplasts (Fenoll et
al., 1988,1990). Similar GC boxes may be present in the WDV
LIR. The 84-bp deletion of mutant pAPS84 removed two
GC-rich stretches 5'to the geminivirus nanonucleotide motif,
and their absence may account for a reduction in virion sense
promoter activity observed. Other enhancer elements also
may be located upstream of the virion sense TATA box. Particularly striking are repeated polypyrimidine tracts (CTTTT)
that may affect DNA curvature (Travers, 1989) and CCAAT
box motifs (Jones et al., 1985, 1987; Cohen et al., 1986) at
nucleotides 2699 and 2708, which are present as imperfect
palindromes in a number of other geminiviruses.
A correlation between replication and virion sense promoter activity was indicated by concomitant increases in CAT
activity and replicon dsDNA forms in protoplasts transfected
with pWDV2CAT. Constructs pWDV3CAT and PECA101 revealed that the C1-C2 protein was necessary for virion sense
promoter activity, whereas mutant pAPS84 indicated that actively replicating DNA monomers were not required. Our
results suggest that the relationship between replication and
virion sense transcription in WDV is mediated by the C1-C2
ORF fusion product, which activates the virion sense promoter in conjunction with, but separate from, its role in replication (Schalk et al., 1989). The AC2 (AL2) ORF of a bipartite,
dicot-infecting geminivirus, tomato golden mosaic virus, has
been shown to trans-activate the virion sense promoter of genome component A and may also trans-activate the virion
sense promoter of genome component 6 (Sunter and Bisaro,
1991). None of the monocot-infecting geminiviruses sequenced to date contains an ORF equivalent to AC2. In WDV,
the C1-C2 ORF product therefore appears to be a multifunctional protein, equivalent to both the AC1 and AC2 proteins
of dicot-infecting geminiviruses. In many ways, the activity of
the C1-C2 protein is analogous to the large T-antigen of simian virus 40, which is another multifunctional protein involved
in the initiation of DNA replication and activation of late promoter activity (Prives, 1990).
It is clear from our data that all virion sense fusions in WDV
require an activating component. In previous studies in which
WDV-based replicons have been used for the development
of plant vectors (Matzeit et al., 1991; Ugaki et al., 1991), the
contribution of replication to the level of reporter gene expression has been estimated by comparison with replication
defective mutants that have disrupted expression of their
complementary sense ORFs. This comparison may have
given an overestimate of the contribution of vector replication
to the level of reporter gene expression because in all cases
the reporter gene was fused to the virion sense promoter. We
note that deletion mutant pAPS84, which has part of the putative geminivirus ori deleted but retains an intact complementary sense transcription unit, gave levels of CAT activity
approximately half that of pWDV2CAT (Figures 4A and 5).
Therefore, under our experimental conditions, the contribution of replication to the total level of CAT activity was no more
than twofold.
Replication and Gene Expression in WDV
Our data do not reveal whether activation of the virion
sense promoter is a direct or an indirect interaction between
the C1-C2 protein and viral sequences. Other host factors
may be involved in a multicomponent complex, or C1-C2 may
regulate host genes, which, in turn, influence viral replication
and virion sense transcription. Figure 6 shows a region of
the C1-C2 ORF, identified as an NTP-binding domain
(Gorbalenya and Koonin, 1989), which contains amino acid
residues that are also conserved in the N-terminal DNA binding domain of the avian myeloblastosis (myb) oncogenehomologous regulatory genes C7 (Paz-Ares et al., 1987) and
P7 (Grotewold et al., 1991) of maize and Hv7 (Marocco et al.,
1989) of barley. Apart from this region of homology, the C1-C2
polypeptide does not show features characteristic of myb-like
gene products in plants and animals, for example, repeat domains (Klempnauer and Sippel, 1987) and evenly spaced
Repeat 2
<----------------------------
DDI..PFKFT
DDI..PFKFC
DDI..PFKFC
DDI..PFKFV
DDVDPTYLKR
DDVTPQYLKL
DDISPNYLKL
DDVDPHYLKH
PNWKCFVGAQ
PCWKQLIGCQ
PCWKQLVGCQ
PCWKGLVGSQ
KHWKHLIGAQ
KHWKELIGAQ
KHWKELIGAQ
..FKEFNGSQ
Figure 6. Amino Acid Sequence Comparison.
The deduced amino acid sequences from the N-terminal region of
myb gene homologs in maize (ZmC7, Paz-Ares et al., 1987; ZmP1,
Grotewold et al., 1991) and barley (Hv7, Marocco et al., 1989) were
compared to a highly conserved domain in the C1-C2 ORF of four
monocot-infecting geminiviruses, WDV, DSV, maize streak virus
(MSV), and chloris striate mosaic virus (CSMV) and an equivalent domain in the C1 ORF of genome component A of four dicot-infecting
geminiviruses, beet curly top virus (BCTV Stanley et al., 1986),
tomato golden mosaic virus (TGMV Hamilton et al., 1984), bean
golden mosaic virus (BGMV; Howarth et al., 1985), and African cassava mosaic virus (ACMV; Stanley and Gay, 1983). Conserved amino
acids and conservative changes with a threshold pair value of >0.2
(calculated according to the scoring system of Gribskov and Burgess,
1986) are boxed. Consensus phosphorylation sites are marked (').
The two convergent ends of repeat sequences characteristic of plant
myb-like genes (Marocco et al., 1989) but not present in the geminivirus sequences are shown at the top of the alignment.
219
tryptophans (Saikumar et al., 1990). It is interesting to note
that proteins encoded by the maize P7 gene arise as a result
of alternative splicing at their 3'ends (Grotewold et al., 1991),
a process similar to the intron skipping observed in the transcription of the complementarysense ORFs of WDV and DSV
(Accotto et al., 1989; Schalk et al., 1989; Mullineaux et al.,
1990; Dekker et al., 1991).
The host cell is thought to play a role in the regulation of
geminivirus replication (Townsend et al., 1986). Despite the
fact that the complementary sense promoter of WDV must be
active on input DNA in einkorn protoplasts (to generate viral
replicons), the onset of virion sense promoter activity appeared to be delayed compared with the CaMV 35s promoter
(Figures 3A and 3B), and WDV2CAT replicon DNA was not
detected immediately after transfection (Figure 2A). We have
shown that the C1-C2 protein plays a pivotal role in viral replication and gene expression; it is, therefore, a likely target for
temporal regulation by host cell factors. These factors could
regulate complementary sense transcription and the splicing
of complementary sense transcripts (Mullineaux et al., 1990;
Dekker et al., 1991), and could modify the activity of their
translated proteins. The ratios of C l and C1-C2 proteins
would be significant, for example, if cooperative binding to viral or host genomes was necessary,such as the heteromeric
binding of the fos and jun protooncogene-encodedtranscription factors (Kouzarides and Ziff, 1989) or if the proteins functioned antagonistically, as do the products of alternatively
spliced fosB transcripts (Murnberg et al., 1991).
At least two potential phosphorylation sites are located in
the putative NTP-binding domain (Gorbalenya and Koonin,
1989) of the C1-C2 protein and are conserved between all the
geminiviruses sequenced to date (Figure 6).RXX$/T is a
consensus site for calmodulin-dependent protein kinases
(Sommer et al., 1990), RIGK was identified using the computer program Scrutineer (Sibbald and Argos, 1990) and is
a consensus site for phosphorylation by protein kinase C.
These and other adjacent Ser and Thr residues may be targets for post-translational modification by the host cell. The
DNA binding activity of the c-myb-encoded protein is regulated by phosphorylation and may be linked to severa1 host
cell signal transduction pathways (Lüscher et al., 1990). Simian virus 40 DNA replication has also been shown to be linked
to the host cell cycle by the activity of cellular phosphatases
and kinases that regulate the phosphorylation status of the
viral T-antigen (McVey et al., 1989; Virshup et al., 1989) and
by cyclin (Prelich et al., 1987), a nuclear protein that is encoded by monocot and dicot plant genomes (Suzuka et al.,
1989). WDV may interact with the host cell in a similar
manner.
METHODS
Molecular Techniques
Plasmids were constructed using standard recombinant DNA procedures (Sambrook et al., 1989). Restriction endonucleases and DNA
220
The Plant Cell
modifying enzymes were obtained from Bethesda Research Laboratories; radioisotope-labeledchemicals were obtained from Du PontNew England Nuclear.
Construction of WDVCAT Transcription Fusions
pJIT34 (Woolston et al., 1988) is a 2750-bp monomer of a Czechoslovakian isolate of WDV inserted into the Kpnl site of pUC18
(Yanisch-Perronet al., 1985). pJIT34 was used as the source of vira1
DNA for pWDV1-59, a WDVCAT dimer. Construction was as follows:
(1) The V2 ORF ATG was removed by digesting pJIT34 with Cfrl
(nucleotide 486) and BstEll (nucleotide 500), rendering the ends
blunt with T4 DNA polymerase, and subsequently ligating the ends
in the presence of 100 M excess of BamHl linker (5’CGGGATCCCG
33. The resulting plasmid, pJIT34A4, contained Saml, BamHI, and
BstEll sites at the deletion junction. This manipulation changed two
predicted amino acid residues of the V1 ORF. (2) The 772-bp Taql
fragment containing the Tn9 CAT coding sequence (Alton and
Vapnek, 1979)from pBR328 was inserted into the Sphl and Kpnl sites
of pUC19 after the ends of the DNA fragments were made flush. This
plasmid was called “pJIT24.” (3) The CAT coding sequence was
released from pJIT24 as a Hindlll-Sacl fragment, the ends were made
flush, and the fragment was inserted into two Alul sites (nucleotides
539 and 630) of pJIT34A4 in a three-step ligation. (4) The resulting
plasmid, pJIT34A4CAT, was digested with Kpnl to release a WDVCAT
monomer, which was self-ligatedand inserted as a tandem dimer into
the Kpnl site of pUC18 to generate pWDV1-59.
The source of WDV DNA for constructs pWDV2 and pWDV2CAT
(Figure 1) was a WDV dimer equivalent of pJIT34A4, called
“pJIT34A4D.” pWDV2 was generated as follows: (1) A Hindll monomer
was released from pJIT34A4D and inserted into the Hindlll site of
pBluescript KS+ (Alting-Meesand Short, 1989) to create pJIT34BS1,
A 1.4-kb Mlul (end-filled, nucleotide 1246)-Ncol (nucleotide 2591)
fragment was released from pJIT34A4D and inserted into pJIT34BSl
at the Ncol (nucleotide 2591) and EcoRV (polylinker) sites to create
pJIT34BS1.4. (2) The V2 ORF was deleted by digesting with Mlul
(nucleotide 1246) and BstEll (nucleotide 500),filling the sites, and
recircularizing the plasmid (BstEll site restored, pJIT34BS1.4AV2).
(3) Restriction sites in the pBluescript KS+ polylinker were removed
in two steps by digestion with Asp718 and Clal, making the ends
flush, and self-ligating (Kpnl restored), then repeatingthe procedure
using the Pstl and Xbal sites to create pJIT34BS1.4AV2Amcs. (4) A
619-bp BamHI-Rsalfragment from pJIT34A4was ligated to a 21-mer
oligonucleotide containing Bglll, Xhol, EcoRV, and Sphl sites. The
resulting 640-bp fragment was inserted into the BamHl and EcoRV
sites of pBluescript SK+ to create pBS2. (5) pBS2 was digested with
Sall, end filled, and subsequently digested with BamHl to release a
663-bp fragment that was inserted into the BamHl and BstEll (filledin) sites of pJIT34BS1.4AV2Amcs to create pWDVl. (6) To prevent interference with translation of inserted sequences fused to the V1
ORF, an oligonucleotide with TAA non-sense codons in all three
ORFs (Mullineaux et al., 1988) was inserted at the Smal site. Translation of the VI ORF would be truncated by nine codons. This plasmid
was called “pWDV2.” pWDV2CAT was generated by recovering a
1.0-kb BstEll-BamHI fragment containing the CAT coding sequence
from pWDV1-59 and inserting it into the BstEll and Bglll sites of
pWDV2 (Figure 1).
pWDV3CAT contains a frameshift mutation in both C2 ORFs obtained by destroying two Ndel sites in the following way: (1) A 2.1-kb
Sacll-EcoRVfragmentwas releasedfrom pWDV2CAT and ligated into
pBluescript SK+, and digested with the same restriction enzymes.
(2) The resulting plasmid, containing only one copy of the C2 ORF,
was digested with Ndel, blunt ended, and recircularized. (3) The
mociified Sacll-EcoRV fragment was reinserted into the same sites of
pWDVPCAT, replacing one of the wild-type C2 ORFs. (4) The remaining Ndel site was removed by linearizing the plasmid with Ndel, making the ends flush and recircularizing to obtain pWDV3CAT.
pWDV3.35SCATNOS was constructed by releasing a 2.3-kb SallXhol fragment from p35SCATNOS and inserting it into the Xhol site
in the polylinker of pWDV3CAT in an antisense orientation with respect to the WDV virion sense promoter. p35SCATNOS is a pUCbased vector containing three fragments: 4.0kb of the NOS 3’polyadenylation sequence from the Sphl site to the Hindlll site (Depicker
et al., 1982); the CAT coding sequence, inserted as a Stul-Sacl fragment from pJIT25 (equivalent to pJIT24, with the CAT coding sequence in the opposite orientation); and the CaMV 35s promoter,
released as a BamHl fragment from pJIT112. pJIT112 is derived from
pJIT58 (Guerineau et al., 1990) and contains the 355 promoter sequence from CaMV (strain Cabb B-JI), corresponding to coordinates
7040 to 7432 of the CaMV Strasbourg strain (Franck et al., 1980).
Deletion Mutants
Deletion mutants were named according to the first two letters of the
5‘restriction site, followed by the first letter of the 3’restriction site and
the size of the deletion in base pairs. pJIT36 is an M13mp18 vector,
containing a BstEll site, which was inserted as an oligonucleotide into
the M13 polylinker. pJIT36 was used as a cloning intermediatefor the
construction of deletion mutants pBSB347, pAVB365, and pAPB514.
An 807-bp Sacl-BstEll fragment was released from pJIT34 and inserted into the corresponding sites of pJIT36. Following digestion
with BamHl and the appropriate second enzyme (BstXI, Avall, and
Apal, respectively), the DNA ends were made flush and self-ligated.
The modified segments were transferred to pWDV2 as Ncol (nucleotide 2591)-BstEll (nucleotide 500) fragments into the corresponding
unique sites in pWDV2. pAPA38 was created by digesting pWDV1-59
with Apal, self-ligating, and transferring the modified segment into
pWDV2 as an NcoCBstEll fragment, as described above. For the construction of mutants pALB288 and pSSB430, Ncol (nucleotide
2591)-AIul (nucleotide 242) and Ncol-Sspl (nucleotide 80)fragments
were released from pJIT34 and inserted into the Ncol and BamHl
(filled) sites of pWDV2. pAPS84 was constructed by sequentially ligating an 80-bp BamHI-Sspl (nucleotide 426) fragment and a 346-bp
Sspl (nucleotides 80 to 426) fragment from pJIT34 into the Sspl and
BamHl sites of pBluescript SK+. A 426-bp partially digested BamHISspl fragment was released and inserted into the Apal (nucleotide
2746) and BamHl sites of mutant pAPA38, to create pAPS84. PECA101
was constructedby releasing an EcoRl (nucleotide 2679, filled)-Ncol
(nucleotide 2591) fragment from pJIT34 and inserting it into the Ncol
and Apl (nucleotide2746) sites of pWDV2. The CAT coding sequence
was introduced into all the deletion mutants as a BstEll-BamHI fragment, released from pWDV1-59 and inserted into the BstEll and Bglll
sites of each mutant.
Protoplast Transfection
The einkorn (Tn’ticum monococcum) suspension culture was obtained originally from Dr. H. Lorz, Max Planck lnstitute (Cologne, Germany). Liquid cultures (40 mL) were shaken continuouslyat 120 rpm,
Replication and Gene Expression in WDV
221
at 25OC in P10 medium (Kao, 1977). A 5-mL packed volume of cells
was transferred to fresh medium every 7 days. Prior to transfection,
a 15-mL packed volume of cells was digested overnight in 100 mL of
filter-sterilized CPW solution (10 mM CaCI2, 10 mM MgS04, 3 mM
2[N-morpholino]ethanesulfonicacid, 1 mM KN03, 0.2 mM KH2P04,
0.5 M mannitol, 0.1% [w/v] potassium dextran sulphate, pH 5.6), containing 1% (w/v) cellulase RS and 0.Wo (w/v) pectolyase Y-23. Protoplasts were filtered through 50-pm meshes and washed in
volumes of CPW. lntact protoplasts were purified
successive 100"
on a 6-mL 0.5 M sucrose cushion in a 0.5-mL volume of CPW after
centrifugation at 1089 in a MSE Mistral 2000 swing out rotor (MSE
Scientific Instruments, Loughborough, United Kingdom). Fifty micrograms of CsCl gradient-purifiedplasmid DNA was added to aliquots
of 2 to 5 x 106 protoplasts and gently resuspended in 0.35 mL of
SM buffer (0.4 M sucrose, 15 mM MgCI2, O.l*/o [wlv] 2[N-morpholino]ethanesulfonicacid, pH 5.6). A 0.65-mL polyethylene glycol solution was added (40% polyethylene glycol 3350, 0.4 M sucrose, 0.1 M
CaN03, pH 9), and the protoplasts were left to incubate at room temperature for 15 min. Five successive 2-mL volumes of 0.2 M CaCIZ
were added at 5-min intervals. Protoplasts were washed twice in 10
mL of CPW and incubated in the dark at 25OC in 4 mL of Murashige
and Skoog (1962) medium (product number 9-300-40, Imperial Labs.,
Andover, United Kingdom), pH 5.8, supplemented with 0.25 M glucose, 0.25 M sorbitol, 90 mM sucrose, 10% (vlv) coconut milk
(Sigma), 0.01% (w/v) myo-inositol, 0.05% (w/v) casein enzymatic
hydrolysate, 2.5 pM 2,4-dichlorophenoxyacetic acid, 2.8 pM indole
acetic acid, 0.9 pM benzylaminopurine, and 0.5 pM zeatin.
to @rs.David Baulcombe and Gary Creissen for critical reading of the
manuscript. This work was supported in part by a grant-in-aidto the
John lnnes lnstitute from the Agriculture and Food Research Council
and by the Agriculture and Food Research Council Plant Molecular
Biology Programme. J.M.I.H. gratefully acknowledges the support of
a John lnnes Foundation Studentship. E.L.D. was supported by a European Molecular Biology Organization (Heidelberg, Germany) fellowship, and C.J.W. was supported by the Department of Trade and
lndustry Plant Gene To01 Kit Consortium (Laboratory of the Government Chemist, Middlesex, United Kingdom).
DNA Gel Blot Analysis
Boulton, M.I., Steinkellner, H., Donson, J., Markham, P.G., King,
D.I., and Davies, J.W. (1989). Mutational analysis of the virionsense genes of maize streak virus. J. Gen. Virol. 70, 2309-2323.
Transfected protoplasts were ground with fine sand in TLES medium
(Woolston et al., 1988) and extracted twice with Tris-saturated phenollchloroform(1:l). RNA was precipitatedfrom total nucleic acids after incubation on ice with an equal volume of 4 M LiCI. Purified DNA
was extracted with phenol/chloroform, and any remaining RNA was
digested with DNase-free RNase at 3PC for 1 hr. DNA was precipitated in ethanol, and 5 pg was loaded in each lane of a 1% agarose
gel in TAE buffer (Sambrook et al., 1989). Following electrophoresis
at 90 mA for 6 hr, DNA was transferred to a nitrocellulosefilter. A fulllength WDV monomer, released as a Ncol fragment from pWDVKlOD
(Woolston et al., 1989), was used to probe the filter.
CAT Assay
Protoplasts were ground with fine sand in extraction buffer (50 mM
Tris-HCI, 50 mM potassium acetate, 1 mM EDTA, 2.5% [v/v] glycerol,
0.5 mM phenylmethyl-sulfonyl fluoride, 3 mM P-mercaptoethanol).
Extracts were incubated at 65OC for 15 min prior to the CAT assay
(Gorman et al., 1982). Acetyl coenzyme A (3.8 mM) and 14C-chloramphenicol (7.4 KBq [specific activity, 2.2 GBqlmmol]) were used in
each reaction, and the products were separated by thin-layer
chromatography.
Received October 11, 1991; accepted December 9, 1991.
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DOI 10.1105/tpc.4.2.213
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