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INTRODUCTION |
Geminiviruses cause economically significant diseases in a wide
range of cereal, vegetable, and fiber crops (1). These viruses have a
single-stranded DNA genome that is replicated in nuclei of infected
cells by a rolling circle mechanism (2, 3). Of the different gene
products encoded by the virus, only AC1, the
replication-associated protein (Rep), is essential for viral DNA
replication. The first step in the replication process involves
recognition of specific DNA sequences referred to as iterons, (4), by
the Rep protein in the common region
(CR)1 of the virus genome.
Most iteron sequences occur as direct repeat motifs of 6-12 base pairs
between the TATA box and the start site of transcription of the
AC1 gene. The iterons serve as high affinity binding sites
of the Rep protein and therefore function as the origin recognition
sequences. Specific regions on the N terminus of Rep protein are
involved in DNA binding and have been identified for Tomato
golden mosaic virus (TGMV) (5, 6), African cassava mosaic
virus (ACMV) (7), and Tomato yellow leaf curl virus (8).
The potential binding site sequences in the common region of the
Tomato leaf curl New Delhi virus (ToLCNDV) (9) genome were
identified by site-directed mutagenesis (10). Further analyses using
gel shift assays confirmed that the Rep protein specifically binds to
the iterated motifs GGTGTCTGGAGTC (nucleotides 2640-2653) in the
origin of replication (11). In the present study, our objective was to
identify the DNA binding domain of the Rep protein and to determine the
nature and contribution of DNA binding and protein oligomerization
properties of the Rep protein to limit viral DNA accumulation in
plants. In two cases, truncated Rep proteins have been shown to confer
resistance to other geminiviruses (7, 12), and the resistance was
specific and limited to the homologous virus. We based our choice of
truncated Rep protein on the knowledge of overlapping sites for DNA
cleavage, domains for DNA binding, and domains for protein
oligomerization (13, 14). We hypothesized that a truncated Rep protein
that was competent for DNA binding and oligomerization domain might
have a greater probability to interfere with the virus replication and
might be effective against both homologous and heterologous viruses.
In this study, we mapped the minimal binding domain on the Rep protein
by electrophoretic mobility shift assays (EMSAs). We also tested the
effect of truncated and full-length AC1 sequences on DNA
replication of ToLCNDV and other geminiviruses in transient assays
using BY2 protoplasts and Nicotiana benthamiana plants. These studies revealed that transient expression of the
ToLCNDV-truncated Rep protein encoding the DNA binding and the
oligomerization domains could significantly inhibit replication of
ToLCNDV viral DNA and to some extent the replication of other
geminiviruses having similar iteron sequences.
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MATERIALS AND METHODS |
Plasmid Constructs--
The full-length AC1 genes
from the severe and the mild strains of ToLCNDV were amplified by PCR
from pMPA1 (DNA-A of the severe strain ToLCNDV) and pMPA2 (DNA-A of the
mild strain ToLCNDV) (15), cloned in the bacterial expression vector
pGEX-4T-3 (Amersham Pharmacia Biotech), and overexpressed in
Escherichia coli cells. The recombinant proteins were named
according to the number of amino acids at the N or C terminus of the
Rep protein. The C-terminal truncations were made by inserting an
in-frame stop codon at positions 2436 (pAC1-(1-52)), 2250 (pAC1-(1-114)), and 2110 (pAC1-(1-160)). The truncated AC1
sequences were subcloned as a BamHI-XhoI
fragment in the pGEX-4T-3 vector, generating pAC1-(1-52),
pAC1-(1-114), and pAC1-(1-160), respectively. At the N terminus, the
first 21 amino acids of the protein were deleted, and an
NheI site was inserted to create an in-frame start codon.
The truncated fragment was cloned as a NheI-XhoI
fragment in the vector pGEX-4T-3 to produce pAC1-(22-360).
The plasmids pAC1-(52-360) and pAC1-(114-360) were produced similarly
but had a deletion of the first 51 and 113 amino acids, respectively,
from the N terminus of the AC1 gene.
Protein Expression and Analysis--
The truncated Rep proteins
were expressed from plasmids mentioned above in E. coli
cells. The glutathione S-transferase (GST)-tagged AC1 fusion
proteins were purified by glutathione affinity chromatography on
glutathione-Sepharose beads according to the manufacturer's recommendations.
Briefly, the cells were grown to a density of 0.75-0.8
A600. The cultures were induced by the
addition of isopropyl-
-D thiogalactoside at a final concentration of
1 mM and grown further for 2 h. The cells were finally
harvested at 4000 rpm (Beckman, JS 10.5 rotor) for 10 min. The pellets
were suspended in ice cold 1× PBS (10 mM
KH2PO4, 100 mM NaCl) and lysed by
sonication. The lysate was clarified at 17,000 × g for
30 min. The resulting supernatant was loaded on a glutathione-Sepharose
4B column (Amersham Pharmacia Biotech) previously equilibrated with 1×
PBS. After repeated washing of the column with 1× PBS, the protein was
eluted with glutathione elution buffer (Amersham Pharmacia Biotech).
The eluted fractions were dialyzed against 1× PBS to remove
glutathione and concentrated using centricon filters (Amicon,
Centricon). Protein concentrations were estimated using Bradford's
reagent (Bio-Rad).
Protein extracts from E. coli cells co-expressing the
untagged, wild type AC1 and GST fusion of truncated Rep proteins were tested for AC1 oligomerization by co-purification on
glutathione-Sepharose. Co-purification of proteins was monitored by
resolving the eluted fractions on SDS-PAGE and by immunoblotting. The
full-length and truncated Rep proteins were detected using the
polyclonal anti-AC1 antibody. A similar procedure was used to assess
the oligomerization of full-length Rep proteins from other
geminiviruses with the truncated Rep protein of ToLCNDV
(pAC1-(1-160)). Protein extracts from E. coli cells
co-expressing wild type AC1 of Pepper huasteco yellow
vein virus (PHYVV), Potato yellow mosaic virus (PYMV), and ACMV and the GST-tagged pAC1-(1-160) were incubated with
GST-Sepharose beads, washed thoroughly with 1× PBS, and eluted with
glutathione elution buffer (Amersham Pharmacia Biotech). The eluate was
resolved on SDS-PAGE gels and then transferred to nitrocellulose
membranes and detected by immunoblotting using polyclonal anti-AC1
antibody and anti-GST antibody.
Construction of Expression Cassettes--
For expression of the
truncated AC1 gene in plant cells, the mutants described
above were subcloned as BamHI fragments in the plant
expression vector pILTAB 350. This placed the DNA fragment 3' of the
cassava vein mosaic virus promoter (16) upstream of the AC1
gene sequences to produce the gene expression cassettes, pILTAB 401 (encoding AC1-(1-52)), pILTAB 402 (encoding AC1-(1-114)), and pILTAB
403 (encoding AC1-(1-160)), respectively.
Constructions of infectious clones of plasmids containing full-length
DNA of ToLCNDV are named pMPA1 and pMPB1 and were previously described
(15). John Stanley (John Innes Institute, Norwich, United Kingdom)
generously provided full-length infectious dimers of ACMV-Kenya, pCLV
1.3A, and pCLV 2B (17). Infectious monomers of PHYVV (18) were kindly
provided by Riviera Bustamante (CINVESTAV, Irapuato, Mexico). The PYMV
clones have been described (19).
EMSAs--
The sequences of the synthetic oligonucleotides used
as probes or competitors in EMSAs are given in Table III. In the case of the severe and mild strains of ToLCNDV, the 18-mer oligonucleotides corresponding to the binding sites of the Rep protein were used as
probe (11). For the geminiviruses, ACMV, PHYVV, and PYMV, fragments of
their CR sequences were synthesized and used as competitors in
EMSAs. All oligonucleotides were synthesized commercially by Life
Technologies, Inc.
The single-stranded 18-mer oligonucleotides containing the potential
binding sites of the Rep protein of ToLCNDV were annealed to their
complementary strands. The oligonucleotides were end-labeled with
[
-32P]ATP using T4 polynucleotide kinase and purified
on polyacrylamide gels. The final concentration of the probes was 500 pM (30,000 cpm). The concentration of competitor DNA used
was 50 nM per reaction. Both the probe and the competitor
DNAs were purified on Sephadex G-25 columns, quantified by
scintillation counting, and diluted to 30,000 cpm for each binding reaction.
The binding assays were performed using purified Rep protein.
Typically, the binding reactions contained 500 ng of pure protein, 1 ng
of labeled DNA, and 0.2 µg of poly(dI-dC). Binding buffer contained
20 mM HEPES, pH 7.5, 60 mM KCl, 1 mM dithiothreitol, and 15% glycerol. Reactions were
incubated at 25 °C for 30 min, and the complexes were resolved on
4% polyacrylamide gels in 0.25× TBE buffer. The gels were dried on
Whatman paper and exposed to x-ray film. Comparative efficiency of
binding was analyzed by quantifying the amount of radioactivity in the
retarded bands using a PhosphorImager (Molecular Dynamics).
Transient Replication Assays in Protoplasts and
Plants--
Protoplasts derived from Nicotiana tabacum BY-2
suspension cultures were used for transfection with viral DNA (20).
Protoplasts were collected from cultures 48 h postinoculation for
DNA isolation and analysis. One million protoplasts were inoculated by
electroporation (250 V, 500 microfarads) with 2 µg each of A and B
component DNAs and 40 µg of sheared herring sperm DNA (10). For
co-inoculation experiments, 2 µg of the plasmid DNA containing the
expression cassettes with truncated AC1 gene sequences were
used. Total DNA from the protoplasts was extracted 48 h after
transfection (21, 22). Viral DNA accumulation was analyzed by Southern
blotting (10).
Total proteins were extracted from the protoplasts 48 h after
transfection by sonication of the cell pellets in ice cold 1× PBS (10 mM KH2PO4, 100 mM
NaCl). The lysate was clarified at 17,000 × g for 15 min, and the resulting supernatant was used for immunoprecipitations. Immunoprecipitations were done by incubating 50 µg of total protein extracts with polyclonal anti-AC1 antiserum (1 mg) overnight at 4 °C. Protein-antibody complexes were incubated with protein
A-agarose for 2 h at 4 °C and then washed with 1× PBS. Bound
proteins were eluted from the agarose beads in SDS-PAGE sample buffer
by boiling at 100 °C for 5 min. Proteins resolved on the gel were
transferred on nitrocellulose membranes and analyzed by immunoblotting
with polyclonal anti AC1 antibody using 3,3'-diaminobenzidine
tetrahydrochloride for colorimetric quantitation of the expressed Rep
levels in the cell extracts.
Two-week-old seedlings of N. benthamiana were grown in
magenta boxes and inoculated with partial tandem dimers of viral DNA using a Bio-Rad helium-driven particle gun (10). Ten plants were
inoculated with each mutant using 0.5 µg each of DNA-A and DNA-B
genomic components per plant. Plants were observed for symptom development, and newly emerging leaves were harvested for Southern blot
analysis 4 weeks postinoculation.
Southern Blot Analysis--
DNA extractions from systemically
infected leaf samples were completed as described by Dellaporta
et al. (21) and from protoplasts by following the procedure
of Mettler (22). Total DNA (4 µg) was fractionated on 1% agarose
gels without ethidium bromide and transferred to nylon membranes. Viral
DNA was detected by using a 900-base pair
AflII-PstI fragment of the A component
containing sequences from the open reading frames of the
AC1, AC2, and AC3 genes or a probe
specific for the B component (878-base pair PCR-amplified BC1 gene). The amount of viral DNA was quantified as
previously described (10). In the case of geminiviruses other than
ToLCNDV, fragments of their AC1 and BC1 genes
were amplified and used as probes to analyze the replication levels of
viral DNA.
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RESULTS |
Determination of a Minimal Binding Domain of the Rep Protein of
ToLCNDV--
The Rep protein binds specifically to a directly repeated
DNA sequence motif in the common region of the ToLCNDV genome (11). Purified Rep proteins truncated at amino acids 160, 114, and 52 were
used to map the C-terminal boundary of the Rep DNA binding domain
in vitro. As a control, full-length Rep protein (amino acids
1-360) was used in all assays. The truncated and full-length Rep
proteins were expressed in E. coli with a GST tag and
affinity-purified on a glutathione-Sepharose 4B column. The
affinity-purified proteins were highly enriched as determined by
Coomassie staining following electrophoresis on SDS-PAGE gels. The
proteins were detected in immunoblots using anti-GST antibody (data not shown).
The purified Rep proteins were tested for their ability to bind a
radiolabeled 18-mer (nucleotides 2632-2653) that contains the Rep
binding site sequence, 5'-GGTGTCTGGAGTC-3'. DNA-protein complexes that
contained Rep-(1-360) and Rep-(1-160) were detected. No binding was
observed for Rep-(1-52) or Rep-(1-114) (Fig.
1A, lanes
1-4). These results located the C-terminal boundary of the DNA binding domain of the Rep protein between amino acids 115 and
160.

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Fig. 1.
Determination of the DNA binding domain of
the Rep protein of ToLCNDV. A, binding of full-length
and C-terminal truncated Rep proteins to origin DNA sequences. Highly
enriched preparations of GST-AC1 fusion Rep proteins were analyzed for
their ability to bind to radiolabeled iteron sequences containing the
ToLCNDV Rep protein binding site in EMSAs. Typically, the binding
reactions contained 500 ng of pure protein, 1 ng of labeled DNA, and
0.2 µg of poly(dI-dC). Binding buffer contained 20 mM
HEPES, pH 7.5, 60 mM KCl, 1 mM dithiothreitol,
and 15% glycerol. Reactions were incubated at 25 °C for 30 min, and
the complexes were resolved on 4% polyacrylamide gels in 0.25× TBE
buffer. Lane 1 represents the binding observed
in vitro with a full-length Rep protein. Lanes
2-4 show the same binding assays with the Rep protein
truncated at its C terminus at amino acids 1-52 (lane
2), 1-114 (lane 3), and 1-160
(lane 4). B, mapping the N-terminal
boundary of the DNA binding domain of the Rep protein of ToLCNDV.
Highly enriched preparations of GST-AC1 fusion Rep proteins were
analyzed for their ability to bind to radiolabeled iteron sequences
containing the ToLCNDV Rep protein binding site in EMSAs. Typically,
the binding reactions contained 500 ng of pure protein, 1 ng of labeled
DNA, and 0.2 µg of poly(dI-dC). Binding buffer contained 20 mM HEPES, pH 7.5, 60 mM KCl, 1 mM
dithiothreitol, and 15% glycerol. Reactions were incubated at 25 °C
for 30 min, and the complexes were resolved on 4% polyacrylamide gels
in 0.25× TBE buffer. Lane 1 represents the
binding observed in vitro with a full-length Rep protein.
Lanes 2-4 show the same binding assays with the
Rep protein truncated at its N terminus at amino acids, 22-360
(lane 2), 52-360 (lane 3),
and 114-360 (lane 4). C,
co-purification of the N-terminal truncated GST-AC1 fusion proteins
with the full-length untagged wild type Rep protein. Bacterial cell
lysates co-expressing a truncated GST-AC1 fusion protein with a
full-length Rep protein were passed over a glutathione-Sepharose 4B
column. After washing, the eluted fractions were resolved on SDS-PAGE
and detected in immunoblots using a polyclonal anti AC1 antiserum.
Lanes 1-4 represent the different GST-tagged Rep
fusion proteins truncated at their N termini that bound with the
untagged full-length Rep protein. Lane 1,
GST-AC1-(1-360); lane 2, GST-AC1-(22-360);
lane 3, GST-AC1-(114-360); lane
4, GST-AC1-(52-360); lane 5, untagged
full-length Rep protein used as a control.
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The N-terminal boundary of the DNA binding domain was determined
in vitro by comparing the binding of full-length
Rep-(1-360) and Rep-(22-360), Rep-(52-360), and Rep-(114-360) to
the 18-base pair iteron sequence, 5'-GGTGTCTGGAGTC-3' in EMSAs. The
DNA-protein complexes were observed in case of full-length Rep protein,
while no DNA-protein complexes were detected for the Rep-(22-360),
Rep-(52-360), or Rep-(114-360) (Fig. 1B, lanes
2-4). These results demonstrated that the sequences within
the first 21 amino acids of the Rep protein are essential for
protein-DNA interactions. Together, these results placed the DNA
binding domain of ToLCNDV Rep protein between amino acids 1 and 160.
To determine if truncations at the N and the C termini of Rep protein
affect its ability to oligomerize, GST-tagged truncated Rep proteins
were co-expressed with untagged wild type full-length Rep protein in
bacterial cells and co-purified on glutathione-Sepharose beads. The
bound fractions were eluted and analyzed in immunoblots using
polyclonal anti-AC1 antiserum. The wild type Rep-(1-360) co-purified
with GST-tagged truncated proteins Rep-(22-360), Rep-(52-360), and
Rep-(114-360) (Fig. 1C, lanes 1-5),
suggesting that truncations made at the N terminus in the Rep did not
affect the ability of the Rep protein to oligomerize with itself,
although each of the truncated proteins was deficient for DNA binding.
ToLCNDV Replication Is Inhibited by Transiently Expressed Rep
Protein--
The effect of Rep protein on viral DNA replication was
investigated by co-inoculating N. tabacum BY2 protoplasts
with DNA-A and various cassettes that express truncated AC1
gene sequences from the CsVMV promoter. ToLCNDV DNA-A replicated in
BY-2 cells and accumulated high levels of single-stranded (ss) and
supercoiled (sc) DNA (Fig. 2A,
lane 1). In contrast, there was a significant decrease in the level of viral DNA replication (78% drop) in the presence of Rep-(1-160) (Fig. 2A, lane
4, Table I). Reduction in
replication was estimated by quantifying the amount of radioactivity using a PhosphorImager (Storm 860; Molecular Dynamics). The reduction in virus replication was not as dramatic in the presence of Rep-(1-52) (Fig. 2A, lane 2) or Rep-(1-114)
(Fig. 2A, lane 3) when compared with
Rep-(1-160) (Fig. 2A, lane 4). EMSAs
showed that the Rep-(1-52) and Rep-(1-114) do not bind DNA (Fig.
1A, lanes 2 and 3),
implying that an intact DNA binding domain and/or a protein
oligomerization domain is essential for inhibition of replication.

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Fig. 2.
Effect of Rep-(1-160) on accumulation of
viral DNA in tobacco protoplasts. A, Southern blot
analysis of total DNA extracted from protoplasts 48 h after
co-transfection with wild type, infectious dimers of the DNA-A of the
severe strain pMPA1 alone (lane 1) or with
Rep-(1-52) (lane 2), Rep-(1-114)
(lane 3), or Rep-(1-160) (lane
4). Lanes 5-8 represent viral DNA
accumulation in protoplasts co-infected with the DNA-A of the mild
strain pMPA2 alone (lane 5) or with Rep-(1-52)
(lane 6), Rep-(1-114) (lane
7), and Rep-(1-160) (lane 8) of the
homologous strain. B, immunoblot analysis of the proteins
extracted from tobacco protoplasts co-transfected with various Rep
constructs and detected by anti-AC1 antibody. 50 µg of total protein
extracts were incubated with 1 mg of anti-AC1 antibody overnight at
4 °C. Bound proteins were recovered from the agarose beads after
extensive washing of protein-antibody complexes in 1× PBS and boiling
the beads in SDS-PAGE sample buffer. Proteins resolved on the gel were
transferred on nitrocellulose membranes and analyzed by immunoblotting
with polyclonal anti-AC1 antibody using the 3,3'-diaminobenzidine
tetrahydrochloride (DAB) that provided a quantitative
estimation of the precipitated protein-antibody complex in the samples.
The protoplasts were transfected with wild type infectious dimer of the
DNA-A of the severe strain pMPA1 with Rep-(1-360) (lane
3), Rep-(1-160) (lane 4),
Rep-(1-114) (lane 5), or Rep-(1-52)
(lane 6). The extracts from the uninfected
protoplasts served as the negative control (lane
2). The molecular masses of the truncated and full-length
Rep (kDa) are shown on the left.
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Table I
Virus replication in BY2 protoplasts and N. benthamiana plants
co-inoculated with truncated Rep protein gene constructs and the viral
DNA of the severe strain of ToLCNDV
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Similar experiments were conducted with the mild strain of ToLCNDV in
which analogous truncated mutations of the Rep gene were co-introduced
in tobacco protoplasts with DNA-A from the mild strain. In these
studies, an analogous inhibition of viral DNA accumulation in BY2
protoplasts was detected (Fig. 2A, lanes 5-8, and Table II).
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Table II
Virus replication in BY2 protoplasts and N. benthamiana plants
co-inoculated with truncated Rep protein constructs and viral DNA of
the mild strain of ToLCNDV
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To determine the relative expression levels of the three truncated Rep
proteins in transfected tobacco protoplasts, total proteins were
extracted from the tobacco protoplasts 48 h after infection and
immunoprecipitated with the anti-AC1 antibody, and the protein-antibody
complexes were resolved on SDS-PAGE gels. All of the three truncated
proteins could be detected in immunoblots from the transfected
protoplasts when developed using 3,3'-diaminobenzidine tetrahydrochloride. 3,3'-Diaminobenzidine tetrahydrochloride produces a
brown precipitate with the peroxidase and thereby provided a direct
measure of the amount of antibody bound to the expressed protein in the
samples, revealing that all three truncated Rep proteins were
expressed stably and in equivalent amounts in the protoplasts (Fig.
2B, lanes 2-6).
Infection of N. benthamiana--
Two-week-old seedlings of
N. benthamiana plants were co-bombarded with 2 µg each of
infectious dimers of ToLCNDV DNA-A and DNA-B in the presence or absence
of genes encoding Rep-(1-160). The plants were observed daily for
symptom development. All of the plants inoculated only with the wild
type virus DNAs developed severe symptoms 5 days after inoculation. In
contrast, plants co-inoculated with the virus and the genes encoding
Rep-(1-160) developed milder symptoms of ToLCNDV infection (Table I).
About 55% of the plants were asymptomatic, 30% showed mild chlorosis, and 15% expressed mild leaf curl symptoms (Table I). None of the
plants showed severe infection or stunted growth as in plants infected
only with ToLCNDV. Most of the plants co-inoculated with Rep-(1-52) and Rep-(1-114) developed severe symptoms by 7 days postinoculation (Tables I and II).
The levels of viral DNA in ToLCNDV-infected plants were analyzed by
Southern blot analysis of young leaves sampled 28 days postinoculation
using probes that detected DNA-A and DNA-B (see "Materials and
Methods" and Fig. 3, A and
B, respectively). The amount of viral DNA ranged from
undetectable to very low (an average of 15% of the wild type levels)
in asymptomatic plants, and the accumulation of both genomic components
increased with increasing severity of symptom expression. Plants
co-inoculated with expression cassettes Rep-(1-52) and Rep-(1-114)
developed intermediate to severe symptoms in most of the plants and
accumulated viral DNA between 85 and 92% of wild type infection.

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Fig. 3.
Expression of the truncated Rep protein
containing the minimal DNA binding domain (Rep-(1-160)) of the Rep
protein from two strains of ToLCNDV interferes in virus accumulation in
N. benthamiana plants. Two-week-old N. benthamiana plants were bombarded with 2 µg each of DNA-A and
DNA-B of ToLCNDV, and plasmids encoding truncated Rep proteins were
expressed from the CsVMV promoter. Total DNA was extracted from
N. benthamiana plants 21 days postinoculation and analyzed
by Southern blots. The blots were probed for accumulation of DNA A
(lanes 1-12) and DNA-B (lanes
13-24) as described under "Materials and Methods" using
AC1- and BC1-specific probes, respectively. The levels of viral DNA
accumulation in plants co-infected with Rep-(1-160) ranged from 8 to
23% as depicted in lanes 3-11 (DNA-A) and
lanes 15-23 (DNA-B). Wild type (WT)
controls are shown in lanes 12 and
24.
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Co-infection with Rep-(1-160) of ToLCNDV (Severe) Reduces the
Viral DNA Accumulation of Other Geminiviruses--
To investigate the
potential of truncated Rep protein to inhibit the replication of other
geminiviruses, we selected examples of viruses that belonged to the Old
World (ACMV) and New World geminiviruses (PHYVV and PYMV-TT). We
reasoned that for the Rep to be able to interfere in replication of
heterologous geminiviruses, it must (a) bind to the origin
sequences of these viruses and (b) oligomerize with their
Rep proteins. For the EMSA studies, fragments of the intergenic region
sequences of the selected heterologous geminiviruses close to the TATA
box were chosen. The coordinates of these sequences are given in Table
III. To determine if the putative iteron
sequences of the other geminiviruses could compete with the cognate
iteron sequences of ToLCNDV for binding to ToLCNDV Rep protein,
synthetic oligonucleotides encoding the CR sequences from each virus
were synthesized and used as competitors in EMSAs. None of the CR
sequences were effective competitors in EMSA with the ToLCNDV Rep
protein (Fig. 4A,
lanes 3-6) and did not affect binding of the Rep
protein with its cognate 13-mer iteron sequences to a significant
degree.

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Fig. 4.
Interaction of ToLCNDV Rep-(1-160) with the
Rep protein of other geminiviruses. A, competition of
heterologous geminivirus CR sequences in EMSAs with the Rep-(1-160) of
ToLCNDV. Each reaction contained 500 ng of purified Rep-(1-160)
incubated with a 100-fold excess of CR sequences of different
geminiviruses for 5 min on ice before the addition of the
32P-labeled probe comprising the iteron sequences of
ToLCNDV. After 30 min, the DNA-protein complexes were resolved on 4%
polyacrylamide gels. Lane 1, the control reaction
containing the iteron sequences of the severe strain of ToLCNDV as the
competitor; lane 2, binding by the Rep-(1-160)
to iteron sequences of the severe strain of ToLCNDV-A1 without any
competitor; lane 3, binding by the Rep-(1-160)
in the presence of CR sequences of ACMV; lane 4,
binding by the Rep1-160 with CR sequences of PHYVV as
competitor; lane 5, binding by the Rep-(1-160)
in the presence of CR sequences from PYMV; lane
6, binding by the Rep-(1-160) in the presence of iteron
sequences of the mild strain of ToLCNDV-A2. B, immunoblots
showing co-purification of Rep protein from other gemeniviruses with
the Rep-(1-160) of ToLCNDV as detected by polyclonal anti-AC1
antibody. Lanes 2 and 7, Rep protein
of ACMV; lanes 3 and 8, Rep protein of
PHYVV; lanes 4 and 9, Rep protein of
PYVMV; lanes 5 and 10, Rep
protein of ToLCNDV, mild strain; lanes 6 and 11, Rep protein of ToLCNDV, severe strain.
Lane 1, control showing the relative mobility of
GST-tagged Rep-(1-160) protein in the gel. C, immunoblots
showing co-purification of Rep protein from other geminiviruses with
the Rep-(1-160) of ToLCNDV as detected by polyclonal anti-GST
antibody. Lanes 2 and 7, ACMV;
lanes 3 and 8, PHYVV; lanes
4 and 9, PYVMV; lanes 5 and
10, ToLCNDV, mild strain; lanes 6 and
11, ToLCNDV, severe strain. Lane 1,
control showing the relative mobility of GST-tagged Rep-(1-160)
protein in the gel.
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The crude lysates of E. coli cells co-expressing wild type
Rep proteins from ACMV, PHYVV, or PYMV and the GST-tagged ToLCNDV Rep-(1-160) were tested for the ability to bind to each other. Crude
protein extracts from bacterial cells co-expressing the target proteins
were loaded on a GST-Sepharose column, washed extensively with 1× PBS,
and eluted with glutathione elution buffer. The resulting fractions
were detected in immunoblots using polyclonal anti-AC1 and anti-GST
antibodies. In immunoblots, the anti-AC1 antibody detected wild type
untagged Rep proteins from ACMV, PHYVV, and PYMV-TT that co-purified
with the ToLCNDV Rep-(1-160) (Fig. 4B, lanes
2-6). When the same blot was washed and reprobed with anti-GST antibody, only the truncated Rep protein of ToLCNDV
(Rep-(1-160)) in each of the samples was detected (Fig. 4C,
lanes 2-6).
To determine if Rep-(1-160) could reduce accumulation of other
geminiviruses, in vivo replication assays were conducted by co-bombarding the N. benthamiana plants with partial/tandem
dimers of full-length A and B components of ACMV, PHYVV, and PYMV-TT with genes encoding Rep-(1-160) of ToLCNDV. Most plants developed typical symptoms of virus infection within 21-27 days postinoculation as opposed to 7-10 days required for the symptoms on control plants to
develop infection. Southern blot analysis of viral DNA extracted from
the plants harvested 28 days postinoculation showed a minor reduction
in the levels of virus accumulation as
compared with the control plants (Table
IV and Fig.
5, A and B).
However, the decrease in replication was not as significant as the
inhibition observed in the case of ToLCNDV DNA.
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Table IV
Regulation of virus DNA replication in BY2 protoplasts by the
N-terminal sequences of AC1 gene of ToLCNDV
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Fig. 5.
Effect of Rep-(1-160) expression on DNA
accumulation of other geminiviruses. Two representative samples
from a set of 30 inoculated plants are shown to reflect the variation
in DNA accumulation. A, Southern blots showing relative
levels of DNA accumulation in N. benthamiana plants
co-inoculated with infectious dimers of DNA-A and DNA-B of ACMV
(lanes 2 and 3), PHV (lanes
5 and 6), and PYMV (lanes 8 and 9) in the presence of Rep-(1-160) and probed with the
AC1 gene sequences of different geminiviruses. Lanes
1, 4, and 7 represent the wild type
(WT) level of DNA-A accumulation in the absence of
Rep-(1-160) for ACMV, PHYVV, and PYMV, respectively. B,
Southern blots showing relative levels of DNA accumulation in N. benthamiana plants co-inoculated with infectious dimers of DNA-A
and DNA-B of ACMV (lanes 2 and 3),
PHYVV (lanes 5 and 6), and PYMV
(lanes 8 and 9) in the presence of
Rep-(1-160) and probed with the BC1 gene sequences of
different geminiviruses. Lanes 1, 4,
and 7 represent the wild type (WT) level of DNA-B
accumulation in the absence of Rep-(1-160) for ACMV, PHYVV, and PYMV,
respectively.
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DISCUSSION |
We determined the nature and the significance of the DNA binding
and protein oligomerization functions of a truncated Rep-(1-160) protein to interfere in DNA accumulation of homologous and heterologous geminiviruses. Our studies show that while both activities of Rep-(1-160) contribute to interference in DNA accumulation, protein oligomerization, unlike DNA binding, is nonspecific and can occur between the Rep proteins of two unrelated geminiviruses.
We mapped the DNA binding domain on the Rep protein of ToLCNDV to amino
acids 1-160 and showed that the transient expression of this Rep
sequence significantly inhibits ToLCNDV DNA accumulation in
inoculated tobacco protoplasts and plants. Of the three C-terminal truncations made in the AC1 gene, only Rep-(1-160) bound
the iteron DNA sequences in vitro, and the two truncations
Rep-(1-52) and Rep-(1-114) were not competent to bind viral DNA
in vitro. None of the N-terminal truncations tested
(i.e. Rep-(22-360), Rep-(52-360), or Rep-(114-360)) bound
viral DNA, indicating that an intact N terminus is required for the Rep
protein to bind the origin sequences. In co-purification assays, each
of the three N-terminal truncated proteins bound with the wild type Rep
protein as detected by immunoblotting of the bound fractions. The
co-immunoprecipitation assays indirectly suggested that the
oligomerization domain might overlap the DNA binding domain. In TGMV,
it is known that the oligomerization domain overlaps the DNA binding
domain (13).
In co-infection studies, the sequences comprising the Rep-(1-52) or
Rep-(1-114) amino acids of the Rep protein did not cause a significant
reduction in ToLCNDV accumulation. However, Rep-(1-160) bound with
high affinity to the iteron sequences and reduced viral replication in
protoplasts and plants. Colorimetric quantification of the truncated
proteins revealed that all three proteins were expressed in equivalent
amounts, ruling out the possibility that poor expression and/or
instability of Rep-(1-52) and Rep-(1-114) in tobacco protoplasts may
have compromised their ability to inhibit virus replication. In related
studies, we observed that N. benthamiana plants co-bombarded
with plasmids that produced Rep-(1-160) and infectious ToLCNDV
produced a range of symptoms from asymptomatic to a mild leaf curl.
None of the plants developed the severe puckering and blistering
associated with wild type virus infection. Southern blot analysis of
the infected plants showed that accumulation of viral DNA in plants
with mild or no symptoms was much less than in plants showing severe
symptoms. More importantly, the degree of inhibition in plants was
similar to those observed in BY-2 protoplasts, indicating that the
impact is probably on virus replication.
Rep-(1-160) contains the DNA binding domain of the ToLCNDV-Rep. By
analogy with the Rep proteins of TGMV and Tomato yellow leaf curl
virus, this fragment is expected to contain the domains for DNA
cleavage and ligation, as well as protein oligomerization domain (13,
23). This region of the Rep protein of ToLCNDV is involved in the
specificity of origin recognition and binding (10). Considering the
various activities that are associated with Rep-(1-160), it is
possible to suggest the mode of action of Rep-(1-160) in limiting
virus DNA accumulation.
One possibility is that the Rep-(1-160) protein reduces replication by
competing with the viral Rep protein for binding the iteron sequences
in the origin. The truncated Rep protein may therefore behave as a
dominant negative mutant (24) and block virus replication.
Another possibility is that the truncated Rep protein does not contain
the NTP binding domain present on the C terminus of the Rep protein.
The NTP binding domain is required for replication (25), and the lack
of this region may interfere with the normal replication process of the virus.
The fact that the Rep protein represses its own transcription may be
yet another explanation for the inhibition of virus replication. Presumably, binding by the Rep protein to the origin is responsible for
the repression of AC1 gene transcription in TGMV (26, 27) and ACMV (7). Constitutive expression of the truncated viral Rep
protein could repress the transcription of the wild type AC1 gene by binding to the origin, thereby influencing viral accumulation levels. Alternately, as suggested in the case of wheat dwarf virus, constitutively expressed Rep may adversely affect the integrity of the
viral DNA by introducing nicks at cryptic motifs (28).
Comparison of co-infection experiments performed using truncated Rep
proteins of the mild and the severe strains of ToLCNDV in protoplasts
and plants suggested that only homologous Rep sequences could reduce
virus accumulation with high efficiency. The Rep-(1-160) from the
severe strain did not significantly restrict the virus replication of
the mild strain and vice versa. Our previous work showed that the mild
and the severe strains of ToLCNDV exhibit selectivity in binding to
their cognate iteron sequences (11). Hence, the inability of
Rep-(1-160) of one strain to limit virus DNA accumulation of the
heterologous strain may reflect specificity of interaction by the Rep
protein for its cognate origin DNA sequences. Since the Rep-(1-160)
can bind to DNA and form oligomers, these results support the
hypothesis that DNA binding and protein oligomerization are important
in inhibition of virus replication by Rep-(1-160).
Notwithstanding that Rep-(1-160) of the severe strain of ToLCNDV had a
modest effect on the replication of the mild strain, we were interested
to know if Rep-(1-160) can interfere with the replication of related
geminiviruses that have similar sequences in their origins of
replication. The reduction in virus accumulation in the case of
unrelated geminiviruses was rather surprising, considering that none of
the CR sequences from these viruses were effective competitors for
Rep-(1-160) in EMSAs. However, the results were not unexpected because
the co-purification experiments revealed the ability of Rep-(1-160) to
interact with the Rep proteins of heterologous geminiviruses. These
data suggest that since the Rep-(1-160) protein does not bind to the
heterologous CR sequences, reduction in virus accumulation may result
from oligomerization of Rep-(1-160) with the Rep proteins of the
unrelated geminiviruses. This level of virus reduction was similar to
the reduction of mild strain ToLCNDV accumulation by the Rep-(1-160).
We suggest that oligomers of Rep-(1-160) could potentially interfere
with the replication complexes formed during infection by PHYVV, PYMV, ACMV, or the mild strain of ToLCNDV. Formation of heteromultimers via
protein-protein interactions (29) has been reported between TGMV and
BGMV. The formation of heteromultimeric complexes might sequester the
wild type Rep oligomers that otherwise would participate in the
formation of a replication complex or might prevent recognition of
origin sequences.
Several approaches to control replication of geminiviruses have been
developed. Transgenic N. benthamiana plants that accumulate defective interfering DNA (30, 31) of ACMV were less susceptible to
ACMV infection, but resistance was confined to closely related strains
of ACMV. Transgenic N. tabacum expressing antisense RNA targeted against TGMV AL1 (32) or Tomato yellow leaf curl virus (33) showed that specificity of resistance depended on the level of homology between the antisense RNA and the target virus sequence. Finally, the possibility of expressing a full-length AC1
transgene in ACMV (34) and the N-terminal sequences of Tomato
yellow leaf curl virus Rep (12) in virus resistance has also been
documented. Our studies demonstrate the potential of using Rep proteins
that are mutated in the oligomerization and DNA binding domain to
interfere with viral DNA replication. Experiments are in progress to
test the stable expression and efficiency of Rep-(1-160) in transgenic tobacco and tomato plants for resistance to ToLCNDV.