Download Localization of the P1 protein of potato Y potyvirus in association

Journal
of General Virology (1998), 79, 2319–2323. Printed in Great Britain
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SHORT COMMUNICATION
Localization of the P1 protein of potato Y potyvirus in
association with cytoplasmic inclusion bodies and in the
cytoplasm of infected cells
Jelena Arbatova,1 Kirsi Lehto,2 Eija Pehu1 and Tuula Pehu1
1
2
Department of Plant Production, PO Box 27 (Viikki), FIN-00014 University of Helsinki, Finland
Department of Biology, Laboratory of Plant Physiology and Molecular Biology, FIN-20014 University of Turku, Finland
The N-terminal P1 proteinase of potato virus Y
(ordinary strain group isolate PVY-O) was expressed in E. coli. Antiserum was raised against the
expressed protein and used to detect the viral
proteins in infected tobacco leaf tissue by Western
blotting and by electron microscopy with immunogold labelling. In the immunogold localization
studies P1 protein was detected in association with
the cytoplasmic inclusion bodies characteristic of
PVY infections and in the cytoplasm of the infected
plant cells. No significant P1 antibody binding with
other plant cell organelles, or with the cell wall
and plasmodesmata, was detected by immunogold
labelling.
Potato virus Y (PVY) is the type member of the family
Potyviridae of positive-sense, single-stranded RNA plant
viruses, the largest of the plant virus groups currently known.
The potyvirus genome encodes a single large polyprotein that
undergoes proteolytic processing, catalysed by virus-encoded
proteinases (Dougherty & Selmer, 1993). Functions or putative
roles have been assigned to most of the mature viral proteins
although additional activities may be associated with polyprotein intermediates (reviewed by Dougherty & Carrington,
1988 ; Riechmann et al., 1992). However, the role of the Nterminal protein P1 in the virus infection cycle has remained
unclear.
The P1 protein is a serine-type proteinase which catalyses
autoproteolytic cleavage at a Tyr-Ser dipeptide between itself
and the helper component proteinase, HC-Pro (Verchot et al.,
1991, 1992). The C-terminal 147 amino acid residues of the P1
protein constitute the complete functional proteinase ; the Nterminal 157 amino acid residues are dispensable for proteinase
activity (Verchot et al., 1992). In addition to its proteolytic
Author for correspondence : Jelena Arbatova.
Fax ­358 9 7085582. e-mail Jelena.Arbatova!Helsinki.Fi
0001-5651 # 1998 SGM
activity, the P1 protein has been shown to exhibit nonspecific
RNA-binding activity (Brantley & Hunt, 1993 ; Soumounou &
Laliberte, 1994). The RNA-binding properties of P1 are similar
to those described for known movement proteins of plant
viruses (Citovsky et al., 1991, 1992 ; Osman et al., 1992, 1993 ;
Schoumacher et al., 1992) and it has been suggested that P1
could also be involved in cell-to-cell transport of virus in plants
(Atabekov & Taliansky, 1990 ; Brantley & Hunt, 1993 ;
Dougherty & Semler, 1993 ; Riechmann et al., 1992). However,
it has been demonstrated by mutational and complementation
analysis for another potyvirus, tobacco etch virus, that the P1
protein plays little, if any, role in virus movement (Verchot
& Carrington, 1995 a, b). Deletion of the entire P1 coding
sequence had only minor effects on cell-to-cell and longdistance transport but considerably reduced genome amplification of TEV mutants, suggesting that the function of P1 is
related to virus replication. To further analyse the role of P1 in
the life-cycle of potyviruses we investigated the subcellular
localization of the PVY P1 protein in infected plant cells.
For antiserum production, the P1 gene from PVY-O was
expressed in E. coli. Cloning of the P1 gene has been described
by Pehu et al. (1995). The cloned P1 sequence was excised by
NdeI and BamHI and ligated into the pET3a expression vector.
The construct was transformed into E. coli BL21(DE3)pLysE
cells (Novagen). Cell cultures in the exponential growth phase
were induced by adding IPTG to a final concentration of
1 mM, and grown for an additional 2±5 h. The cells were
pelleted and lysed by the boiling method (Sambrook et al.,
1989) for analysis by SDS–PAGE. After staining with
Coomassie blue, a band of the expected size for the P1 protein
(31 kDa) was clearly visible in the gel. This band was not
observed in samples from the non-induced controls (data not
shown).
For large-scale production, protein expressed in E. coli was
purified as inclusion bodies. The collected cells were lysed with
lysozyme (100 µg}ml in 50 mM Tris, pH 8±0, 2 mM EDTA) at
30 °C for 15 min. After lysing, the suspension was sonicated to
shear the DNA. Inclusion bodies were collected by centrifugation at 10 000 g for 10 min at 4 °C. The pelleted inclusion
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J. Arbatova and others
A
B
C
Fig. 1. Western blot analysis of antiserum raised against PVY-O P1 protein.
Lanes : A, pET3a transformed BL21(DE3)pLysE cells ; B, pET3a/P1
transformed, induced BL21(DE3)pLysE cells ; C, P1 protein purified as
inclusion bodies. Ten µl from each sample (an aliquot equivalent to 100 µl
of bacterial culture) was loaded on the gel. The position of P1 protein is
indicated to the right and the positions of marker proteins (kDa) to the
left.
bodies were washed several times in 50 mM Tris, pH 8±0,
2 mM EDTA. After washing and solubilization of inclusion
bodies, the inclusion body protein was purified by preparative
electrophoresis on a 10 % SDS–polyacrylamide gel (Laemmli,
1970). The bands were visualized by soaking the gel in ice-cold
1 M KCl for 1 min.
A gel slice containing approximately 500 µg of the
expressed protein was homogenized in Freund’s incomplete
adjuvant (Difco) and injected subcutaneously into a rabbit.
Injections were repeated three times at 14 day intervals ; 10 ml
of blood was collected 14 days after the last injection, and the
bleeding was repeated several times at 10 day intervals. Four
rabbits were immunized.
The antiserum against P1 protein was tested by Western
blotting. Aliquots from non-induced and induced E. coli cultures
and the purified P1 protein in 2¬ sample buffer (100 mM
Tris–HCl, pH 6±8 ; 4 % SDS ; 20 % glycerol ; 0±2 % bromphenol
blue ; 200 mM dithiothreitol) were boiled for 5 min and
analysed by SDS–PAGE on a 10 % gel. Electrophoresis was
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A
B
Fig. 2. Detection by Western immunoblotting of the P1 protein in the
extracts from PVY-O infected N. tabacum leaf tissue, 7 days postinoculation. Lanes : A, 10 µl sample of extract from healthy tobacco plant ;
B, 10 µl sample of extract from PVY-O-inoculated tobacco plants. The
position of the P1 protein is indicated to the right and the positions of
marker proteins (kDa) to the left.
done as described by Laemmli (1970). After electrophoresis,
proteins were transferred to PVDF membrane (Immobilon-P
from Millipore) at 30 V overnight using a Mini Trans-Blot
Electrophoretic Transfer Cell (Bio-Rad). The membranes were
incubated with the P1 antiserum diluted 1 : 1000 in TBST
(20 mM Tris–HCl, pH 7±5 ; 150 mM NaCl, 0±05 % Tween 20).
Goat anti-rabbit IgG–alkaline phosphatase conjugate diluted
1 : 3000 in TBST was used as the secondary antibody.
Detection of immunocomplexes was carried out using the
ProtoBlot Western Blot AP System (Promega) according to the
manufacturer’s instructions. In the induced E. coli, and in the
purified P1 protein samples, P1 antiserum reacted with a
protein of the expected size, 31 kDa (Fig. 1).
The antiserum produced against P1 protein was used for
detection of the P1 protein in infected leaf material. Total
protein samples were prepared from 500 mg of leaf tissue from
PVY-O-infected (7 days post-inoculation) or healthy tobacco
(Nicotiana tabacum). The samples were ground in liquid
nitrogen and homogenized in 1 ml of ES buffer (75 mM
Tris–HCl, pH 6±1, 4±5 % SDS, 9 M urea, 7±5 % β-mercaptoethanol, 5 mM PMSF). An equal amount of 2¬ sample
Localization of the PVY P1 protein
buffer was added to the plant samples, which were then
boiled for 5 min ; 10 µl from each protein sample was analysed
by SDS–PAGE on a 10 % gel. After electrophoresis, proteins
were transferred to Immobilon-P membrane, and the Western
blot detection of the P1 protein was done as described above.
In the Western blots, a protein with a relative molecular mass
of 31 kDa was detected in the samples prepared from infected
plants, but not in the samples prepared from healthy plants
(Fig. 2), suggesting that this virus-specific protein was P1. The
P1 antiserum also cross-reacted with plant proteins on Western
blotting membranes (Fig. 2), and in an attempt to reduce this
background we preadsorbed P1 antiserum with acetone
powder of healthy tobacco tissue. However, preadsorption
reduced not only the background but also lowered the intensity
of the specific signal with P1 protein, suggesting that the P1
antibodies recognize epitopes shared by the PVY P1 protein
and some tobacco plant proteins. Western blotting showed
that in the sample from infected tobacco leaves two very weak
protein bands of lower molecular mass are present ; these could
be products of proteolysis of the P1 protein (Fig. 2, lane B).
Next, we used the antiserum raised against the P1 protein
in conjunction with the immunogold labelling technique. For
immunogold labelling, PVY-O-infected (7 days post-inoculation) and comparable healthy N. tabacum plants were placed
in the dark overnight prior to tissue processing, to reduce the
number of starch grains found in chloroplasts. Leaf strips of
about 0±5¬0±5 mm from systemically infected or healthy
leaves were fixed overnight in 2 % (w}v) paraformaldehyde
and 3 % glutaraldehyde in phosphate–citrate buffer, pH 7±2, at
4 °C. Dehydration and embedding of plant tissue in LR Gold
resin at low temperature were performed according to van
Lent et al. (1990). Thin sections of the plant tissue were
harvested on Formvar-coated Ni-grids and treated for 30 min
on drops of 1 % BSA in TBS (Tris 20 mM, NaCl 500 mM,
pH 7±5) to block nonspecific binding of antibodies. The primary
P1 antibodies were concentrated by precipitation with ammonium sulfate according to a published protocol (Cooper &
Paterson, 1993), and diluted with TBS to a concentration of
50 µg}ml. Sections were incubated with the primary antibodies
for 2 h at room temperature or overnight at 4 °C in a humid
chamber. After incubation with primary antibodies, sections
were washed 3¬10 min with TBS, incubated for 1±5 h at room
temperature with 10 nm protein A–gold (pAg) (Zymed)
diluted 1 : 30 with 50 mM Tris, pH 7±4, 150 mM NaCl
containing 1 % BSA. Then sections were washed 6¬5 min
with TBS and 2¬5 min with distilled water before air drying.
After immunogold labelling, thin sections were stained with
uranyl acetate and alkaline lead citrate and examined with a
JEOL JEM-1200EX transmission electron microscope.
Although the P1 antiserum gave a cross-reaction with
some plant proteins on Western blotting membranes, no gold
label was found in thin sections from non-infected plants
incubated with P1 antibodies (Fig. 3 a). Most probably, the
plant protein epitopes which cross-reacted with the P1
antiserum on Western blotting membranes were masked or
modified during fixing and embedding in LR Gold resin.
Likewise, no gold label was found on thin sections from
infected plants incubated with preimmune serum in spite of the
presence of cytoplasmic inclusions within the cells (Fig. 3 b).
The occurrence of inclusions in potyvirus-infected cells
makes it easy to determine with the electron microscope which
cells are infected. PVY encodes a cylindrical inclusion protein
that aggregates in the cytoplasm of infected cells to form
inclusions resembling pinwheels (Fig. 3 b–d) or bundles when
seen in longitudinal section (Fig. 3 e). Immunogold labelling
with P1 polyclonal antibodies localized the P1 protein both in
association with cytoplasmic inclusion bodies and in the
cytoplasm of infected plant cells of tobacco leaves (Fig. 3 c–e).
Immunolabelling of sections of PVY-O-infected leaves showed
essentially no antibody binding to the plant cell organelles,
including the nucleus, mitochondria, chloroplasts, microbodies
or cell wall in the cells with clear labelling in the cytoplasm (Fig.
3 c–f). Plasmodesmata were observed in some sections, but no
gold label was associated with them (data not shown).
There are several possible explanations why P1 protein is
colocalized with cytoplasmic inclusion bodies. A first possibility is that P1 protein localized in association with inclusion
bodies because it was synthesized there. It has been shown
previously that the coat protein and P3 protein of tobacco vein
mottling potyvirus (TVMV) are associated with cytoplasmic
inclusions in infected tobacco leaves and protoplasts (Ammar
et al., 1993, 1994 ; Rodriguez-Cerezo et al., 1993). Ammar et al.
(1994) suggested that synthesis of all the potyviral proteins
might occur in, on or near these inclusions, which are usually
associated with the rough endoplasmic reticulum.
However, there are other possibilities. With the demonstration of the helicase activity of the cylindrical inclusion
protein of a potyvirus (Lain et al., 1990), the replication of
potyviral RNA is also likely to be connected with cytoplasmic
inclusions. The role of P1 protein in virus infectivity beyond its
autoproteolytic activity is still not determined completely.
Using tobacco etch potyvirus (TEV) mutants lacking the entire
P1 coding region Verchot & Carrington (1995 a, b)
demonstrated that P1 protein activity is not essential for virus
infectivity and that the P1 protein may contribute to but is not
strictly required for the TEV RNA amplification. Their data
suggest that the P1 protein stimulates viral genome amplification and does not effect cell-to-cell movement. If cytoplasmic inclusions are indeed sites of potyviral RNA replication, the present results on the localization of the P1 protein
on the cytoplasmic inclusion bodies are consistent with the
hypothesis of Verchot & Carrington that P1 protein could
participate in virus replication. However, further experiments
will be necessary to determine the role of P1 protein in the
virus infection cycle more precisely. Finally, the absence of the
PVY P1 protein from the cell wall and plasmodesmata is also
consistent with the results of Verchot & Carrington on the P1
protein of TEV, suggesting that P1 is not involved in virus or
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J. Arbatova and others
Fig. 3. Immunolocalization of the P1 protein in thin sections of cells from systemically infected N. tabacum leaf tissue, 7 days
post-inoculation with PVY-O. (a) Section from healthy tobacco leaf treated with antibodies to PVY P1 protein. (b) Section from
infected tobacco leaf incubated with preimmune rabbit serum. (c–f) Thin sections of infected cells incubated with antibodies to
PVY P1 protein. CI, cytoplasmic inclusions ; Ch, chloroplast ; CW, cell wall ; M, mitochondrion ; Mb, microbody ; N, nucleus ; Nu,
nucleolus ; S, starch grain ; V, vacuole. Bar, 200 nm.
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Localization of the PVY P1 protein
viral RNA movement through the plasmodesmata of PVYinfected cells.
We are indebted to Dr Jan van Lent (Agricultural University of
Wageningen, the Netherlands) for introducing us to the method of
immunogold labelling of antigens in thin sections for electron microscopy. We thank our colleagues from the Electron Microscopic Unit,
Institute of Biotechnology, University of Helsinki for the help with
electron microscopy.
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Received 28 April 1998 ; Accepted 3 June 1998
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