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Virology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y v i r o
Department of Entomology and Plant Pathology, Noble Research Center, Oklahoma State University, Stillwater, OK 74078, USA
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA
The Samuel Roberts Noble Foundation 2510 Sam Noble Pky., Ardmore, OK 73401, USA
d
Biology Department, University of Wisconsin-LaCrosse 1725 State St. La Crosse, WI 54601, USA
e
Department of Statistics, Oklahoma State University, Stillwater, OK 74078, USA
b
c
a r t i c l e
i n f o
Article history:
Received 13 May 2009
Returned to author for revision 9 July 2009
Accepted 2 August 2009
Available online 3 September 2009
Keywords:
Potexvirus
Potato virus X
Triple gene block
Replicase
a b s t r a c t
Potato virus X (PVX) infection leads to certain cytopathological modications of the host endomembrane
system. The subcellular location of the PVX replicase was previously unknown while the PVX TGBp3 protein
was previously reported to reside in the ER. Using PVX infectious clones expressing the green uorescent
protein reporter, and antisera detecting the PVX replicase and host membrane markers, we examined the
subcellular distribution of the PVX replicase in relation to the TGBp3. Confocal and electron microscopic
observations revealed that the replicase localizes in membrane bound structures that derive from the ER. A
subset of TGBp3 resides in the ER at the same location as the replicase. Sucrose gradient fractionation
showed that the PVX replicase and TGBp3 proteins co-fractionate with ER marker proteins. This localization
represents a region where both proteins may be synthesized and/or function. There is no evidence to
indicate that either PVX protein moves into the Golgi apparatus. Cerulenin, a drug that inhibits de novo
membrane synthesis, also inhibited PVX replication. These combined data indicate that PVX replication relies
on ER-derived membrane recruitment and membrane proliferation.
Published by Elsevier Inc.
Introduction
Successful virus infection is a consequence of specic interactions
with host subcellular machinery to enable essential events such as
viral protein synthesis, replication, and cell-to-cell spread. The ability
of viruses to interact with host components underlies the appropriate
subcellular targeting of viral proteins and nucleic acids (Ahlquist,
2006; Buck, 1999a; Hwang et al., 2008). It is often presumed that a
viral movement protein (MP) carries infectious material into
uninfected cells in the form of a ribonucleoprotein complex, although
the exact mechanism by which viral genomes are transferred from the
site of synthesis to the plasmodesmata has not been clearly shown
(Haywood et al., 2002; Kiselyova et al., 2001; Lucas, 2006; Nelson,
2005; Verchot-Lubicz et al., 2007).
For most positive strand RNA viruses, viral replicases cause
membrane rearrangements to create a container or protected
environment for genome replication (Mackenzie, 2005; Salonen et
Corresponding author. Fax: +1 405 744 6039.
E-mail addresses: dbamu001@ucr.edu (D. Bamunusinghe),
cindy_hemenway@ncsu.edu (C.L. Hemenway), rsnelson@noble.org (R.S. Nelson),
sanderfo.anto@uwlax.edu (A.A. Sanderfoot), Verchot.lubicz@okstate.edu
(J. Verchot-Lubicz).
1
Current address for Devinka Bamunusinghe is University of California- Riverside,
Department of Plant Pathology and Microbiology, 3401, Watkins Drive, Boyce Hall,
Riverside, CA 92521.
0042-6822/$ see front matter. Published by Elsevier Inc.
doi:10.1016/j.virol.2009.08.002
al., 2005; Sanfacon and Zhang, 2008; Schwartz et al., 2004; Villanueva
et al., 2005). There are wide ranging reports of plant-infecting RNA
viruses associating with invaginations of the endoplasmic reticulum
(ER), chloroplast outer membrane, vacuolar membranes, mitochondria, nuclear envelope, or peroxisomes (Goodin et al., 2007; Hwang et
al., 2008; Jonczyk et al., 2007; Kim et al., 2006; Prod'homme et al.,
2003; Rubino et al., 2001; Turner et al., 2004; Wei and Wang, 2008).
Grapevine fanleaf virus (GFLV), Cowpea mosaic virus (CPMV), Red
clover necrotic mosaic virus (RCNMV) and Tobacco mosaic virus (TMV)
are a few recent examples of viruses whose replication requires
proliferation and/or invagination of ER membranes to sustain virus
replication (Carette et al., 2000; Heinlein et al., 1998; Mas and Beachy,
1999; Ritzenthaler et al., 2002; Turner et al., 2004). CPMV in particular
provides an example of a virus that stimulates host cells to increase
membrane synthesis resulting in specic expansion of the ER with no
obvious change in the Golgi apparatus (Carette et al., 2000), although
little is known about how viruses are able to induce synthesis of
distinct types of cellular membranes.
Tobacco mosaic virus (TMV) replicase is located on the ER or ER
modications (Buck, 1999b; Ishikawa and Okada, 2004). For TMV,
irregular shaped membrane bound structures containing the 183and 126-kDa replication-associated proteins and viral genomes serve
as a vehicle for trafcking virus within and between neighboring
cells (Kawakami et al., 2004; Liu et al., 2005). A current model is that
the TMV replication associated proteins and/or MP, through an
273
Fig. 1. Infectious clones of PVX.GFP and PVX.TGBp3GFP show green uorescent foci. (A) Schematics of the viral genomes with GFP included. PVX.GFP has GFP expressed as a free
protein from a duplicated subgenomic RNA. PVX.TGBp3GFP has GFP fused to the 3 end of the TGBp3 coding sequence. (B, C) Infection foci containing PVX-GFP and PVX.TGBp3GFP,
respectively, were photographed at 35 dpi and were similar in dimension. Both viruses spread systemically in N. benthamiana. Scale bar is 1 mm.
274
spheres at the vertices of the connecting tubules (Ju et al., 2005, 2007;
Krishnamurthy et al., 2003; Samuels et al., 2007). Immunouorescence and immunogold labeling in this study was carried out using
infection foci that were similar in size to those presented in Fig. 1.
To clearly represent the polygonal nature of the ER network, N.
benthamiana leaves were bombarded with plasmids expressing GFPer, which has GFP fused to ER targeting and retention signals (Haseloff
and Amos, 1995). Fig. 2A shows GFP-er uorescence highlights
interconnecting tubules that form a meshwork lattice around the cell.
Furthermore, leaf epidermal cells bombarded with plasmids expressing TGBp3GFP (Haseloff and Amos, 1995; Krishnamurthy et al.,
2003) show a similar network, although the tubules appear shorter in
comparison to GFP-er expressing cells, and there are cisternae at the
intersection of connecting tubules (Fig. 2B). Intriguingly, the pattern
of spherical bodies seen in PVX.TGBp3GFP infected cells is not
exactly reproduced in cells expressing TGBp3GFP alone (compare
Figs. 2B and C), suggesting that there may be other PVX factors
Fig. 2. Localization of PVX replicase and TGBp3 proteins through uorescence imaging of N. benthamiana leaf epidermal cells after infection. (A and B) Cells were bombarded with
pRTL2-GFPer or -TGBp3GFP, respectively. Arrows in panel B point to examples of ER cisternae. (C) PVX.TGBp3GFP infected cell shows similar polygonal tubular network as seen in
panels A and B. Arrowheads point to vesicles. Bars represent 10 m. (DI) Immunolabeling of PVX.TGBp3GFP infected leaf epidermal cell (DF) and mesophyll cells (GI) shows
green uorescence due to TGB3GFP fusion and red uorescence indicating PVX replicase. (F, I) represent overlaid images show yellow highlighting where red and green
uorescence overlap. (DF) Bars represent 10 m. (GI) Bars represent 200 m. (JL) Mock inoculated leaf epidermal cell treated with replicase antisera. (J, K) Green and red
uorescence images, respectively show no indication of signal in mesophyll cell. Outline of mesophyll cell is shown in white. Bars represent 10 m.
275
276
277
Fig. 4. Electron micrographs of thin sections through PVX.TGBp3GFP infected leaf segments. (AC) Low magnication images showing a region of neighboring cells, and major
structures: Chloroplasts (Chl), mitochondria (M), cell wall (CW) and vacuole (Vac). (B and C) Samples were treated with PVX replicase and BiP antisera showing few or no gold
particles in these organelles. (D) PVX.TGBp3GFP inoculated sample treated with PVX replicase and BiP antisera. Lines point to 10 nm gold particles which correspond to BiP antisera
and arrows point to 20 nm gold particles corresponding to PVX replicase. (F) Mock-inoculated samples show rough ER treated with PVX replicase antisera. No gold particles were
detected in mock samples. (G) Higher magnication of area represented by box in panel D shows 10 and 20 nm gold particles. Scale bars represent 500 nm.
Fig. 3. Localization of PVX replicase and TGBp3 proteins through uorescence imaging of BY-2 protoplasts. (A, D) PVXGFP infected protoplast shows green cytosolic and nuclear
uorescence at 24 h post inoculation. The vacuoles (v) and nucleus (n) are indicated. (B) Red uorescence shows replicase in punctae throughout the protoplast. (C and E) Overlay of
red and green uorescence shows red spherical bodies occur in sites that are devoid of green uorescence. (F, G) GFP-er expressing protoplast show tubular network extending from
perinuclear region to the plasma membrane. (HJ) PVX.TGBp3GFP infected protoplast shows the tubular ER network at 12 h post inoculation. The tubular strands are shorter than in
control samples although the polygonal pattern is maintained. Red uorescent spherical bodies overlay the network. The yellow orange uorescence highlights regions containing
both TGBp3GFP and replicase. (KM) PVX.TGBp3GFP infected protoplast shows the tubular ER network at 44 h post inoculation (green tubular haze). Spherical bodies contain
TGBp3GFP and replicase. (NP) Mock inoculated protoplast treated with replicase antisera. (N) Transmitted light image of protoplast following immunolabeling procedure. (O and P)
Green and red uorescence images, respectively show no indication of signal. Bars in all panels represent 10 m.
278
Table 1
Distribution of immunogold labeling with GFP, RdRp, and BiP antisera in N. benthamiana cells.
Sample
Treatment
# Fields
Cell wall
Cytoplasm
PVX.TGBp3GFP
RdRp + BiP
RdRp + BiP
buffer
GFP
35
35
35
35
0.57 0.10 cd
0.86 + 0.14 c
0.10 0.02 a
0.43 0.08 c
1.00 0.17
0.31 0.05
0.54 0.09
0.97 0.16
Mock
RdRp + BiP
RdRp + BiP
buffer
GFP
35
35
35
35
0.00 + 0.00 a
0.03 + 0.01 b
0.09 + 0.01 a
0.36 + 0.1 a
0.00 + 0.00 a
0.00 + 0.00 b
0.00 + 0.00 a
0.10 0.02 a
cd
c
a
bc
ER
Chloro
2.40 0.41 ab
4.43 0.75 b
0.06 0.01 a
2.80 0.47 a
0.00 0.00 d
0.29 0.05 c
NO
NO
0.14 + 0.02 a
2.57 + 0.43 a
0.06 + 0.01 a
0.00 0.00 a
0.00 + 0.00 a
0.32 + 0.06 b
0.00 + 0.00 a
NO
PVX.TGBp3GFP infected N. benthamiana leaf segments were embedded, sectioned, and analyzed by immunogold labeling and electron microscopy to assess the subcellular
accumulation of TGBp3GFP and replicase. Control samples included mock inoculated healthy leaves. Immunogold labeling was conducted using commercially available full-length
mouse monoclonal AV antiserum to detect GFP, BiP antiserum, PVX RdRp antisera, or no primary antiserum (buffer). Samples were treated with 10-nm gold-conjugated anti-mouse
to detect GFP. Samples were dual-labeled using antiserum to detect RdRp and BiP and were treated with 10 and 20 nm of gold-conjugated anti-goat and anti-rabbit sera. Fields are
dened as areas of 1 m2 (using an ultrastructure size calculator) that contain gold particles. Gold particles in each eld were counted manually. The total numbers of elds analyzed
for each plant sample are indicated. The numbers of gold particles detected in the cell wall (CW), cytoplasm, ER, vacuole, and chloroplast (Chloro) were determined for all elds
treated with each antiserum. Since certain samples were treated with two antisera, we highlighted the antisera treatment that was scored in the row in bold italics to under the
treatment heading. Zeros indicate subcellular domains with no label. NO indicates that the structure was not observed. Statistical analysis was conducted as detailed in Materials and
methods. Bolded values represent means and standard errors within the same row that show the greatest amount of immunogold label and were signicantly above zero. Statistical
signicant differences are represented by different letters next to each standard error.
MBBs. Infected (10 day post inoculation) and uninfected leaf extracts
were subjected to homogenization and centrifugation to achieve
separation of organelles and nuclei from other cellular components.
Post nuclear extracts from PVX.TGBp3GFP infected and mockinoculated N. benthamiana leaves were separated by ultracentrifugation of continuous sucrose gradients. Sixteen 0.5 ml-fractions were
collected from the top of the gradient and then examined for the
presence of relevant marker proteins by immunoblot analysis.
Membranes associated with PVX replicase and TGBp3GFP proteins,
respectively, were identied using replicase and GFP antisera and
other membranes were examined using antiserum to previously
established marker proteins. For most marker proteins, fractions were
examined by SDS-PAGE, though the replicase fractions were examined by slot blots and then treated with PVX replicase antisera.
Because of the low levels of replicase in these fractions, we used slot
blots which allowed us to load greater quantities of each fraction into
each lane prior to immunodetection. There was no non-specic
labeling observed from healthy tissue extracts run through sucrose
gradients and probed with PVX replicase and GFP antisera (data not
shown).
Immunoblot analysis of PVX.TGBp3GFP infected leaf extracts
determined that PVX replicase, TGBp3GFP, and BiP (ER lumenal
binding protein) co-fractionated near the top of the sucrose gradient
(fractions 16; Fig. 6A), although replicase and TGBp3 do not
completely coincide in their peak fractions. These results reect a
broader association of TGBp3 with the ER while the PVX replicase
resides in a subcompartment of ER membranes. Slot blot analysis
Table 2
Association of immunogold labeling vesicles and organelles in N. benthamiana leaves.
Sample
Treatment
Fields
Coated vesicles
MBBs
Mitochondria
Peroxisomes
Golgi
Plasmodesmata
PVX-TGBp3 infected
RdRp + BiP
RdRp + BiP
buffer
GFP
6 to 35
6 to 35
NO
2029
0.70 0.12bc
0.80 0.14bc
NO
0.08 0.02b
3.10 0.58a
6.17 1.15a
NO
1.90 + 0.42a
0.00 0.00c
0.09 0.02c
NO
NO
0.00 0.00c
0.20 0.03c
NO
0.07 0.01b
0.00 0.00c
0.00 0.00c
NO
NO
0.00 0.00c
0.00 0.00c
0.00 0.00
NO
Healthy
RdRp + BiP
RdRp + BiP
buffer
GFP
27 to 35
27 to 35
NO
17
NO
NO
NO
NO
NO
NO
NO
NO
0.09 0.01a
0.09 0.01a
NO
NO
0.00 0.00a
0.17 + 0.00a
NO
0.00 + 0.00
0.00 0.00a
0.00 0.00a
NO
NO
NO
NO
NO
NO
PVX.TGBp3GFP infected and non-inoculated healthy N. benthamiana leaf segments were embedded in LR White, sectioned, and analyzed by immunogold labeling and electron
microscopy as in Table 1. Fields are dened as numbers of vesicles or organelles identied in a broad range of samples and blocks. Gold particles in each eld were counted manually.
The total numbers of elds analyzed for each plant sample are indicated. Zeros indicate subcellular domains with no label. NO, none observed. Statistical analysis was conducted as
detailed in Materials and methods. Bolded values represent means and standard errors, within the same row that show the greatest amount of immunogold label and were
profoundly above zero. Statistical relatedness is represented by letters next to each standard error.
279
Fig. 5. Electron micrographs of PVX.TGBp3GFP and mock inoculated leaf segments. (AC) MBBs found in PVX.TGBp3GFP infected leaf tissues but are absent from mock-inoculated
samples. These are PVX-induced structures which contain virions, ER membranes, and granules which resemble ribosomes. Bodies label with PVX replicase, BiP and GFP antisera.
Images presented here show lines pointing to 10 nm gold particles which correspond to BiP antisera and arrows pointing to 20 nm gold particles corresponding to PVX replicase.
Arrowheads point to virion particles. Boxed areas are shown as insets at higher magnication to show virions and gold particles (D) Example of peroxisomes. These failed to label
with antisera. (E, F) Examples of coated vesicles which were more abundant in PVX.TGBp3GFP infected samples but were rarely seen in mock inoculated samples. Statistically, the
amount of replicase and GFP labeling these vesicles was not signicant (Table 2). These resemble TGBp2 containing structures reported in Ju et al., (2005). (G) Example of Golgi
apparatus. Scale bars represent 500 nm.
280
Fig. 6. (A) Presence of viral proteins in sucrose gradient fractions of membranous rich
extracts from PVX.TGBp3GFP infected plants. Fractions were analyzed by slot or
Western blot using a variety of antisera. The identity of each antiserum is on the top left
of each panel. Fractions 116 are identied next to each lane. Fraction 1 is from the top
of the gradient and fraction 16 is near the bottom. The bar on top of each Western blot
indicates fractions 16 which contain PVX replicase, TGBp3GFP and BiP. The
remaining fractions contain post-ER membranes. (B) Box plots representing qRT-PCR
analysis of host transcript levels in healthy and virus infected leaves. PVX-3D and PVX9D indicate PVX.TGBp3GFP infected leaves which were harvested at 3 and 9 day post
inoculation. Box plots represent the range of values obtained for 20 samples at each
time point and represent the variability of gene expression. The boundaries of each box
represent the 25th and 75th percentiles, and the horizontal line within the box
represents the median values (i.e. 50 percentile). The spacing of components within the
box indicates the degree of dispersal or skewness in the data. The lines at the top and
bottom of the box (whiskers) represent the sample minimum and maximum. Longer
lines at the top indicate a positive skewness. Outliers are indicated by x.
281
et al., 2007; Suhy et al., 2000; Villanueva et al., 2005). Recent cell
biological investigations with RCNMV and GFLV revealed extensive
redistribution of the ER as a result of virus infection (Ritzenthaler et
al., 2002; Turner et al., 2004). Both viruses cause accumulation of ER
aggregates or vesicles in the perinuclear region. In this study,
immunolabeling of leaf cells and BY-2 protoplasts showed PVX
replicase and TGBp3GFP both localized to spherical bodies occurring
along the ER network. In protoplasts, MBBs containing PVX replicase
and TGBp3GFP were observed between 12 and 48 h indicating that
formation of these structures is an early event. The MBBs observed in
leaf cells at 7 dpi appeared larger than in protoplasts, which could
result from increasing modications and distortion of the ER over
time, due to the presence and accumulation of viral products.
Fractionation experiments provide further indication that both the
PVX replicase and TGBp3GFP proteins coincide with the ER marker
BiP, and are excluded from Golgi and endosomal vesicles, pointing to
the ER as the primary site for PVX replication.
Localization of a viral MP and replicase in the same membrane
bound complexes was rst reported for TMV (Heinlein et al., 1998;
Kahn et al., 1998). The TMV MP is an integral membrane protein that
causes distortions in the ER network (Reichel and Beachy, 1998;
Reichel et al., 1999). Thus, the data reported in this study provide an
example of another virus whose replicase and MP appear in the same
membrane bound complexes. These similarities between TMV and
PVX suggest that certain interactions between plant viral replicases,
or replication-associated proteins, and MPs could be conserved.
Although it is logical that this localization represents a region where
both proteins are synthesized and/or function, evidence presented
here showing the proximity of the PVX replicase and the TGBp3
movement protein in particular subcellular sites provides the basis for
future work to examine whether the PVX replicase and TGBp3 act in
concert to promote virus infection and movement.
In leaf cells and protoplasts, the primary site for TGBp3GFP
accumulation is the cortical ER (Ju et al., 2005; Krishnamurthy et al.,
2003) and the same spherical bodies containing PVX replicase. Thus,
TGBp3GFP associates broadly with the ER while the PVX replicase is
restricted to a subcompartment containing ER membranes. Given that
early studies revealed that deleting TGBp3 from the viral genome does
not hamper virus replication, TGBp3 is not likely to function as the
membrane anchor for the PVX replicase (Beck et al., 1991; Verchot et
al., 1998). Typical membrane-anchoring proteins are essential and
knockout mutations would eliminate virus replication. Mutational
analysis has shown that TGBp3 binding to the ER is essential for virus
intercellular transport although there has been little progress toward
clarifying the role for the ER in modulating virus cell-to-cell
movement. Thus, the exact role of TGBp3 in the ER is unknown,
although we have recently proposed that TGBp3 could play a role in
modulating viral protein turnover (Ju et al., 2008). Moreover, we
chose to examine the amount of TGBp3GFP in the cytoplasm in this
study based on our recently reported results (Ju et al., 2008) which
suggested that TGBp3GFP is turned over by ER-associated degradation pathways (ERAD). TGBp3GFP is dislodged from the ER for
cytosolic degradation by the 26S proteasome (Brandizzi et al., 2003; Ju
et al., 2008; Meusser et al., 2005). Given the role of TGBp3 in viral
protein turnover and virus movement, it is possible that TGBp3 may
function to target the PVX replicase for degradation and acquiring
viral RNAs for transfer to plasmodesmata. Further research is needed
to determine if the stability of the PVX replicase is altered by the
presence of TGBp3.
In this study, electron microscopy of virus-infected tissues
identied MBBs, which were lled with ER strands and virions.
Immunolabeling detected PVX replicase, TGBp3 and the ER marker
protein, BiP, in these bodies. Since no other ER associated bodies were
detected in virus infected cells, it is reasonable to consider these MBBs
to be the spherical bodies seen using confocal microscopy. Virus
infected cells also showed an abundance of coated vesicles that were
282
not detected in healthy tissues. The identity of these vesicles was not
further examined in this study because they failed to show
immunoreactivity with GFP or replicase antisera. However, they
have some resemblance to TGBp2 induced vesicles reported in a
related study (Ju et al., 2005). The TGBp2-related vesicles are similar
in size and show the same granular appearance along the outer
membrane. GFLV polyprotein precursors were reported to associate
with membranous vesicles that resembled the COP-coated vesicles
which trafc from the ER to the Golgi apparatus. Researchers
proposed that GFLV might employ the COPII budding machinery to
recruit membrane scaffolds for the viral replicase (Ritzenthaler et al.,
2002). The COP vesiculation machinery has been shown to be
essential for poliovirus replication (Belov et al., 2005; Cuconati et
al., 1998; Fogg et al., 2003). While we found no evidence that coated
vesicles (Fig. 5E and F) are COP-related structures their abundance in
PVX.TGBp3GFP infected cells warrants further investigations.
Through treatment with the lipid biosynthesis antagonist, cerulenin, we determined that membrane synthesis was necessary for PVX.
TGBp3GFP replication (Fig. 7). Cerulenin was also shown to inhibit
replication of GFLV, CPMV, and poliovirus (Carette et al., 2000; Fogg et
al., 2003; Ritzenthaler et al., 2002). Thus, PVX belongs to a class of
viruses that require de novo membrane synthesis to support virus
replication. The moderate increase in BiP expression seen in PVX
infected cells may correlate with expansion of the ER or may reect a
moderate level of stress exerted upon the ER as the result of virus
infection. Viral pathogenesis can sometimes trigger the unfolded
protein response (UPR), leading to increased cellular synthesis of ER
resident chaperones necessary to restore ER homeostasis. BiP is one ER
resident chaperone that is known to play a vital role in UPR (Kamauchi
et al., 2005; Urade, 2007). Similar events have been reported to occur
during mammalian virus infection. Specically, aviviral nonstructural proteins trigger UPR signaling, which, for viruses such as Hepatitis C
virus, leads to restoration of ER homeostasis necessary for a persistent
virus infection (Tardif et al., 2005). Increased expression of ER resident
chaperones ensures proper protein folding and processing in the ER, as
a result of the increased demand on the cellular machinery. In addition,
misfolded proteins can be relocated to the cytoplasm for proteasomal
degradation. The role for TGBp3 or PVX replicase in regulating
accumulation of ER resident chaperones has not yet been examined,
although we have reported TGBp3 accumulation is monitored by the
26S proteasome (Ju et al., 2008).
Taken together, the data demonstrate that the ER-derived MBBs
contain both the PVX replicase and TGBp3 proteins. PVX replicase and
TGBp3 do not exactly coincide throughout the ER or in membrane
fractions. While the top fractions 15 contain BiP, which is a marker for
ER membranes, replicase was predominantly in fraction 1 while peak
levels of TGBp3 were in fraction 5. These results are reective of
evidence that the replicase does not occur throughout the cortical ER but
are restricted to a subcompartment of the ER, such as MBBs. Particles
resembling PVX virions were also seen inside MBBs, although further
studies are needed to learn how many PVX-encoded proteins associate
with these structures. Importantly, this is the rst report to demonstrate
a spatial relationship between the PVX TGBp3 and replicase. Given the
close relationship of the TMV replicase and movement protein, it is
worth considering that interactions between the PVX replicase and
TGBp3 may serve analogous functions. However, further dynamic
investigations are necessary to reveal whether TGBp3 is similar to the
TMV movement protein and drives trafcking of membrane bound viral
replication complexes toward the plasmodesmata.
Materials and methods
Infectious clones, in vitro transcription, and plant inoculations
PVXGFP and PVX.TGBp3GFP are infectious clones of PVX
described in prior studies (Fig. 1; Krishnamurthy et al., 2003; Mitra
four times (5 min per wash) in 25 mM PIPES buffer (pH 7.0) and then
transferred into secondary xatives [2% (w/v) OsO4 and 0.5% (w/v)
potassium ferrocyanide in 25 mM PIPES buffer (pH 7.0)] for 2 h at
room temperature. Samples were washed twice with 25 mM PIPES
buffer (pH 7.0) for 15 min and then twice with ddH2O for another
15 min. Samples were transferred to 2% (w/v) aqueous uranyl acetate
for 16 h at 4 C and washed twice with ddH2O for 15 min. Samples
were dehydrated in an acetone series (10%, 20%, 40%, 60%, 80%, and
100%) and inltrated with acetone/Spurr's (1:1) or LR-White resin
overnight and then embedded in Spurr's or LR-White resin.
Ultra thin sections (700 nm) were cut using a diamond knife on a
Sorvall MT 6000 ultra microtome. Sections were mounted on formvarcoated nickel grids (Electron Microscopy Science, Hateld, PA) and
used for immunogold labeling.
283
Cerulenin treatment
Sucrose gradient fraction of plant extracts containing PVX replicase
Following electroporation, protoplast viability was measured
using 0.1% uorescein diacetate (FDA) in 1 mL of 0.05 M phosphate
buffer. Cerulenin (Sigma-Aldrich Co., St. Louis, MO) was dissolved in
dimethyl sulfoxide and added to the protoplast culture medium to a
nal concentration of 0, 15, 30, and 45 M (Carette et al., 2000;
Ritzenthaler et al., 2002). Protoplasts were incubated for 24 h and
harvested. A haemocytometer was employed to calculate the
proportion of GFP expressing protoplasts. The proportions of viable
protoplasts that were virus infected were recorded.
Immunogold labeling of LR-White or Spurr's resin-embedded
plant material
Immunogold labeling of tissues were conducted using monoclonal
GFP (BD Living Colors; Clontech Laboratories, Mountain View, CA)
and PVX replicase polyclonal goat antisera as previously described
(Mitra et al., 2003). Grids were incubated in blocking solution
consisting of PBS, pH 7.5 (130 mM NaCl, 7.0 mM Na2HPO4, 3.0 mM
NaH2PO4) plus 2% BSA (w/v) for 15 min, and then was incubated with
5% normal sheep sera (Sigma-Aldrich Co.) in PBS plus 2% BSA for
15 min. Then samples were incubated with GFP monoclonal antisera
diluted 1:500 in PBS plus 2% BSA (w/v), PVX replicase goat antisera
diluted 1:500 in PBS plus 0.1% Tween (v/v), or buffer containing no
primary antisera for 2 h. Grids were then washed ve times for 5 min
with PBS and then with PBS plus 2% sh gelatin (v/v) for 15 min. Grids
were then incubated for 1 h with either 10 or 20 nm gold-conjugated
goat antisera (EY Labs, San Mateo, CA) diluted 1:10 in PBS plus 2% sh
gelatin.
Some grids were dual labeled with antiserum to detect replicase
and GFP and a longer procedure was followed (Mitra et al., 2003).
Grids were rst incubated with replicase goat antisera diluted 1:500
in PBS plus 0.1% Tween (v/v) for 2 h and then with the 20 nm gold
conjugated rabbit anti-goat serum diluted 1:10 in PBS plus 2% sh
gelatin for 2 h as described above. Samples were washed twice for
10 min with PBS and then incubated for 30 min with GFP monoclonal
antisera diluted 1:500 in PBS. Grids were washed ve times for 5 min
with PBS, then for 15 min with PBS with 2% sh gelatin, and were
incubated 30 min with 10 nm gold-conjugated goat anti-mouse
serum (EY Labs) diluted 1:10 in PBS plus 2% sh gelatin. Grids were
washed three times for 5 min with ddH2O, and stained with a solution
of 2.5% uranyl acetate and 70% methanol (v/v) for 30 min, and then
with a solution of 2% Reynold's lead citrate pH 12.0 (in ddH2O) for
20 min. Samples were washed with mildly warm ddH2O three times
for 5 min and then dried. Resin embedded sections were consecutively labeled with PVX replicase goat primary, anti-goat secondary
and GRP78 (BiP) rabbit primary and anti-rabbit secondary antisera
(ABR-Afnity Bioreagents Inc., Rockford, IL) using established protocols (Ju et al., 2005). Grids were stained with uranyl acetate and
Reynolds lead citrate.
Table 3
Primers for qRT-PCR.
Genes
Accession
Primers
Primer sequences
18S rRNA
AJ236016
BiP
FJ463755
SYP21
L41651
SYP41
NM_001036873
SYP61
NM_001036025
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
5-ATGGCCGTTCTTAGTTGGTGGAGC-3
5-AGTTAGCAGGCTGAGGTCTCGAAC-3
5-AGCTTTGAGCAGTCAACACCAAGT-3
5-AAAACGTGCCCGAGTAAGTGGTTC-3
5- CCGATTTACGCGACAAGTTGCACA-3
5- ACTCGATGATCTGTTTCGCTGGCT-3
5- TTCAGTGAACTGCAGACGACCACT-3
5- TAGAACGACCCGGCATGAATCCAA-3
5- ACTTGTGGAAGCATTGAGTGGCAG-3
5- TTCACATTCCGCACCTGTGTCCTA-3
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