1933
J. gen. Virol. (1989), 70, 1933-1942. Printed in Great Britain
Key words: viroid/PR protein~tomato plants
'Pathogenesis-related' Pl(pl4) Protein. Vacuolar and Apoplastic
Localization in Leaf Tissue from Tomato Plants Infected with Citrus
Exocortis Viroid; in vitro Synthesis and Processing
By P. V E R A , 1 J. H E R N A N D E Z - Y A G O
1,2 AND V. C O N E J E R O
1Departamento de Biotecnologia, Universidad Politbcnica de Valencia, Camino de Vera s/n,
46021-Valencia and 2Instituto de Investigaciones Citol6gicas, Amadeo de Saboya 4,
46010-Valencia, Spain
(Accepted 20 April 1989)
SUMMARY
Tomato 'pathogenesis-related' Pl(pl4) protein was synthesized in vitro, mRNAs
were isolated from leaves showing characteristic symptoms of viroid infection,
followed by chromatography on oligo(dT)-cellulose and the poly(A)+ fraction was
translated with a rabbit reticulocyte lysate system. No significant differences were
found between the levels of [3SS]methionine incorporation directed by mRNA
preparations from healthy and viroid-infected leaves. Only mRNA from infected
leaves incorporated label into a protein that could be immunoprecipitated with rabbit
IgG specific for tomato P 1(p 14) protein. Analysis by SDS-PAGE of the immunoprecipitated protein from the in vitro translation system revealed that P 1(p 14) was translated
as a precursor protein of 2K to 3K larger than the Pl(pl4) that accumulated in vivo.
This protein was converted to the mature form when translation was carried out in the
presence of canine pancreatic microsomal membranes. By using Protein A-gold
immunocytochemistry we have detected Pl(pl4) concentrated in dense inclusion
bodies within the vacuole as well as in association with electron-dense material present
in the intracellular spaces. The presence of the additional polypeptide sequence in
the newly synthesized protein indicates that Pl(pl4) undergoes post-translational
processing. The additional sequence is probably the signal peptide that directs its
transport through the endoplasmic reticulum into the vacuolar compartment of tomato
leaf cells and/or the intercellular space.
INTRODUCTION
Infection of plants with pathogens results in the induction and accumulation of a family of
proteins named pathogenesis-related (PR) proteins. The production of these proteins may be
part of a general mechanism of response, programmed by the plant genome, which is activated
after pathogen attack or after treatment with different stress-causing agents (van Loon, 1985).
Recent studies have shown that some PR proteins are hydrolytic enzymes:/~-l,3-glucanases
(Kauffmann et al., 1987; Kombrink et al., 1988), chitinases (Kombrink et al., 1988; Legrand et
al., 1987; Nasser et al., 1988) and a protease (Vera & Conejero, 1988a, b). These enzymes are
considered to play an important role in defence reactions.
Tomato plants respond to citrus exocortis viroid (CEV) infection with the synthesis and
accumulation of a set of basic PR proteins (Granell et al., 1987; Vera et al., 1988; Vera &
Conejero, 1988a, b). One of these proteins, Pl(pl4), has also been connected with natural
senescence in healthy plants (Camacho Henriquez & S~inger, 1982). Consistent with this
observation, Pl(pl4) has recently been located in association with disorganized cytosol of
certain decaying cells and in the intercellular spaces (or apoplasts) of healthy (non-infected) l e a f
tissue. Consequently, the idea that Pl(pl4) is involved in cell degeneration has been proposed
(Vera et al., 1988).
0000-8919 © 1989 SGM
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1934
P. VERA, J. HERN~,NDEZ-YAGO AND V. CONEJERO
This paper reports the immunocytochemical localization of P1 (p14) in CEV-infected tomato
leaf tissue by using the same immunocytochemical approach described previously (Protein A gold electron microscopy) (Vera et al., 1988). We show that Pl(pl4) protein is located in the
apoplast and in the central cell vacuole. We have also determined that P 1(p14) is synthesized in
the form of a precursor protein (pre) as observed for other plant proteins which also accumulate
in vacuoles (Nelson & Ryan, 1980; Broglie et al., 1986; Cleveland et al., 1987). In addition, we
show that prePl(pl4) can be processed to its mature form when canine microsomal membranes
are present during translation.
METHODS
Plant mawriaL Tomato plants (Lycopersioon esculentum Mill, CV. Rutgers) were grown from seeds in a
greenhouse at 25 to 30 °C. Inoculation with a purified preparation of CEV was performed at an early young stage
as described previously (Granell et al., 1987).
Isolation o f P l ~ 1 4 ) and preparation of antiserum. PR protein Pl(pl4) was purified from viroid-infected tomato
plants using a procedure described elsewhere (Vera et al., 1988). An antiserum was produced in rabbits and its
specificity has already been described (Vera et al., 1988).
Electrophoresis andfluorography. Electrophoresis was performed in 14~ polyacrylamide gels containing SDS as
described (Conejero & Semancik, 1977). The gels were then impregnated with Amplify (Amersham), dried, and
then exposed to Kodak XAR5 film. [35SlMethionine was quantified by liquid scintillation.
Immunocytochemistry. Leaves showing characteristic symptoms of viroid infection were collected 20 days after
inoculation. The leaves were cut under water into 2 x 1 mm strips and fixed with 19/ooformaldehyde~-0"7~
glutaraldehyde in potassium phosphate buffer, 1 mM-CaC12 (pH 7.2) for 2 h at 4 °C as previously described (Vera et
al., 1988). Tissue blocks were dehydrated, infiltrated with Lowicryl K4M resin and polymerized under u.v. light as
described (Vera et al., 1988). Thin sections were cut and mounted on 200-mesh nickel grids covered with carbon
film and floated on 1 ~ ovalbumin in phosphate-buffered saline (PBS) pH 7-2 for 30 rain at 20°C. The sections
were transferred to Pl(pl4) antiserum diluted 100-fold in PBS-ovalbumin and processed for Protein A-gold
labelling as previously described (Vera et al., 1988). The sections were stained in the dark for 20 rain with 5%
aqueous uranyl acetate and for 45 s with alkaline lead citrate.
Controls were treated either with preimmune serum diluted 100-fold, Protein A-gold alone or with anti-P l (pl 4)
serum saturated with a large excess of purified P1 (p 14). All these controls were treated and processed as described
above and demonstrated a very low level of non-specific background labelling. The sections were examined and
micrographs were recorded in a Philips 300 electron microscope.
Determination of Pl(p14) density. Electron micrographs of sections were taken and printed at a magnification of
x 40000. P 1(p 14)-associated gold particles were counted and attributed to each cell compartment. A transparent
sheet with a grid (lcm-spaced paraUed lines) was superimposed on the prints and the number of gold particles per
unit area was determined. The proportion of gold particles in each cell compartment was used as a measure of
Pl(pl4) density.
Extraction ofpoly(A) ÷ mRNA. Total RNA was extracted from leaf tissues according to a procedure derived from
Chirgwin et al. (1979). Tomato leaves (25 g) from either healthy or CEV-infected plants were frozen in liquid
nitrogen and ground in a mortar. The powder was mixed rapidly with 100 ml of 50 mM-Tris-HC1 pH 8.0,
containing t0 mM-EDTA, 5 M-guanidinium thiocyanate, 2 ~ sodium N-lauroylsarcosine and 5 ~ (v/v) 2mercaptoethanol. After incubation at 60°C for 10 rain and centrifugation at 20000g for 30 rain, the supernatants
were adjusted to 0-1 g/ml CsCI and layered on 12 ml cushions of 5.7 M-CsCI, 50 mM-EDTA, pH 8-0, in 30 ml
polyallomer tubes. The tubes were centrifuged for 24 h at 25000 r.p.m, in an SW27 rotor at 18°C. The R N A pellets
were then dissolved in a small volume of 25 raM-sodium citrate, 7-5 M-guanidine hydrochloride, 5 mM-dithiothreitol
and RNA was precipitated by adding 0-025 volumes of 1 M-acetic acid and 2.5 volumes of ethanol. The final pellets
were dispersed in ethanol and centrifuged for 5 min at 6000 r.p.m. The firm pellets were vacuum-dried and the
RNA was dissolved in 1 ml sterile distilled water and precipitated by adding 0.1 volumes of 2 M-potassium acetate,
pH 5.0 and 2-5 volumes of ethanol, This was left overnight at - 20 °C. The yield was generally in the range of 200 to
300 ~tg total RNA per g of fresh tissue. Poly(A)÷ mRNA was purified by two cycles of selection on an oligo(dT)cellulose column (Sigma) according to the procedure of Maniatis et al. (1982).
Cell-free translation of mRNAs, processing and immunoselection of the translation products. Total poly(A) + mRNAs
were translated in a rabbit reticulocyte cell-free translation system (Amersham) using [3sS]methionine (900
Ci/mmol, 1 mCi/ml) (Amersham). Approximately 0.1 p.g poly(A) ÷ m R N A and 1.2 ~tCi [35S]methionine were
added per ~tl of lysate. After incubation for 1 h at 35°C, aliquots of the translation mixture were analysed by SDSPAGE. Cell-free translation products were processed by the addition of canine pancreatic microsomes (New
England Nuclear), at the indicated concentration, to the reaction mixture containing the reticulocyte lysate
system. Translation products corresponding to 50 ~tl of lysate were immunoselected for Pl(pl4) as follows. The
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PR protein in viroid-infected tomato plants
1935
translation products were diluted by the addition of 100 ~tlof buffer A (10 mM-Tris-HCl pH 7.3, 150 mM-NaC1,10
mM-EDTA, 1% Triton X-100). The samples were centrifuged at 100000 g for 45 min and 15 ~tl of anti-Pl(pl4)
serum was added to the supernatants. Following incubation for 12 h at 4°C, 100 ~tlof Protein A-Sepharose CL-4B
(Pharmacia) (10 mg/ml) prepared in buffer A was added and incubation was continued for 1 h at 20 °C, after which
the Protein A-Sepharose with the attached antigen-antibody complexes were sedimented by centrifugation
through a cushion of 1 M-sucroseprepared in buffer A, followed by three washes in buffer A. The pellets were
analysed by SDS-PAGE.
In vivo labellingof Pl(pl4). In vivolabelling was performed by incubation of CEV-infected tomato leaf discs (0.5
cm in diameter) with [35S]methionine (1000 Ci/mmol, 200 ~tCi/ml; in vivo labelling grade, Amersham) for 3 h.
After labelling the discs were homogenized in 84 raM-citric acid-32 mM-NaHPO4 pH 2.8, containing 15 m.M-2mercaptoethanol. The homogenates were centrifuged for 20 min at 20000g and the supernatants were adjusted to
pH 7.3 with 1 M-Tris-HC1. Radiolabelled Pl(pl4) was immunoprecipitated with anti-Pl(pl4) serum as described
above.
RESULTS
lntracellular localization of the PR protein P1(p14)
Immunocytochemical localization showed that Pl(pl4) is located in dense inclusion bodies
present in the cell vacuole (Fig. 1 a to f). Control sections treated with preimmune serum (Fig. 2e
and Fig. 3), Protein A-gold alone or anti-Pl(pl4) antiserum saturated with a large excess of
P l ( p l 4 ) protein (not shown) had no specific labelling in the vacuolar inclusion bodies. N o
significant labelling of the cytoplasm was found for P l ( p l 4 ) (see Table 1 and compare the gold
particle distribution on nuclei, mitochondria, chloroplasts, microbodies and cell walls).
The distribution of gold label within vacuolar inclusion bodies was prominent and no
significant variations could be found between cells. These inclusion bodies were found to be
present in 30 to 40% of the ceils analysed on every section of parenchyma leaf tissue from CEVinfected plants. We have not found these inclusion bodies in leaf tissue from healthy (noninfected) plants. N o traces of Pl(pl4) as a free protein in vacuoles could be found (data not
shown). In healthy plants, P l ( p l 4 ) protein has been detected in association with only a
disorganized cytosol found in a few cells of the parenchyma tissue and also in some intercellular
spaces of mature tomato leaves (Vera et al., 1988).
Intercellular localization of P l(p l 4) protein
Immunogold labelling of CEV-infected leaf sections revealed that P 1(p14) was also present in
intercellular spaces, and always in association with electron-dense material. This material has
also been found associated with Pl(pl4) protein in preparations from healthy (non-infected)
tomato leaf tissue (Vera et al., 1988). It must be emphasized that in CEV-infected preparations,
the abundance of both electron-dense material and the anti-Pl(pl4) gold label in intercellular
spaces was dramatically enhanced with respect to healthy preparations [see Table 1 and Fig. 2,
and compare with those previously published (Vera et al., 1988)]. Control sections treated with
preimmune serum (Fig. 2 e, 3 a to c and Table 1), presaturated antiserum or Protein A-gold alone
(not shown) showed no specific labelling in these compartments.
In vitro synthesis of tomato PR protein Pl(pl4)
R N A was extracted from healthy and CEV-infected tomato leaves 20 days after inoculation
with CEV. At this early growth stage, leaf tissue from healthy plants had no detectable P1 (p 14)
content. This protein however, is normally found in older leaves (Camacho Henriquez & S~inger,
1982; Vera et al., 1988). One percent to 1.3 % of the total R N A in either healthy or CEV-infected
leaves was present as poly(A) + m R N A . Maximum incorporation of radioactivity from
[35S]methionine was achieved with 3 to 4 ~tg poly(A) ÷ m R N A per translation assay. For analysis
of immunoprecipitable material, 50 Ixl of translation mixture was used. About 0.12% of
radioactivity incorporated into protein was recovered in anti-Pl(pl4) immunoprecipitation
assays containing poly(A) + m R N A from CEV-infected tomato leaves; negligible radioactivity
was obtained with poly(A) ÷ m R N A from healthy leaves.
Further analysis by S D S - P A G E and fluorography revealed that the immunoprecipitable
material from the translation assays was concentrated in a single band of Mr 16000 to 16500
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0
0
Z
~q
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Z
~ I~~
I ~ ~
0
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i
Z
Z
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PR protein in viroid-infected tomato plants
(d)
r~
~" .
1937
(e)
V
V
V
V
g
[
? -:..
Fig. 2. (a to e) Immunocytochemical localization of P1 (pl 4) on thin sections of tomato mesophyll cells.
Sections were incubated with P1 (p14) antiserum (a to d) or with preimmune control serum (e), followed
by incubation with Protein A-colloidal gold as described in Methods. Key, as described for Fig. 1. Bar
markers represent 1 ~tm•
Fig. 1. (a to J) Immunocytochemical localization of Pl(pl4) in vacuolar inclusion bodies of palisade
parenchyma ceils from CEV-infected tomato leaves• Vacuolar inclusion bodies (denoted by arrows in a)
in different cells differ in size, shape and complexity (a to3'). P 1(p 14) is indicated by the gold particles
concentrated over the inclusion bodies (IB). M, mitochondrion; V, vacuole; CHL, chloroplast; IS,
intercellular space. Bar markers represent 1 p.m.
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1938
P. VERA, J. HERN.~,NDEZ-YAGO AND V. CONEJERO
(a)
,T'
Fig. 3. (a to c) Control sections incubated with preimmune serum and Protein A-colloidal gold,
showing essentially no staining either in vacuolar inclusion bodies or in intercellular spaces. Key as
described for Fig. 1. Bar markers represent 1 ~tm.
Table 1. Density of gold labelling on palisade parenchyma cells of CEV-infected tomato leaves
using anti-P1(p14) and control sera
Density of labelling (gold particles/~tm2)
r--
Cell compartment
Intercellular space
Cell wall
Nucleus
Chloroplast
Mitochondrion
Vacuole (inclusion body)
Ground cytosol
.X
Anti-Pl(pl4)
Control serum
Corrected densit~
140 + 12
2.5 + 1.0
11 + 3.1
14 ___2.4
1.2 + 0.3
127 + 8.4
7-4 + 3-5
3-2 + 0.6
3.4 + 1"3
8 + 0.7
9 + 3.2
4.5 + 2.0
6.3 + 3.3
6-2 _ 2.7
137 + l 1
120 + 4
-
* For each antiserum, gold labelling was examined in each cell compartment in a number of samples accounting
for a total surface area exceeding 140 ixm2. Values are means (n = 3) + S.D.
I" Corrected value is the labelling density for anti-Pl(pl4) minus the labelling density for control antiserum.
(Fig. 4). As immunoprecipitation of translation products in the presence of 3 ~tg of unlabelled
P l ( p l 4 ) (Fig. 4, lane 6) or with p r e i m m u n e serum (lane 8) produced no radioactive band, we
conclude that the band represents a precursor protein larger that P l ( p l 4 ) , the mature form of
which has an Mr of approximately 14000 (Fig. 4, lane 4) as determined after immunoprecipitation and S D S - P A G E of P l ( p l 4 ) synthesized in vivo.
The m R N A s isolated from leaves of CEV-infected and those from healthy plants were
virtually identical in their capabilities to direct the incorporation of pSS]methionine into the
bulk of proteins. However, only the m R N A isolated from leaves of CEV-infected plants
directed the incorporation of [35S]methionine into immunoprecipitable P l ( p l 4 ) in the in vitro
translation system (Fig. 4, lanes 5 and 7). This suggests that CEV infection, as well as other
stress-causing agents and even senescence, induces synthesis of new m R N A s . This is in
agreement with the discovery that some tobacco and parsley P R proteins synthesized on
m e m b r a n e - b o u n d polysomes are regulated at the level of transcription (Carr et al., 1985;
Somssich et al., 1986; Hooft v a n Huijsduijnen et al., 1985).
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PR protein in viroid-infected tomato plants
1
2
3
4
5
6
1939
7
8
LL~ .....................
i
• prePl(pl4)
• Pl(pl4)
Fig. 4. SDS-PAGE and fluorography of proteins synthesized in vitro and in vivo. Lanes 1 and 2
correspond to the in vitro synthesized proteins in reticulocyte lysates programmed with poty(A)÷ mRNA
from healthy and CEV-infected plants respectively. Lanes 3, 4, 5 and 6 represent antigen-antibody
competition experiments to determine the relationship of in vivo (lanes 3 and 4) and in vitro (lanes 5 and
6) [programmed with poly(A)÷ RNAs from CEV-infected plants] synthesized P 1(p14). Immunoprecipitation of Pl(pl4) was performed in the presence (lanes 3 and 6) or absence (lanes 4 and 5) of 3 p.g of
purified P 1(p 14). Lane 7 represents immunoprecipitation with anti-P 1(p 14) serum of in vitro translation
products when poly(A)÷ RNAs from healthy plants were used to programme the reticulocyte synthesis
reaction. Lane 8 represents immunoprecipitation with preimmune serum of in vitro translation products
when poly(A)÷ RNAs from CEV-infected plants are used to programme the synthesis.
Processing of preP l(p l4) by microsomal membranes
The Pl(pl4) protein detected by the immunological method and synthesized in the
reticulocyte system in the presence or absence of canine microsome membranes is shown in Fig.
5. The results clearly demonstrate that the precursor protein was processed to the mature form
when microsomes were present during translation. This result strongly suggests that the P1 (p14)
protein has a signal peptide which is removed during its translocation into the lumen of the
endoplasmic reticulum.
DISCUSSION
Using Protein A-gold immunocytochemistry and an antibody against tomato leaf protein
Pl(pl4) we have found that the latter accumulates in two different locations of the CEV-infected
leaf tissue: in intercellular spaces, as observed for other PR proteins (Parent & Asselin, 1984;
Carr et al., 1987; Dumas et al., 1988; Hosokawa & Ohashi, 1988) and, without precedent, in the
cell vacuole.
The clear-cut localization of P 1(p 14) within vacuoles, and always in association with inclusion
bodies, introduces further aspects on the interpretation of the physiological role of PR proteins.
The possibility that inclusion bodies found in vacuoles are artefacts of tissue preparation can be
excluded because they have not been detected in similarly prepared leaf tissue sections from
healthy plants (data not shown; see Vera et al., 1988). The nature of these inclusion bodies is as
yet unknown. They have also been found in tobacco cell vacuoles during adaptation to high
osmotic stress, associated with 'osmotin', a protein which is highly homologous to a tobacco
mosaic virus-induced PR protein in tobacco plants (Singh et al., 1987). By analogy, it can be said
that vacuolar inclusion bodies may function within vacuoles as a type of storage compartment
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1940
P. VERA, J. HERN.~NDEZ-YAGO AND V. CONEJERO
1
2
3
4
--67K
--43K
--30K
--20K
prePl(pl4) I~ ~
Pl(pl4)~-
~
~°~
~"
--14K
Fig. 5. Processing of prePl(p14) in the in vitro
translation system. Reticulocyte lysate cell-free
translation systemscontained 0.1 (lane 1), 1 (lane
2) and 2.5 (lane 3) A26o units of canine pancreas
microsomes. Lane 4 corresponds to a co-electrophoresis of in vivo and in vitro (absence of
microsomes) synthesized and immunoprecipitated Pl(pl4) (equal amounts of radioactivity).
Poly(A)÷ RNA from CEV-infected plants was
used to programme the lysates as described in
Methods.
for PR proteins, particularly P 1(p 14). In fact, none of these vacuolar inclusion bodies has been
found without P l(pl4). This localization has also been suggested for the salt-induced NP-24
protein of tomato plants (King et al., 1988) and for the sweet-tasting protein 'thaumatin' (Edens
& van der Wel, 1985), both highly homologous to the tobacco protein osmotin. In addition,
Pl(pl4)-like PR proteins have been found in several plant species under stress from different
agents (White et al., 1987). Taking all this into account the possibility exists that vacuolar
inclusion bodies are a general feature of the reaction of plants to biological or non-biological
afflicting agents.
The fact that P 1(pl4) is synthesized as a higher Mr precursor is in agreement with the idea that
this protein must be transported within the cell. It implies that Pl(pl4) undergoes posttranslational modification to generate the shorter mature protein found to accumulate in vivo.
This argument is validated by the finding that microsomal membranes are able to convert
prePl(pl4) to its mature form, thus being consistent with the observation on other vacuolar
proteins which lose an N-terminal domain on their journey to the vacuole [e.g. tomato and
potato proteinase inhibitors (Nelson & Ryan, 1980; Cleveland et al., 1987; Graham et aL, 1985)
or chitinase (Broglie et al., 1986; Boller& Vogeli, 1984)], and also with the fact that a Pl(p14)like PR protein of the tobacco plant is derived from a precursor by the removal of an N-terminal
signal peptide of 30 amino acids (Hooft van Huijsduijnen et al., 1985). The mechanism
accounting for the localization of Pl(pl4) in both vacuoles and intercellular spaces is still a
matter of conjecture. One possible explanation, as suggested for bean chitinase (Boller& V6geli,
1984), is the occurrence of vacuoles breaking and discharging their contents into intercellular
spaces after dissolution of the cell (e.g. in a hypersensitive reaction) as part of the pathogenetic
process. Also, the possibility that once in the endoplasmic reticulum, P l(p 14) can be sorted via
alternative pathways either to the apoplast or to the vacuole cannot be disregarded. The
elucidation of these questions may be fundamental to our understanding of the mechanism of
compartmentation of PR proteins and of their biological role.
This research was supported by grant 2509/84 from CAICYT, Ministerio de Educaci6n y Ciencia of Spain and
by a grant from Diputaci6 Provincial de Valencia (Generalitat Valenciana).
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P R protein in viroid-infected tomato plants
1941
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