Journal of General Virology (2012), 93, 2290–2298
DOI 10.1099/vir.0.044420-0
The movement protein encoded by gene 3 of
rice transitory yellowing virus is associated with
virus particles
Akihiro Hiraguri,1 Hiroyuki Hibino,1 Takaharu Hayashi,1 Osamu Netsu,13
Takumi Shimizu,1 Tamaki Uehara-Ichiki,1 Toshihiro Omura,1
Nobumitsu Sasaki,2 Hiroshi Nyunoya2 and Takahide Sasaya1
Correspondence
1
Takahide Sasaya
2
[email protected]
Received 17 May 2012
Accepted 13 July 2012
National Agricultural Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8666, Japan
Gene Research Center, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu,
Tokyo 183-8509 Japan
Gene 3 in the genomes of several plant-infecting rhabdoviruses, including rice transitory yellowing
virus (RTYV), has been postulated to encode a cell-to-cell movement protein (MP). Transcomplementation experiments using a movement-defective tomato mosaic virus and the P3
protein of RTYV, encoded by gene 3, facilitated intercellular transport of the mutant virus. In
transient-expression experiments with the GFP-fused P3 protein in epidermal leaf cells of
Nicotiana benthamiana, the P3 protein was associated with the nucleus and plasmodesmata.
Immunogold-labelling studies of thin sections of RTYV-infected rice plants using an antiserum
against Escherichia coli-expressed His6-tagged P3 protein indicated that the P3 protein was
located in cell walls and on virus particles. In Western blots using antisera against E. coliexpressed P3 protein and purified RTYV, the P3 protein was detected in purified RTYV, whilst
antiserum against purified RTYV reacted with the E. coli-expressed P3 protein. After immunogold
labelling of crude sap from RTYV-infected rice leaves, the P3 protein, as well as the N protein,
was detected on the ribonucleocapsid core that emerged from partially disrupted virus particles.
These results provide evidence that the P3 protein of RTYV, which functions as a viral MP, is a
viral structural protein and seems to be associated with the ribonucleocapsid core of virus
particles.
INTRODUCTION
Transitory yellowing disease of rice (Oryza sativa L.) was
first described in Taiwan (Chiu et al., 1965) and later
in Japan and Thailand (Inoue et al., 1986). The main
symptoms are yellowing of lower leaves and fewer tillers,
resulting in a loss in grain yields. On the basis of inoculation
experiments with insect vectors (Nephotettix nigropictus, N.
cincticeps and N. virescens) and electron microscopy of the
diseased rice cells, the agent of transitory yellowing disease
was identified as a rhabdovirus and named rice transitory
yellowing virus (RTYV) (Chiu et al., 1965, 1968, 1990;
Shikata & Chen, 1969). Another rhabdovirus, rice yellow
stunt virus (RYSV), causes yellow stunt disease of rice, a
serious problem in central and southern China (Chen et al.,
1979; Fan et al., 1965). The virus is transmitted by the same
insect vectors as for RTYV. As judged from the intracellular
3Present address: Laboratory of Plant Pathology, Department of
Biological and Environmental Science, Shizuoka University, 836 Ohya,
Shizuoka 422-8529, Japan.
A supplementary table is available with the online version of this paper.
2290
distribution of RTYV and RYSV, both viruses were assigned
to the genus Nucleorhabdovirus. The complete nucleotide sequences of RTYV and RYSV indicated that both are
strains of the same virus, rather than distinct viruses;
however, symptoms on rice induced by RTYV infection
differ slightly from those caused by RYSV infection
(Hiraguri et al., 2010).
Rhabdoviruses have a non-segmented, negative-sense
ssRNA genome that contains a 39-leader sequence (39-le),
five genes and a 59-trailer sequence (tr-59) in the order
39-le2N2P2M2G2L2tr-59 (Dietzgen et al., 2011). In
general, rhabdovirus particles are composed of a lipid
envelope derived from host membranes and a ribonucleocapsid core consisting of a viral RNA genome bound to
complexes of the nucleocapsid protein (N), the phosphoprotein (P) and polymerase (L), which are encoded by
genes N, P and L, respectively. The glycoprotein (G),
encoded by gene G, protrudes from the exterior of the
lipid envelope, and the matrix protein (M), encoded by
gene M, connects the envelope to the ribonucleocapsid
core (Dietzgen et al., 2011). The genome organization of
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RTYV MP is associated with virus particles
RTYV (synonym of RYSV) is unique in having two genes
in addition to the basic gene order (Hiraguri et al., 2010;
Huang et al., 2003). The small P6 protein of RYSV,
encoded by gene 6 between genes G and L, was detected in
purified virus preparations by immunoblot analysis using
antiserum against the glutathione S-transferase (GST)–P6
fusion protein (Huang et al., 2003). Gene 3, located
between genes P and M, has been identified in the genomes
of all plant-infecting rhabdoviruses, but not in animalinfecting rhabdoviruses (Scholthof et al., 1994; Tanno
et al., 2000; Tsai et al., 2005; Wetzel et al., 1994). The P3
protein, encoded by gene 3, has been postulated to be a
movement protein (MP; Melcher, 2000), and its function
as an MP has been demonstrated for RYSV (Huang et al.,
2005). However, the involvement of the P3 protein in virus
particles has not been adequately shown.
Primary infection of plants, from the entry of the viruses
into a plant cell with a mechanically damaged cell wall and
plasma membrane, is mostly confined to a single cell. MPs
are then responsible for transporting viral genomes or
particles to adjacent uninfected cells through plasmodesmata (PD). Plant viruses encode one or more MPs.
Although MPs are highly variable in their amino acid
sequences and protein structures, they share the ability to
localize at PD and increase the size-exclusion limit of PD
(Benitez-Alfonso et al., 2010; Lucas, 2006; Melcher, 1990).
The requirement of MPs for cell-to-cell movement was
first demonstrated in studies on a non-structural, 30 kDa
protein (30K) of tobacco mosaic virus (TMV) in the genus
Tobamovirus (Atabekov & Dorokhov, 1984). By a reversegenetics approach using in vitro-constructed mutants with
a frame shift in the 30K protein gene, the 30K protein has
been revealed to be involved in cell-to-cell movement
(Meshi et al., 1987). Additionally, a potexvirus mutant
defective in cell-to-cell movement can spread when
infectious RNA of the mutant virus was co-bombarded
with a separately cloned 30K protein gene of TMV
(Morozov et al., 1997). Several viral movement proteins
were characterized as belonging to the ‘30K’ superfamily of
MPs, with primary and secondary structural similarity to
the 30K protein of TMV (Melcher, 2000).
Analyses of primary and secondary protein structures of the
P3 protein of RYSV suggested that the P3 protein is a
member of the ‘30K’ superfamily and can trans-complement
cell-to-cell movement for a movement-deficient potato
virus X (PVX) mutant in Nicotiana benthamiana leaves
(Huang et al., 2005). In the present study, to demonstrate
the ability of the P3 protein of RTYV to act as an MP, we
used a particle bombardment-mediated trans-complementation assay with a cell-to-cell movement-defective tomato
mosaic virus (ToMV) mutant and transient expression
experiments with the GFP-fused P3 protein (P3–GFP). In
Western blot analyses of purified virus and immunogold
electron microscopy of the crude sap from RTYV-infected
rice leaves, we found that the P3 protein is associated with
virus particles, in particular the ribonucleocapsid core of
virus particles.
http://vir.sgmjournals.org
RESULTS
Trans-complementation of a movement-defective
ToMV mutant by the RTYV P3 protein
To investigate trans-complementation for the P3 protein as
a cell-to-cell movement protein of the ‘30K’ superfamily,
we used a particle bombardment-mediated trans-complementation assay with a ToMV-based chimeric virus. Gene 3
of RTYV was inserted into a plant expression plasmid,
pE7133-GW (Hiraguri et al., 2011), then the plasmid was
introduced into mature leaves of N. benthamiana via particle
bombardment, together with a plasmid piLMRd.erG3 (Fig.
1a) (Hiraguri et al., 2011). When piLMRd.erG3 that
encoded an endoplasmic reticulum (ER)-targeting GFP
(erGFP) as a reporter was used alone to bombard N.
benthamiana leaves, GFP fluorescence was restricted to
single, isolated epidermal cells at 96, 100 and 100 % of
transfected sites at 1, 2 and 4 days post-bombardment,
respectively [Fig. 1b(i), c], indicating that the ToMV mutant
was able to express GFP in initially infected cells, but was
defective in cell-to-cell movement due to the lack of viral
MP. In contrast, when piLMRd.erG3 was co-bombarded
with pE7133-RTYV P3, clusters of green-fluorescing cells at
1 day post-bombardment [Fig. 1b(ii)] and the number of
fluorescing clusters (Fig. 1c) increased over time. In 76 % of
infection foci, the movement-defective virus had spread
from the initially infected cells across one or more cell
boundaries at 4 days post-bombardment. These results
suggested that the defect in cell-to-cell movement of the
ToMV mutant from piLMRd.erG3 was complemented by
co-expression of the RTYV P3 protein. Similar results were
obtained in a positive control: after piLMRd.erG3 was cobombarded with plasmid pE7133-ToMV MP, ToMV MP
was then expressed [Fig. 1b(iii)]. However, we noted that the
ToMV MP was more efficient than the P3 protein in
mediating cell-to-cell movement of the movement-defective
virus (Fig. 1c).
Subcellular localizations of RTYV P3-GFP in N.
benthamiana epidermal cells
To clarify the subcellular sites of the RTYV P3 protein in N.
benthamiana epidermal cells, we bombarded leaves with an
expression plasmid encoding the P3 protein fused with
GFP (pE7133-RTYV P3 : GFP) (Fig. 2a). As a control for
the expression of free GFP, pE7133-GFP was used. In
N. benthamiana leaves bombarded with pE7133-RTYV
P3 : GFP, green fluorescence from the fusion protein was
detected predominantly as punctate structures at the cell
periphery, presumably indicating PD [Fig. 2b(i)]. These
punctate structures were stationary and occurred at
discrete regions of the epidermal cell walls, regardless of
the age or position of leaves used for the bombardments.
RTYV P3 protein fusions were also detected in the nucleus,
but the intensity of the fluorescence was much lower than
that for free GFP. In contrast, when plasmid pE7133-GFP
was used to bombard N. benthamiana leaves, green
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A. Hiraguri and others
(a)
ToMV genome
126 kDa
184 kDa
MP
pE7133-RTYV P3
35S
RT YV P3
NOS
pE7133-ToMV MP
35S
ToMV MP
NOS
CP
piLMRd.erG3
35S
126 kDa
184 kDa
erGFP
NOS
(b)
(i)
(ii)
(c)
Frequency distribution of
fluorescing epidermal cells (%)
100%
(iii)
4
90%
23
80%
43
13
70%
60%
50%
16
96
100
52
58
74
73
6
6
13
15
15
4
100
21
40%
9
10
15
30%
48
20%
32
10%
–
1 day
p.b.
24
10
23
RT YV P3
2 days 4 days
p.b.
p.b.
1 day
p.b.
ToMV MP
2 days 4 days
p.b.
p.b.
1 day
p.b.
2 days 4 days
p.b.
p.b.
Four or more cells
0
0
0
7
12
24
18
40
29
Three cells
0
0
0
4
1
4
3
3
0
Two cells
1
0
0
5
6
7
3
3
5
Single cells
27
33
29
15
9
11
7
8
6
Fig. 1. Trans-complementation of a movement-defective ToMV mutant by the RTYV P3 protein. (a) Schematic diagrams of an
infectious ToMV expression plasmid (left) and viral MP expression plasmids (right). Genomic organization of wild-type ToMV is
at the top. Open boxes represent ORFs of encoded proteins; a box delineated with broken lines represents an untranslatable
sequence derived from the ToMV MP gene. Pentagons indicate the 35S RNA promoter of cauliflower mosaic virus (CaMV)
(35S) and the nopaline synthase terminator (NOS). (b) Confocal laser micrographs of GFP in leaves of N. benthamiana 2 days
post-bombardment with piLMRd.erG3 alone (i), piLMRd.erG3 and pE7133-RTYV P3 (ii) and piLMRd.erG3 and pE7133-ToMV
MP (iii). Bars, 50 nm. (c) Frequency distribution of fluorescing epidermal cells in leaves of N. benthamiana bombarded with
piLMRd.erG3 and various viral MP expression plasmids at 1, 2 and 4 days post-bombardment (p.b.). Numbers of sites with
fluorescence from a single cell or from two, three or four or more cells are shown in the table, with corresponding percentages
plotted on the graph. The experiment was repeated three times; the values shown are from a single representative experiment.
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Journal of General Virology 93
RTYV MP is associated with virus particles
(a)
pE7133-GFP
35S
GFP
NOS
pE7133-RTYV P3 : GFP
35S
RT YV P3
GFP
NOS
(b)
(i)
(ii)
with anti-P3 serum, then treated with gold-conjugated
secondary antibody. Numerous gold particles localized in
the cell walls of infected leaves. This gold labelling pattern
is similar to those reported for other viral MPs (Li et al.,
2004; Xiong et al., 2008). Interestingly, gold particles were
also observed on the virus particles in the host cells, which
we inferred to be the result of an association of RTYV P3
protein with the virus particles [Fig. 2c(i)]. Gold particles
were not observed in the cell walls in thin sections of
healthy rice leaves probed with the anti-P3 serum [Fig.
2c(ii)] or in the cell walls or on the virus particles in thin
sections of RTYV-infected rice leaves probed with a preimmune serum (data not shown).
Detection of RTYV P3 and N proteins in purified
virus by Western blot
(c)
(i)
RTYV particles have been reported to constitute five
proteins encoded by the RTYV genome: N, P, M, G and L.
However, the association of the P3 protein with the virus
particles has not been adequately demonstrated. To clarify
the association of the RTYV P3 protein with virus particles,
we used Western blot analysis of purified RTYV and
antisera against E. coli-expressed viral proteins or the
purified virus. When anti-P3 serum was used, RTYV P3
protein (32.8 kDa) was detected in the purified virus and
in RTYV-infected rice extracts (Fig. 3, lanes 2 and 4), as
well as in E. coli-expressed P3 protein (Fig. 3, lane 1). Due
to the addition of the His6 tag, the migration of P3 protein
(ii)
(a)
Fig. 2. Subcellular localization of the RTYV P3 protein. (a)
Schematic diagram of plasmids used for transient expression of
the free GFP (pE7133-GFP) and the RTYV P3–GFP fusion
protein (pE7133-RTYV P3 : GFP) in leaf epidermal cells of N.
benthamiana. (b) Confocal laser micrograph of the RTYV P3–GFP
fusion protein (i) and the free GFP (ii) in epidermal cells of N.
benthamiana at 2 days post-bombardment. Bars, 50 nm. Open
arrowheads mark fluorescing nuclei. (c) Electron micrographs
showing immunogold labelling of the RTYV P3 protein in the cell
walls of RTYV-infected (i) and healthy (ii) rice leaves treated with
anti-P3 serum, followed by treatment with gold-conjugated
secondary antiserum. Bar, 250 nm.
fluorescence from free GFP was generally detected in the
nucleus and the cytoplasm of epidermal cells, but cell-wallassociated punctate structures were not observed [Fig.
2b(ii)].
The subcellular location of the RTYV P3 protein was
also revealed by immunogold electron microscopy using
antiserum against the Escherichia coli-expressed His6tagged P3 protein (anti-P3 serum) (Fig. 2c). Thin sections
from RTYV-infected rice leaves were prepared and treated
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1
2
3
4
5
6
7
8
His-P3
P3
M
30
(b)
1
2
3
75
50
4
5
6
7
8
His-N
N
Fig. 3. Western blot analysis of P3 and N proteins from purified
RTYV. (a) Detection of RTYV P3 protein with anti-P3 serum (lanes
1–4) and with anti-RTYV serum (lanes 5–8). Total protein from E.
coli-expressed P3 protein (lanes 1 and 5), purified virus (lanes 2
and 6), healthy rice leaves (lanes 3 and 7) and RTYV-infected rice
leaves (lanes 4 and 8) was separated by SDS-PAGE (12 %
polyacrylamide) followed by immunoblot analysis. (b) Detection of
RTYV N protein with anti-N serum (lanes 1–4) and with anti-RTYV
serum (lanes 5–8). Total protein from E. coli-expressed N protein
(lanes 1 and 5), purified virus (lanes 2 and 6), healthy rice leaves
(lanes 3 and 7) and RTYV-infected rice leaves (lanes 4 and 8) was
used. Positions of His6-tagged P3 protein (His-P3), RTYV P3
protein (P3), RTYV M protein (M), His6-tagged N protein (His-N)
and RTYV N protein (N) are marked on the right; molecular size
markers are on the left (in kDa).
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A. Hiraguri and others
extracted from E. coli was slower than those of the P3
proteins from purified virus and from RTYV-infected
rice. Antiserum against purified virus (anti-RTYV serum)
reacted with E. coli-expressed His6-tagged P3 protein (Fig.
3a, lane 5). Furthermore, a weak band (32.8 kDa) with a
molecular size slightly larger than that of RTYV M protein
(31.3 kDa) was recognized as the P3 protein from the
purified virus (Fig. 3a, lane 6). Similar results were
obtained from experiments to detect E. coli-expressed or
intact N proteins with antiserum against the E. coliexpressed His6-tagged N protein (anti-N serum). RTYV N
protein was detected in purified virus and in RTYVinfected rice extracts (Fig. 3b, lanes 2 and 4), as well as in
the E. coli-expressed N protein (Fig. 3a, lane 1). Anti-RTYV
serum reacted with E. coli-expressed N protein (Fig. 3b,
lane 5) and RTYV N protein from purified virus and
RTYV-infected rice extracts (Fig. 3b, lanes 6 and 8).
Considering that N protein is a virus structural protein and
one of the components of a ribonucleocapsid core, these
results suggested that the RTYV P3 protein, similarly to the
RTYV N protein, is associated with virus particles and
might be a viral structural protein.
Detection of RTYV P3 and N proteins in disrupted
virus particles
Immunogold electron microscopy of crude saps from
RTYV-infected rice leaves was conducted to determine the
location of the P3 protein in RTYV particles. When crude
saps were smeared onto membrane-covered grids and
observed by electron microscopy, bullet-shaped virus
particles with enveloped structures (Chiu et al., 1990;
Shikata & Chen, 1969) were observed (Fig. 4a). However,
when the crude saps were treated with antiserum
solutions, the virus particles were unstable and disintegrated easily (Fig. 4b). When the crude saps were treated
with anti-P3 serum and then labelled with immunogold,
the gold particles associated with the RTYV particles; in
particular, gold particles were observed near the outer
(a)
(b)
(c)
(d)
Fig. 4. Immunogold labelling of disrupted RTYV particles from
crude sap of RTYV-infected rice leaves. RTYV particles from crude
sap were not treated (a) or treated with pre-immune antiserum (b),
anti-P3 serum (c) or anti-N serum (d), then treated with goldconjugated secondary antiserum and negatively stained in 2 %
uranyl acetate. RTYV particles were partially disrupted after the
treatment with the antisera. Bars, 100 nm.
2294
shells of partly disrupted RTYV particles (Fig. 4c). In
contrast, no gold particles were observed on the disrupted
viral shells. Similar results were obtained when the crude
saps were treated with anti-N serum, a component of the
ribonucleocapsid core, which is located in virus particles
(Fig. 4d). These results apparently indicated that these
gold labels were on the ribonucleocapsid that had spilled
from the disrupted RTYV particles and that the RTYV P3
and N proteins were associated with virus particles,
specifically with the ribonucleocapsid core inside virus
particles.
DISCUSSION
Most plant viruses have evolved to encode one or more
MPs, which facilitate viral cell-to-cell movement through
the PD of susceptible hosts. Among viruses of the family
Rhabdoviridae, several plant-infecting rhabdoviruses, including RTYV, have one or more additional genes between
genes P and M. A possible MP role for these non-structural
proteins encoded by the genes was first proposed for
the sc4 protein of sonchus yellow net virus (SYNV)
(Scholthof et al., 1994). On the basis of secondary structural predictions, the sc4 protein and related proteins, such
as the P3 proteins of RYSV and maize mosaic virus, P4
protein of maize fine streak virus and 4b protein of
lettuce necrotic yellows virus, are possible members of the
‘30K’ superfamily (Huang et al., 2005; Melcher, 2000). In
transient-expression experiments, the SYNV sc4 protein
was localized at the periphery of epidermal cells of N.
benthamiana (Goodin et al., 2002) and formed punctate
structures at the periphery of SYNV-infected cells (Goodin
et al., 2007), suggesting that the protein may act as an MP
at or near the PD. The first direct evidence for the function
of a non-structural protein as an MP was provided by
trans-complementation with the RYSV P3 protein of a
movement-deficient PVX mutant that contained a frame
shift in the p25 gene, which encodes one of the three MPs
that comprise the triple-gene block of PVX (Huang et al.,
2005). Our data provide evidence here that the RTYV P3
protein can trans-complement cell-to-cell movement of a
movement-deficient ToMV mutant. We have also shown
that P3 protein fused with GFP can accumulate at the cell
periphery and form punctate structures in the absence of
other viral proteins, similar to the subcellular accumulation
observed for fluorescent-protein-fused MPs of the ‘30K’
superfamily of TMV and ToMV (Crawford & Zambryski,
2000; Sasaki et al., 2009). Furthermore, we showed that the
RTYV P3 protein was localized in the cell walls of virusinfected rice plants, which is a common feature of MPs.
However, the RTYV P3 protein appeared to be less effective
than the ToMV MP for trans-complementation of movement-defective ToMV. This difference might be due to
RTYV primarily infecting rice, a monocotyledonous plant;
the RTYV P3 protein may not interact efficiently with
specific host component(s) in a dicotyledonous plant such
as N. benthamiana.
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Journal of General Virology 93
RTYV MP is associated with virus particles
During virus movement in infected cells, a virally encoded
MP interacts with a virus genome to be transported from
virus replication sites along the cytoskeleton and/or ER
networks to the PD in the cell wall of infected cells. Then,
the virus moves through the PD in the form of an MPassociated ribonucleoprotein or virus particle complex
(Lucas, 2006; Scholthof, 2005). In the case of plantinfecting rhabdoviruses, virus particles are at least one
order of magnitude larger than PD, with a diameter of
approximately 5 nm. Therefore, transit of intact particles
would require enormous PD alterations, that ought to be
easily visible by electron microscopy. Although numerous
ultrastructural studies have focused on different rhabdoviruses, such intact particles within enlarged PDs have not
been observed. Similar to other negative-sense RNA viruses
with a ribonucleocapsid complex as a minimal infectious
unit (Jackson et al., 2005; Kormelink et al., 2011; Scholthof,
2005), formation of an MP-associated ribonucleocapsid
may be involved in cell-to-cell movement for plantinfecting rhabdoviruses. A specific interaction between
genes 3 and N of RYSV was revealed in a GST pull-down
assay with E. coli-expressed recombinant proteins, and a
region between aa 61 and 90 of the P3 protein was shown
to be involved in binding to the N protein (Huang et al.,
2005). Our immunogold electron microscopy results
suggest that the RTYV P3 protein is associated with the
ribonucleocapsid core of RTYV. In contrast, the SYNV sc4
protein interacted specifically with the G protein and not
with the N protein in bimolecular fluorescence complementation experiments among all pairwise interactions of
SYNV-encoded proteins (Min et al., 2010). SYNV sc4
protein was considered to interact with the G protein,
together with the SYNV M protein and several cytoplasmtethered transcription activators, and to form an ER- and
microtubule-associated movement complex. Protein Y, a
putative MP, of potato yellow dwarf virus (PYDV) in the
genus Nucleorhabdovirus interacts with the M protein, but
not the N protein, of PYDV (Bandyopadhyay et al., 2010).
A comparison of protein–protein interaction data between
virus-encoded proteins for these three nucleorhabdoviruses
suggests that strategies for cell-to-cell movement may differ
even among closely related viruses in the genus Nucleorhabdovirus. To form the postulated ER- and microtubuleassociated movement complex, the P3 protein of RTYV
and RYSV may bind directly to the N protein. In contrast,
the SYNV sc4 protein and PYDV Y protein may interact
directly with either the G or M protein and may not bind
directly to the N protein.
The virus particles of the plant-infecting rhabdoviruses
consist of five virus-encoded proteins, and the MPs have
been believed to be non-structural proteins (Dietzgen et al.,
2011; Jackson et al., 2005; Kormelink et al., 2011). In
the case of RTYV and RYSV, these virus particles have
been reported to consist of five virus-encoded structural
proteins, N, P, M, G and L, according to SDS-PAGE
analysis of purified viruses (Chiu et al., 1990; Fang et al.,
1994; Hayashi & Minobe, 1987). However, when these
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SDS-PAGE profiles of purified RTYV and RYSV were reverified, a weak band with a molecular mass that differed
slightly from that of the M protein, probably corresponding to the P3 protein, was noted. Our Western blot analysis
using anti-RTYV serum and anti-P3 serum revealed definitively that P3 protein was in purified virus. Furthermore,
our immunogold electron microscope observation of the
thin sections and the crude saps from the virus-infected rice
plants provided evidence that the P3 protein of RTYV was
associated with the virus particles and seemed to be localized
in the viral inner ribonucleocapsid core, rather than in the
outer surface of virus particles. The association of an MP
with virus particles was also shown in an analysis of
disrupted SYNV particles that were fractionated by sucrosegradient centrifugation; the sc4 protein co-sedimented with
envelope-associated viral structural proteins G and M. In
contrast to RTYV, the sc4 protein of SYNV is considered to
be incorporated into the virus envelope that is derived by
budding through the inner nuclear membrane (Scholthof
et al., 1994). In addition, the small P6 protein of RYSV,
encoded by a gene between genes G and L, was detected in
purified virus and thought to be incorporated into virus
particles (Huang et al., 2003). However, an additional gene
such as gene 6 has not been found in the SYNV genome.
These results indicated that the particle structures of SYNV
and RTYV, as well as RYSV, may differ, even though these
viruses are related closely in the same genus of the family
Rhabdoviridae.
Although the virus particle structures of SYNV and RTYV
may differ, the MPs can be associated with virus particles
and can also be a structural protein. The presence of MPs
in virus particles may yield significant advantages for rapid
virus spread from the initially infected cell, where the
enveloped virus particles are uncoated. In the infection
process, numerous virus particles are thought to be injected
into the plant cells. Some of these virus particles may start
translation or replication in the infected cells. Others, if
they are ready to move to neighbouring cells, will form cellto-cell movement complexes with the assistance of the MP
in the virus particles and could be released rapidly into
neighbouring cells, which would provide multiple possibilities for the onset of viral infection.
METHODS
Particle bombardment-mediated trans-complementation assay.
Plasmid piLMRd.erG3, a derivative of piL.erG3, was constructed
previously for trans-complementation experiments with a movementdefective ToMV mutant (Hiraguri et al., 2011). The plasmid lacks the
start codon (from ATG to ACG) and a large part (18–557 nt) of the MP
gene and contains the ER-targeting GFP (erGFP) gene instead of the
ToMV CP gene. The plasmid generates an erGFP fluorescence
replication-competent but MP- and CP-defective ToMV (L strain)
mutant under the control of the 35S RNA promoter of cauliflower
mosaic virus (CaMV) after transfection by particle bombardment (Fig.
1a). Infection and spread of the ToMV mutant derived from
piLMRd.erG3 can be monitored by observing the distribution of
erGFP fluorescence.
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A. Hiraguri and others
For preparing a 35S promoter-driven cDNA clone of RTYV gene 3
and the ToMV MP gene, RTYV gene 3 and the ToMV MP genes were
amplified from a cDNA library of RTYV and plasmid pTLW3 by PCR
as described previously (Hiraguri et al., 2010) with appropriate
primer sets (Table S1, available in JGV Online). The PCR products
were cloned into pDONR221 (Invitrogen) using Gateway technology
according to the manufacturer’s instructions. Then, RTYV gene 3
and the ToMV MP gene were moved into pE7133-GW (Hiraguri
et al., 2011) downstream of the 35S promoter to generate the
plant expression vectors pE7133-RTYV P3 and pE7133-ToMV MP,
respectively.
harvested from infected rice plants were homogenized in an
extraction buffer composed of 0.1 M sodium phosphate, pH 7.4,
0.5 % 2-mercaptoethanol and 10 mM EDTA. The homogenate was
centrifuged at 7000 r.p.m. for 10 min in a Hitachi R10A2 rotor. The
supernatant was centrifuged at 45 000 r.p.m. for 90 min in a Hitachi
P50A2 rotor. The virus pellet was suspended in the same buffer as
above and the suspension was subjected to rate zonal centrifugation at
16 000 r.p.m. for 90 min in 5–20 % Percoll gradients in a Hitachi
SRP28SA rotor. Fractions containing virus particles were pooled, and
the virus was recovered by fractionation on a Sepharose CL-4B
column (GE Healthcare) (Chiu et al., 1965).
These plasmids were introduced into epidermal cells of N.
benthamiana via particle bombardment using the PDS-1000/He
system (Bio-Rad Laboratories) as described previously (Sasaki et al.,
2009). Mature leaves (6–9 cm long) of N. benthamiana (4–7 weeks
old) were cut and placed on an empty plate at a target distance of
6 cm. Three milligrams of 1 mm gold particles were coated with 1 mg
plasmid DNA and the resultant particles were divided into three equal
parts, each of which was used to bombard leaves with a rupture disc
of 900 p.s.i. (6.21 MPa). Bombarded leaves were incubated at 25 uC
in the dark for 1–4 days. GFP signals were observed with an LSM510
confocal laser microscope (Carl Zeiss). An excitation light of 488 nm
produced by the argon laser and an emission filter of 505–530 nm
allowed the detection of GFP-specific fluorescence. For each of the
plasmids used in this research, the bombardment was repeated at least
three times.
Western blot analysis. To elucidate whether RTYV P3 protein is a
viral structural protein, we used Western blot analysis of the purified
virus as described previously (Sasaya et al., 2001). E. coli-expressed P3
protein, purified virus protein and total proteins from healthy and
RTYV-infected rice leaves were separated by SDS-PAGE (12 %
polyacrylamide). The gels were transferred to PVDF membranes
(Millipore) at 50 mA for 50 min. After blocking overnight with Trisbuffered saline (TBS) (20 mM Tris/HCl pH 7.5, 500 mM NaCl)
containing 4 % skimmed milk powder, the membranes were probed
for 1 h on a shaker at room temperature with anti-P3 serum, anti-N
serum or anti-RTYV serum diluted 1 : 5000. Subsequently, the
membranes were incubated with alkaline phosphatase-conjugated
anti-rabbit IgG (H+L) diluted 1 : 10000 (Jackson ImmunoResearch)
for 1 h at room temperature. The detection signal was developed with
nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate
(BCIP).
Localization of RTYV P3 protein in N. benthamiana cells. To
localize RTYV P3 protein in cells, we constructed pE7133-RTYV
P3 : GFP, an expression plasmid for RTYV P3 fused with GFP (P3–
GFP), which was expressed through the activity of a CaMV 35S RNA
promoter. The GFP gene and the RTYV gene 3 lacking the stop codon
were amplified by PCR with pBI-OX-GFP (inPLANTA) and pE7133RTYV P3 as templates, respectively, using appropriate primer sets
(Table S1). To construct pE7133-RTYV P3 : GFP, we cloned the
RTYV gene 3 into pE7133-GW GFP, which was altered by modifying
pE7133-GW to express GFP fused to the C-terminal of arbitrary
proteins (Fig. 2a) (Hiraguri et al., 2011). As a control for the
expression of free GFP, the GFP gene was cloned into pE7133-GW
using the Gateway technology to create pE7133-GFP (Hiraguri et al.,
2011). These plasmids were used individually to bombard N.
benthamiana epidermal leaf cells as already described. The bombarded
N. benthamiana leaves were incubated at 25 uC in the dark for 24 h.
GFP signals were observed with a LSM510 confocal laser microscope
(Carl Zeiss).
Purification of recombinant RTYV P3 protein and preparation
of antisera. For producing anti-P3 serum or anti-N serum, genes 3
and N of RTYV were respectively amplified from a DNA library of
RTYV, described previously (Hiraguri et al., 2010), by PCR with a
specific primer set (Table S1). Amplified PCR fragments were ligated
into plasmid pDONR221 and were transferred into plasmid pDEST17
using an LR clonase reaction to yield pDEST17-P3, in which the N
and P3 sequences are fused in frame to the His6 tag sequence. The
His6-tagged N fusion protein and the His6-tagged P3 fusion protein
were expressed in E. coli strain BL-21AI (Invitrogen) by induction of
arabinose and were purified with an Ni-NTA Purification Module kit
according to the instructions provided by the suppliers (Qiagen).
Approximately 1 mg of the purified His6-tagged N fusion protein or
the His6-tagged P3 fusion protein was emulsified in Freund’s
complete adjuvant and injected subcutaneously into New Zealand
white rabbits. Four injections were given at intervals of 2 weeks;
antisera were collected from bleeds 15 days after the final injection.
Purification of virus. RTYV was purified from RTYV-infected rice
plants as described previously (Hayashi & Minobe, 1987). Leaves
2296
Immunogold electron microscopy of thin sections of RTYVinfected rice leaves. Small leaf samples were excised from healthy
and RTYV-infected rice plants and fixed in 50 mM PBS (pH 7.2)
containing 1 % glutaraldehyde and 2 % formaldehyde for 3 h at 4 uC.
Fixed samples were dehydrated through a graded ethanol series at
220 uC and embedded in Lowicryl K4M resin (Electron Microscopy
Sciences). Polymerization was allowed to proceed for 72 h at 220 uC.
Samples were sectioned on an ultramicrotome (LKB Nova) with a
diamond knife and then mounted on 200-mesh nickel collodion
grids. The sections were placed on drops of anti-P3 serum diluted
1 : 100 in TBS, for 30 min at room temperature. After washing with
TBS, the grids were probed with immunogold-labelled goat
antibodies against rabbit IgG with 20 nm gold particles (British
Bifocals International). The grids were stained in 2 % uranyl acetate
and lead citrate solutions and then observed with an H-7000 electron
microscope (Hitachi).
Immunogold electron microscopy of crude sap from RTYVinfected rice leaves. Leaves harvested from infected rice plants were
homogenized in TBS, and the homogenate was centrifuged at
7000 r.p.m. for 10 min in a Hitachi R10A2 rotor. The supernatant
was dropped onto membrane-coated 200-mesh nickel collodion grids.
The grids were placed on drops of anti-P3 serum diluted 1 : 50 or antiN serum diluted 1 : 500 in TBS for 1 h at 37 uC. After washing with
TBS, the grids were placed on drops of the immunogold-labelled goat
antibodies against rabbit IgG with 10 nm gold particles diluted 1 : 50
for 1 h at 37 uC. After staining in 2 % (w/v) uranyl acetate, the virus
particles were observed with the electron microscope.
ACKNOWLEDGEMENTS
The authors are grateful to Dr A. Tamai (Ishikawa Prefectural
University, Ishikawa, Japan) and Dr T. Meshi (National Institute of
Agrobiological Sciences, Tsukuba, Japan) for providing piL.erG3. This
work was supported by a grant from the Program for Promotion of
Basic Research Activities for Innovative Biosciences of the Biooriented Technology Research Advancement Institution (BRAIN) of
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Journal of General Virology 93
RTYV MP is associated with virus particles
Japan and a Grant-in-Aid for Young Scientists (B) from the Ministry
of Education, Culture, Sports, Science to N. S. (no. 20780030).
Hiraguri, A., Netsu, O., Shimizu, T., Uehara-Ichiki, T., Omura, T.,
Sasaki, N., Nyunoya, H. & Sasaya, T. (2011). The nonstructural
protein pC6 of rice grassy stunt virus trans-complements the cell-tocell spread of a movement-defective tomato mosaic virus. Arch Virol
156, 911–916.
REFERENCES
Huang, Y., Zhao, H., Luo, Z., Chen, X. & Fang, R.-X. (2003). Novel
Atabekov, J. G. & Dorokhov, Y. L. (1984). Plant virus-specific
transport function and resistance of plants to viruses. Adv Virus Res
29, 313–364.
Bandyopadhyay, A., Kopperud, K., Anderson, G., Martin, K. &
Goodin, M. (2010). An integrated protein localization and interaction
map for Potato yellow dwarf virus, type species of the genus
Nucleorhabdovirus. Virology 402, 61–71.
Benitez-Alfonso, Y., Faulkner, C., Ritzenthaler, C. & Maule, A. J.
(2010). Plasmodesmata: gateways to local and systemic virus
infection. Mol Plant Microbe Interact 23, 1403–1412.
Chen, S. X., Yuan, Y. L., King, D. D., Chen, K. Y., Lin, R. T. & Kao, T. M.
(1979). Studies on the development and epiphytotris of the rice
yellow stunt disease. Acta Phytopathol Sin 9, 41–54.
Chiu, R. J., Lo, T. C., Pi, C. L. & Chen, M. H. (1965). Transitory
yellowing of rice and transmission by the leafhopper Nephotettix
apicalis. Bot Bull Acad Sin 6, 1–18.
Chiu, R. J., Jean, J. H., Chen, M. H. & Lo, T. C. (1968). Transmission of
transitory yellowing virus of rice by two leafhoppers. Phytopathology
58, 740–745.
Chiu, R. J., Hsu, Y. H., Chen, M. J., Chen, C. C., Lee, R. C. R., Lin,
M. C., Lin, S. M. & Kuo, T. T. (1990). Purification and partial
structure of the genome of Rice yellow stunt virus: identification of the
gene 6-encoded virion protein. J Gen Virol 84, 2259–2264.
Huang, Y.-W., Geng, Y.-F., Ying, X.-B., Chen, X.-Y. & Fang, R.-X.
(2005). Identification of a movement protein of rice yellow stunt
rhabdovirus. J Virol 79, 2108–2114.
Inoue, H., Omura, T., Hayashi, T., Morinaka, T., Putta, M.,
Chettanachit, D., Parejarearn, A., Disthaporn, S. & Kadkao, S.
(1986). Rice transitory yellowing virus. In Virus Diseases of Rice and
Legumes in the Tropics (Technical Bulletin of the Tropical Agriculture
Research Center of Japan vol. 21) , pp. 65–77. Edited by T. Kajiwara &
S. Konno. Yatabe, Japan: Tropical Agriculture Research Center.
Jackson, A. O., Dietzgen, R. G., Goodin, M. M., Bragg, J. N. & Deng, M.
(2005). Biology of plant rhabdoviruses. Annu Rev Phytopathol 43, 623–
660.
Kormelink, R., Garcia, M. L., Goodin, M., Sasaya, T. & Haenni, A. L.
(2011). Negative-strand RNA viruses: the plant-infecting counter-
parts. Virus Res 162, 184–202.
Li, Y., Bao, Y. M., Wei, C. H., Kang, Z. S., Zhong, Y. W., Mao, P., Wu, G.,
Chen, Z. L., Schiemann, J. & Nelson, R. S. (2004). Rice dwarf
phytoreovirus segment S6-encoded nonstructural protein has a cellto-cell movement function. J Virol 78, 5382–5389.
characterization of rice transitory yellowing virus. Phytopathology 80,
777–783.
Lucas, W. J. (2006). Plant viral movement proteins: agents for cell-to-
Crawford, K. M. & Zambryski, P. C. (2000). Subcellular localization
Melcher, U. (1990). Similarities between putative transport proteins
determines the availability of non-targeted proteins to plasmodesmatal transport. Curr Biol 10, 1032–1040.
Dietzgen, R. G., Calisher, C. H., Kurath, G., Kuzmin, I. V., Rodriguez,
L. L., Stone, D. M., Tesh, R. B., Tordo, N., Walker, P. J. & other authors
(2011). Family Rhabdoviridae. In Virus Taxonomy: Classification
and Nomenclature. Ninth Report of the International Commitee
on Taxonomy of Viruses, pp. 686–713. Edited by A. M. Q. King,
M. J. Adams, E. B. Carstens & E. J. Lefkowitz. San Diego, CA:
Academic Press.
Fan, H., Zeng, D. S., Ye, B. J. & Lu, B. C. (1965). The preliminary
cell trafficking of viral genomes. Virology 344, 169–184.
of plant viruses. J Gen Virol 71, 1009–1018.
Melcher, U. (2000). The ‘30K’ superfamily of viral movement
proteins. J Gen Virol 81, 257–266.
Meshi, T., Watanabe, Y., Saito, T., Sugimoto, A., Maeda, T. & Okada, Y.
(1987). Function of the 30 kd protein of tobacco mosaic virus:
involvement in cell-to-cell movement and dispensability for replication. EMBO J 6, 2557–2563.
Min, B. E., Martin, K., Wang, R., Tafelmeyer, P., Bridges, M. & Goodin,
M. (2010). A host-factor interaction and localization map for a plant-
investigation on rice yellow stunt disease in Guangdong. Plant Prot 3,
143–145.
adapted rhabdovirus implicates cytoplasm-tethered transcription
activators in cell-to-cell movement. Mol Plant Microbe Interact 23,
1420–1432.
Fang, R. X., Wang, Q., Xu, B. Y., Pang, Z., Zhu, H. T., Mang, K. Q., Gao,
D. M., Qin, W. S. & Chua, N. H. (1994). Structure of the nucleocapsid
Morozov, S. Y., Fedorkin, O. N., Jüttner, G., Schiemann, J.,
Baulcombe, D. C. & Atabekov, J. G. (1997). Complementation of a
protein gene of rice yellow stunt rhabdovirus. Virology 204, 367–
375.
potato virus X mutant mediated by bombardment of plant tissues
with cloned viral movement protein genes. J Gen Virol 78, 2077–
2083.
Goodin, M. M., Dietzgen, R. G., Schichnes, D., Ruzin, S. & Jackson,
A. O. (2002). pGD vectors: versatile tools for the expression of green
and red fluorescent protein fusions in agroinfiltrated plant leaves.
Plant J 31, 375–383.
Goodin, M. M., Chakrabarty, R., Yelton, S., Martin, K., Clark, A. &
Brooks, R. (2007). Membrane and protein dynamics in live plant
nuclei infected with Sonchus yellow net virus, a plant-adapted
rhabdovirus. J Gen Virol 88, 1810–1820.
Sasaki, N., Ogata, T., Deguchi, M., Nagai, S., Tamai, A., Meshi, T.,
Kawakami, S., Watanabe, Y., Matsushita, Y. & Nyunoya, H. (2009).
Over-expression of putative transcriptional coactivator KELP interferes with Tomato mosaic virus cell-to-cell movement. Mol Plant
Pathol 10, 161–173.
Sasaya, T., Ishikawa, K. & Koganezawa, H. (2001). Nucleotide
Hayashi, T. & Minobe, Y. (1987). Serological studies on rice transitory
sequence of the coat protein gene of Lettuce big-vein virus. J Gen Virol
82, 1509–1515.
yellowing virus (RTYV) in rice plants and green leafhoppers. Jpn Agric
Res Q 21, 90–95.
Scholthof, H. B. (2005). Plant virus transport: motions of functional
equivalence. Trends Plant Sci 10, 376–382.
Hiraguri, A., Hibino, H., Hayashi, T., Shimizu, T., Uehara-Ichiki, T.,
Omura, T. & Sasaya, T. (2010). Complete sequence analysis of rice
Scholthof, K.-B. G., Hillman, B. I., Modrell, B., Heaton, L. A. &
Jackson, A. O. (1994). Characterization and detection of sc4: a
transitory yellowing virus and its comparison to rice yellow stunt
virus. Arch Virol 155, 243–245.
sixth gene encoded by sonchus yellow net virus. Virology 204, 279–
288.
http://vir.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 16:36:19
2297
A. Hiraguri and others
transitory yellowing virus. J Virol 3, 261–264.
subcellular localization of maize fine streak virus proteins. J Virol 79,
5304–5314.
Tanno, F., Nakatsu, A., Toriyama, S. & Kojima, M. (2000). Complete
Wetzel, T., Dietzgen, R. G. & Dale, J. L. (1994). Genomic organization
nucleotide sequence of Northern cereal mosaic virus and its genome
organization. Arch Virol 145, 1373–1384.
Xiong, R., Wu, J., Zhou, Y. & Zhou, X. (2008). Identification of a
Tsai, C. W., Redinbaugh, M. G., Willie, K. J., Reed, S., Goodin, M. &
Hogenhout, S. A. (2005). Complete genome sequence and in planta
movement protein of the Tenuivirus rice stripe virus. J Virol 82,
12304–12311.
Shikata, E. & Chen, M. J. (1969). Electron microscopy of rice
2298
of lettuce necrotic yellows rhabdovirus. Virology 200, 401–412.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 16:36:19
Journal of General Virology 93