Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai 625021, India
Department of Genetics, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel
Received 11 January 2006; received in revised form 12 July 2006; accepted 12 July 2006
Available online 24 August 2006
Abstract
Bhendi yellow vein mosaic disease (BYVMD) is caused by a complex consisting of a monopartite begomovirus BYVMV and a satellite DNA
component. BYVMV represents a new member of the emerging group of monopartite begomoviruses requiring a satellite component for symptom
induction. Here we report the results of the transient expression of green fluorescent protein (GFP) fused with the C1 and coat protein (CP)
coding regions, in the epidermal cells of Nicotiana benthamiana. GFPCP was found to be targeted into the nucleus whereas GFPC1 was localized
towards the periphery of the cell. The sub-cellular localization of the C1 protein has been compared with that of the CP in yeast cells using a
genetic system for detection of protein nuclear import and export. Expression of C1 ORF in transgenic N. benthamiana under the control of the
Cauliower mosaic virus 35S promoter produced severe developmental abnormalities in the plant, like distorted stem, leaves and stunting of the
plant. We also present the results on the interaction of CP and C1 proteins using yeast two hybrid analysis, suggesting a collaborative role in the
inter- and intracellular dynamics of BYVMD.
2006 Elsevier B.V. All rights reserved.
Keywords: Begomovirus; Bhendi yellow vein mosaic virus; C1; Coat protein; Nuclear localization; Nuclear export
1. Introduction
Bhendi yellow vein mosaic disease (BYVMD) complex
consists of a begomovirus Bhendi yellow vein mosaic virus
(BYVMV) DNA A component and a satellite BYVMD DNA
component (Jose and Usha, 2003). This DNA depends on
BYVMV for its replication and encapsidation. DNA , along
with BYVMV induces typical yellow vein mosaic symptoms in
A. esculentus and severe leaf curl and stunted growth in Nicotiana benthamiana (Jose, 2004). Systemic infection in bipartite
begomoviruses, like Squash leaf curl virus (SLCV), is achieved
by movement protein (MP = BC1) and the nuclear shuttling
protein (NSP = BV1) encoded by the DNA B component of
the genome (Sanderfoot and Lazarowitz, 1995). In contrast,
the monopartite begomoviruses such as BYVMV do not code
for NSP and MP homologs. Therefore, some other proteins
must fulfill some or all of the functions provided by MP and
Corresponding author. Tel.: +972 3 968 3471; fax: +972 3 968 3471.
E-mail address: ygafni@volcani.agri.gov.il (Y. Gafni).
0168-1702/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.virusres.2006.07.007
128
Leaves of N. benthamiana were infiltrated with Agrobacterium strains containing the above constructs (Llave et al.,
2000). After 48 h of infection the leaves were harvested and
observed under a laser scanning confocal microscope for detecting the fluorescence. The images were acquired by an Olympus IX81/FV500 confocal microscope using an argon laser
(488 nm), a green helium/neon laser (543 nm) and the following objectives: PLAPO60Xoil/NA 1.4 WD 0.15 mm, or
PLAPO60XW/LSM/NA1.00 WD 0.15 mm. Image analysis was
carried out using the MICA software (Cytoview, Israel).
2.3.1. Propidium iodide staining
Leaf samples were stained with propidium iodide (PI) after
fixing with PME buffer (50 mM PIPES pH 6.9, 5 mM EGTA,
2 mM MgSO4 ) containing 3% paraformaldehyde, 0.05% Triton X-100, 0.25% DMSO, 50 M PMSF and incubated for 1
h. After the incubation, leaf samples were washed three times
each for 5 min in phosphate buffered saline (PBS). The leaf samples were then transferred to freshly prepared PI solution (final
Table 1
Primers used for PCR
Primers
Sequence
Location
BspCP.F
PCP.R
PC1.F
BspC1.R
BC1.F
HC1.R
SalCP.F
SalC1.R
EcoC1.R
EcoCP.F
XhoC1.F
XhoCP.R
EcoNC1
XhoCC1
5 -TGT
ACATGTCGAAGCGAGCTG-3
5 -CTG CAGTCAATTCGTTACAGAGTC 3
5 -CTG CAGTTAAATTATTATCTTATTATCAATAG-3
5 -TGTACATGAAAATATCTATACATTTCATC-3
5 GGATCCTTAAATTATTATCTTATTAT CAATAG-3
5 AAGCTTATGAAAATATCTATACATTTCATC-3
5 -GTCGACCTATGTCGAAGCGAGCTG-3
5 -GTCGACCTATGAAAATAT CTATACATTT C-3
5 -GAATTCATGAAAATAT CTATACATTT C-3
5 -GAATTCATGTCGAAGCGAGCTG-3
5 -CTCGAGAATTATTATCTTATTATCAATAG-3
5 -CTCGAGATTCGTTACAGAGTC 3
5 -GAATTCATG CAG ATC AGT TCA ACA AG-3
5 -CTCGAGAAGTCGAATGGAATG-3
129
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Table 2
Comparison of known leucine-rich nuclear export signals and BYVMDC1
nuclear localization of the Y10 C1 of TYLCCNV as predicted by the NLS (45 PALAKKK51 ) using PSORT analysis
was confirmed experimentally by Cui et al. (2005). However,
a similar PSORT analysis of BYVMD C1 did not show
any NLS in the sequence. Interestingly PSORT analysis of
a number of C1 sequences revealed that there are at least
two distinct types of C1 proteins, those with a NLS and
those with a NES sequence. The C1 proteins associated with
Tomato yellow leaf curl virusChina (TYLCCNV, CAI96493,
Tomato leaf curl virusChina (ToLCCNV, CAG28792),
Tomato leaf curl virusPune (ToLCNDV, AAV98446), Mungbean yellow mosaic India virus (MYMIV, AAZ78309), and
Papaya leaf curl virus (PaLCuCNV, NP 821073) have a
monopartite NLS. The NLS sequence shows minor variation
between C1 proteins. It is 45 PALAKKK51 in TYLCCNV,
45 PALVKKK51 in MYMIV, PaLCuCNV and ToLCNDV and
45 PAMIKRR51 in ToLCCNV. Online prediction using, NetNES
(http://www.cbs.dtu.dk/index.shtml) revealed the presence of a
nuclear export signal (NES), 105 LEEDIIHMVDI115 towards
the carboxy terminus of BYVMDC1. A Similar sequence has
been found to be present in the C1 proteins from Okra leaf
curl virus (OLCuV, CAC87055), Cotton leaf curl virus (CLCuV,
ABA40413, CAC44025, AAX21514, AAX21495), Malvastrum
yellow vein virus (MYVV, CAH10920, CAI96762) and Ageratum yellow vein virus (AYVV, CAD89717, CAD65761 and
CAC87053). The NES of C1 associated with BYVMD has
been found to share some similarity with already characterized
NES of HIV Rev (Fischer et al., 1995; Pollard and Malim, 1998
and Rhee et al., 2000), inhibitor of protein kinase A (PKI) (Wen
et al., 1995), transcription factor III A (Fridell et al., 1996; Ward
and Lazarowitz, 1999) and BV1 of SLCV (Ward and Lazarowitz,
1999). Table 2 shows the comparison of predicted NES of C1
associated with BYVMD with other characterized NESs. Multiple alignments of these sequences clearly show the distribution
of NLS and NES among C1 proteins (data not shown). The
variation in the distribution of NLS and NES sequences among
C1 correlate with that of the diversity and geographical distribution of DNA already reported (Briddon et al., 2003; Bull et
al., 2004)
131
Fig. 1. Sub-cellular localization of GFP alone and GFP fusion proteins during transient expression in N. benthamiana. The fluorescence was visualized using confocal
microscopy. GFP fluorescence (green channel), bright field channel, and channel overlays of green channel either with red (auto fluorescence of plant tissue or PI
fluorescence) channel or with bright field channels are shown. Expression of GFP alone (Panel A1A4), GFPCP (Panel B1B4), GFPNLSCP (Panel C1C4) &
(Panel D1D4) and GFPC1 (Panel E1E4) & (Panel F1F4). Figures in Panel D and Panel F are stained with propidium iodide. Panels D2 and F2 show PI stained
nuclei. The co-localization of GFP and PI fluorescence is shown in panel D3 (channel overlay of PI fluorescence with GFP fluorescence), whereas the panel F3 did
not show any co-localization of PI with GFP. GFPC1 is localized only in the cytoplasm.
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Fig. 3. One hybrid results: (a) -galactosidase assay, (b) histidine prototrophy on
minimal medium deficient for both tryptophan and histidine supplemented with
100 mM 3AT. (a) pNEA (1), pNEARev (2), pNEACP (3), pNEAC1 (4), pNIACP (5), pNIAC1 (6), and pNIA (7). (b) pNEA (1), pNEARev (2), pNEACP
(3), pNEAC1 (4), pNIACP (5).
suggests that C1 may have a nuclear export or peripheral localization function. Reports show the NES is not conserved motif
but it follow a consensus L X2-3 (F, I, L, V, M) X2-3 (L, I) X
(L, I) (Sekimoto et al., 2005). Sequence analysis revealed that
C1 has a stretch of Leu and Ile-rich region towards its C terminus: 105 LEEDIIHMVDI115 (Table 2). Possibly, this region
may be responsible for the nuclear export or cytoplasmic localization activity of C1, which needs to be further confirmed by
mutational analysis. The two-hybrid protein interaction stud-
133
Fig. 4. Two hybrid results: (a) -galctosidase assay, (b) leucine prototrophy on minimal medium deficient for, tryptophan, histidine, uracil and
leucine. (a) pEGCP-pJGCP (1), pEGCP-pJGLeKAP1 (2), pEGC1-pJGC1
(3), pEGC1-pJGLeKAP1 (4), pEGCP-pJGC1 (5), pEGLam-pJGCP (6),
pEGLam-pJGC1 (7), pEGLam-pJGLeKAP1 (8). (b) pEGCP-pJGCP (1),
pEGCP-pJGLeKAP1 (2), pEGC1-pJGC1 (3), pEGC1-pJGLeKAP1 (4),
pEGCP-pJGC1 (5).
ies showed that C1 interacts with itself, also with CP and the
tomato protein karyopherin . Since CP and C1 have nuclear
import and export functions, respectively, the interaction of CP
with C1 might be involved in the nuclear transport of the
virus analogous to the cooperative interaction of NSP and MP
of bipartite begomoviruses (Sanderfoot and Lazarowitz, 1995;
Gafni and Epel, 2002). Yeast-two-hybrid analysis of the mutant
C1 protein showed that the domain of C1 interacting with
CP is at the N terminal half whereas the domain(s) of C1
interacting with itself and karyopherin are at the C terminal
half. Karyopherins are soluble transport receptors that interact
with basic NLS sequences and help in nuclear import (Gorlich
and Kutay, 1999). The interaction of C1 with karyopherin ,
despite lacking nuclear localization needs to be explored further
to understand its significance. It may be speculated that karyopherin , which is a recycled protein, after carrying its cargo and
emptying it into the nucleus, is brought out of the nucleus by its
Fig. 5. Two hybrid results of mutant C1: (a and b) -galactosidase assay, (c)
leucine prototrophy on minimal medium deficient for, tryptophan, histidine,
uracil and leucine. (a) pEGNC1-pJGNC1 (1), pEGNC1-pJGC1
(2), pEGNC1-pJGLeKAP (3), pEGNC1-pJGCC1 (4) pEGNC1pJGCP (5) pEG Lamin C-pJGNC1 (6). (b) pEGCC1-pJGCP
(1), pEGCC1-pJGC1 (2), pEGCC1-pJGLeKAP (3), pEGCC1pJGNC1 (4), pEGCC1-pJGCC1 (5), pEG Lamin C-pJGCC1 (6).
(c) Lane pEGNC1-pJGNC1 (1), pEGNC1-pJGC1 (2), pEGNC1pJGLeKAP1 (3), pEGCC1-pJGCP (4).
134
Fig. 6. (a) N. benthamiana plants showing abnormal phenotype upon expressing the C1 gene in sense orientation. Plants expressing C1 in sense orientation (ad),
C1 in antisense orientation (e), non-transformed plants (f). (b) Northern analysis of transgenic N. benthamiana plants. Lane 1: non-transformed plant, lanes 25:
C1 sense plants, lane 6 and 7: C1 antisense plants.
135
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