Download Induction by Salicylic Acid of Pathogenesis

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gen. Virol. (1986), 67, 2135-2143.
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Key words: salicylic acid/resistance/pathogenesis-relatedproteins/alfalfa mosaic virus
Induction by Salicylic Acid of Pathogenesis-related Proteins and Resistance
to Alfalfa Mosaic Virus Infection in Various Plant Species
By R. A. M. H O O F T V A N H U I J S D U I J N E N , *
S. W. A L B L A S , R. H. D E
R I J K AND J. F. BOL
Department o f Biochemistry, State University o f Leiden, P.O. Box 9505, 2300 RA Leiden, The
Netherlands
(Accepted 12 June 1986)
SUMMARY
Spraying tobacco plants with salicylic acid induces both the synthesis of
'pathogenesis-related' (PR) proteins and resistance to viruses that can induce necrotic
lesions. We show that spraying Samsun NN tobacco with salicylic acid induced the
production of PR-1 mRNAs and inhibited the systemic multiplication of alfalfa mosaic
virus (A1MV) by 90%. Salicylic acid treatment also induced the synthesis of PR
proteins in bean and cowpea plants, and reduced by 75 % the production of local lesions
in A1MV-infected bean plants. Salicylic acid inhibited the replication of A1MV in
cowpea protoplasts by up to 99%, depending on the mode of application. In A1MVinoculated cowpea protoplasts, the production of viral minus-strand RNA, plus-strand
RNA and coat protein was abolished, indicating that salicylic acid inhibits an early
step in the A1MV replication cycle. The viability of the cells and the synthesis of host
proteins were not affected by salicylic acid. Another aromatic compound, p-coumaric
acid, induced neither PR proteins nor resistance to virus infection.
INTRODUCTION
In at least 16 plant species, the hypersensitive response to virus infection is accompanied by
the de novo synthesis of 'pathogenesis-related' (PR) proteins (for review, see Van Loon, 1985).
The association of these proteins with acquired systemic resistance led to the suggestion that
they function in a defence mechanism (Kassanis et al., 1974; Van Loon, 1975). This hypothesis
is supported by the observation by White (1979) that spraying tobacco plants with salicylic acid
or acetylsalicylic acid induces both the synthesis of PR proteins and resistance to infection with
tobacco mosaic virus (TMV). Moreover, a tobacco hybrid that produces PR proteins
constitutively is highly resistant to TMV infection (Ahl & Gianinazzi, 1982). To learn more
about the functions of PR proteins, we cloned and sequenced DNA copies of the mRNAs for the
PR- 1 proteins of tobacco (Hooft van Huijsduijnen et al., 1985; Cornelissen et al., 1986 a). This
revealed a 90% amino acid sequence homology between PR-la, -lb and -lc, and showed that
PR-1 proteins are derived from precursors by removal of a signal peptide of 30 amino acids. This
is consistent with the observation that PR proteins accumulate in the intercellular spaces of the
leaf (Parent & Asselin, 1984). A 14 000 mol. wt. (14K) protein of tomato (p 14) which is induced
by infection with TMV or viroids (Camacho-Henriquez & S/inger, 1982; Lucas et al., 1985) has a
60% amino acid homology with the tobacco PR-Ib protein (Cornelissen et al., 1986a). An
antiserum against p14 was shown to cross-react with tobacco PR-1 proteins and a PR protein
from cowpea (Nassuth & S/inger, 1986), indicating that PR proteins from different plant species
may be closely related.
Studies on induced resistance have mainly focused on the inhibition of the formation of local
lesions by the challenging virus (see Van Loon, 1985). A few conflicting results on the inhibition
of a systemic infection have been reported. Van Loon & Dijkstra (1976) showed that induction
of resistance in Samsun tobacco by infection with tobacco necrosis virus, resulted in a 40 to 80%
inhibition of a subsequent systemic TMV infection. More recently, White et al. (1983) reported
0000-7224 © 1986 SGM
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R.A.M.
HOOFT VAN H U I J S D U I J N E N AND OTHERS
that treatment with acetylsalicylic acid of Samsun tobacco reduced the replication of TMV by
up to 94%. On the other hand, Fraser (1978) observed that upon induction of resistance in White
Burley tobacco by infection with TMV vulgare, the systemic infection of TMVflavum was not
impaired.
The aim of the present investigation was to shed light on the step in viral replication that is
inhibited by salicylic acid treatment. In order to find a system permitting such an analysis, we
studied the effect of salicylic acid on the replication of alfalfa mosaic virus (AIMV) in tobacco
leaves (systemic infection), bean leaves (production of necrotic lesions) and cowpea protoplasts
(single-step replication cycle). The production of infectious virus was monitored in all three
systems. Subsequently, the protoplast system was used to study the effect of salicylic acid on
viral RNA and protein synthesis.
To permit a correlation with induced resistance, we analysed the effect of salicylic acid on the
synthesis of PR proteins and mRNAs in tobacco, bean and cowpea plants. Recently, it was
reported that aromatic compounds which act as intermediates in the phenylpropanoid pathway
do not induce PR proteins in tobacco (Jamet, 1985), and we have compared the effects of one of
these compounds, p-coumaric acid, with those of salicylic acid on A1MV replication in tobacco
leaves, bean leaves and cowpea protoplasts.
METHODS
Plants and viruses. Tobacco plants (Nicotiana tabacum cv. Samsun NN) were grown for 9 to 10 weeks in a
glasshouse. Inoculation of these plants with A1MV (strain 425) was done by rubbing carborundum-dusted leaves
with a homogenate of infected leaves made in PEN buffer (10 mM-NaH2PO4, 1 mM-EDTA, 1 mM-NaN3, pH 7.0).
TMV (strain WU1) was inoculated on tobacco as a purified solution at 6 ptg/ml. PR proteins were extracted from
A1MV- and TMV-infected tobacco plants 7 days after inoculation.
Bean plants (Phaseolus vulgaris L. cv. Berna) were grown in a glasshouse for 9 to 10 days before use. For
induction of PR proteins, half-leaves were inoculated with A1MV at a concentration of 10 ~tg/ml in PEN buffer,
yielding approximately 900 lesions per half-leaf. PR proteins were isolated 7 days after inoculation. Salicylic acidtreated bean leaves were inoculated with A1MV at a concentration (1 ~tg/ml) that induced 50 to 200 lesions per halfleaf of the controls.
Cowpea plants (Vigna unguiculata L. Walp cv. Blackeye Early Ramshorn) were grown in a growth chamber.
Protoplasts were isolated and inoculated with A1MV as described by Nassuth et al. (1981). PR proteins were
induced by inoculating cowpea plants with 75 ~tg/ml AIMV, yielding about 300 lesions per half-leaf. PR proteins
were isolated 7 days after inoculation.
Tomato plants (Lycopersicon esculentum cvs. Rutgers and Moneymaker) were inoculated with 10 I-tg/ml TMV
when 3 months old.
Local lesion assay. Homogenates were made of A1MV-infected tobacco leaves or cowpea protoplasts and these
were inoculated at several dilutions on seven half-leaves of bean plants according to Kleczkowski (1950). Unless
mentioned otherwise, lesions were counted 3 days after inoculation.
Analysis of PR proteins. PR proteins were isolated from the intercellular spaces of tobacco, bean and cowpea
leaves by the method of Parent & Asselin (1984). Freshly collected leaves were infiltrated in vacuo with cold (4 °C)
25 mM-Tris-HC1 pH 7.8, 0.5 M-sucrose, 10 mM-MgC12, 10 mM-CaCI2, and 5 mM-2-mercaptoethanol. The leaves
were blotted dry, rolled up and centrifuged 30 min at 1000g in 5 ml syringes. The samples, about 0-5 ml per gram of
leaf, were stored at - 20 °C. The compositions of the PR preparations were analysed by electrophoresis at alkaline
pH in non-denaturing cylindrical 10% polyacrylamide gels (Davis, 1964). The samples (400 ~1) were supplemented
with 5 ~tl 0.3 % bromophenol blue and electrophoresed for 1 to 2 h at 5 mA/gel. The gels were fixed for several hours
in 3.5 % perchloric acid (PCA) and stained for 1 h at 37 °C in 0.04% (w/v) Coomassie Brilliant Blue G-250 (Serva)
in 3.5% PCA, as described by Reisner et al. (1975). Subsequently, the gels were incubated for at least 12 h in 7.5%
acetic acid at room temperature.
Treatments with salicylic and p-coumaric acids. Both salicylic acid and (trans) p-coumaric acid, purchased from
Janssen Chimica, were prepared as 0.25 M stock solutions adjusted to pH 7.0 with KOH. Plants were sprayed with
5 mM solutions on 2 successive days prior to the isolation of PR proteins, the isolation of protoplasts or virus
inoculation. Two h after spraying, the leaves were thoroughly rinsed with tap water. Salicylic acid or p-coumaric
acid was added to cowpea protoplasts to a concentration of 1 mM, after the final washing step of the inoculation
procedure.
Northern blot hybridizations. Extraction of RNA from leaves, poly(U)-Sepharose chromatography, agarose gel
electrophoresis and Northern blot hybridization were performed as described previously (Hooft van Huijsduijnen
et al., 1985). The PR-lb probe was clone 69 described by Cornelissen et al. (1986a), labelled by nick-translation
according to Rigby et al. (1977). RNA from cowpea protoplasts was isolated by the procedure of Kubo et al. (1975).
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Radiolabelled probes for the specific detection of A1MV plus- and minus-strand RNAs were prepared as
described by Nassuth & Bol (1983). Unless stated otherwise, the blots were washed for 60 rain in 0.1 × SSC, 0-!
SDS at 50 °C. For non-stringent hybridizations, blots were washed for 60 rain in 1 × SSC, 0.1 ~ SDS at 20 °C.
In vivo radiolabelling of AlMV coat protein. After virus inoculation, samplesof 106 protoplasts were incubated in
standard medium (0.5 i-D-mannitol, 10 m~-CaC12, 0.2 mg/ml chloramphenicol, pH 6.5) supplemented with 10
~tCi/ml t.-[3sS]methionine (1000 Ci/mmol, Amersham). After incubation for 24 h at 25 °C under continuous
illumination at about 2500 lx, the protoplasts were collected, washed, and their protein content was analysed by
SDS-PAGE as described by Nassuth et al. (1983).
RESULTS
Fig. 1 shows the proteins in the intercellular fluid from cowpea, bean and tobacco plants that
had been sprayed with water or salicylic acid. As controls, the intercellular fluid of plants
sprayed with p-coumaric acid and virus-infected plants were analysed. The set of PR proteins
that is known to be induced in tobacco by the hypersensitive reaction to TMV infection is shown
in lane 13 of Fig. 1. A subset of these proteins, notably PR proteins la, lb, 2, N and O, was also
induced by spraying tobacco plants with salicylic acid (Fig. 1, lane 1 l). On the other hand,
systemic replication of A1MV in tobacco plants did not induce the synthesis of PR proteins (Fig.
1, lane 12). Moreover, the composition of the intercellular fluid from tobacco plants sprayed
with p-coumaric acid (Fig. 1, lane 10) was similar to that of untreated plants (Fig. 1, lane 9).
Spraying cowpea plants with salicylic acid induced the accumulation of a protein with an RF
value of 0-57 (Fig. l, lane 3) which was absent from the intercellular fluid of untreated cowpeas
(Fig. 11 lane 1). A similar protein was induced upon inoculation of cowpea leaves with AIMV,
which results in the formation of local lesions (Fig. 1, lane 4). No significant amount of protein
was induced by spraying cowpea plants with p-coumaric acid (Fig. 1, lane 2).
In salicylic acid-treated bean plants, a protein accumulated (Fig. 1, lane 7) that had an RF
value of 0-60. This protein was absent from p-coumaric acid-sprayed or untreated bean plants
(Fig. 1, lanes 6 and 5, respectively). Infection of bean plants with A1MV, which resulted in the
production of local lesions, led to the accumulation of three proteins which gave prominent
bands with Rv values of 0.17, 0.47 and 0.55, in addition to the RF 0.60 protein (Fig. 1, lane 8).
Induction o f PR-1 m R N A s by salicylic acid
Polyadenylated RNA was isolated from tobacco and tomato plants infected with TMV, and
from cowpea and bean plants infected with A1MV. In addition, polyadenylated R N A was
isolated from these plant species after spraying them with salicylic acid. Fig. 2 shows a Northern
blot of polyadenylated R N A from healthy tobacco (lane H), tobacco sprayed with salicylic acid
(lane S) and tobacco infected with TMV (lane T). The blot was probed with the P R - l b clone.
The results demonstrate that spraying with salicylic acid induced the synthesis of PR-1 m R N A s
although the increase was slightly lower than that induced by TMV infection. A similar analysis
of polyadenylated R N A from salicylic acid-sprayed or virus-infected tomato, cowpea or bean
plants, failed to detect any cross-hybridization with the tobacco PR-1 probe, even when the
hybridization was done at low stringency (result not shown). Although PR proteins from
tobacco, tomato and cowpea are known to have related amino acid sequences and to be related
serologically (see Introduction), the homology between the m R N A s was apparently too low to
be detected.
Salicylic acid-induced resistance
Table 1 shows that spraying tobacco plants with salicylic acid reduced the systemic
replication of A1MV by 90~. Spraying bean plants with salicylic acid resulted in a 7 5 ~
reduction of the number of local lesions obtained after inoculation with AIMV. Moreover, the
lesions appeared later (counting was done 5 days after inoculation instead of 3) and were much
smaller than lesions in non-sprayed bean leaves.
The effect of salicylic acid on the replication of A1MV in cowpea protoplasts was assayed in
three different ways (Table 1). When salicylic acid was sprayed on the leaves of the cowpea
plants before isolation of the protoplasts, virus production was reduced to 18 ~ of the untreated
control. Addition of salicylic acid to the protoplasts after inoculation with A1MV resulted in a
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R. A. M, HOOFT VAN HUIJSDUIJNEN AND OTHERS
H
1
C
2
S
3
A
4
H
5
C
6
S
7
A
8
A
H
C
9
10
S
11
A
12
T
13
.................................'S
Fig. 1. Accumulation of PR proteins in the intercellular fluid of cowpea (lanes 1 to 4), bean (lanes 5 to 8)
and tobacco (lanes 9 to 13) after various treatments. Plants were sprayed with water (lanes H), pcoumaric acid (lanes C), salicylic acid (lanes S), or inoculated with AIMV (lanes A) or TMV (lane T).
Samples of the intercellular fluid were electrophoresed in non-denaturing polyacrylamide gels; the
position of the bromophenol blue marker is indicated by arrowheads. The nomenclature of the tobacco
PR proteins is according to Van Loon (1982).
9 6 ~ i n h i b i t i o n of virus multiplication. W h e n the two t r e a t m e n t s were c o m b i n e d , the replication
of A1MV was i n h i b i t e d by 99 ~ or more. S t a i n i n g the living protoplasts with fluorescein diacetate
(Wildholm, 1972) at the end of the 24 h i n c u b a t i o n period showed that the presence of 1 mMsalicylic acid did n o t influence their viability, w h i c h was a p p r o x i m a t e l y 9 5 ~ for b o t h
treatments.
To test the specificity of the effect of salicylic acid on A1MV replication, p - c o u m a r i c acid was
included as a control. T a b l e 1 shows that no significant i n h i b i t i o n of virus m u l t i p l i c a t i o n was
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Salicylic acid-induced resistance to A I M V
H
S
T
(a) (b) (c) (d)
(e) (f)
(g) (h)
i iil !iii i
Fig. 2.
Fig. 3.
Fig. 2. Induction of PR-1 mRNAs in tobacco. A Northern blot was prepared with 10 ~g
polyadenylated RNA from the leaves of healthy plants (lane H), plants sprayed with salicylic acid (lane
S) or plants inoculated with TMV (lane T). The blot was hybridized to radiolabelted PR-I cDNA.
Fig. 3. Effect of salicylic acid and p-coumaric acid on A1MV RNA synthesis. A Northern blot was
prepared with RNA from healthy protoplasts (d, h) and A1MV-inoculated cowpea protoplasts
incubated for 24 h in standard medium (a, e), in medium containing 1 raM-salicylicacid (b, f), or in
medium containing 1 mM-p-coumaricacid (c, g). RNA from 5 × 105 protoplasts was used per lane. The
blot was hybridized to 3zp-labelled AIMV RNA to detect viral minus-strand RNAs (a to d); after
autoradiography the probe was washed off and the blot was hybridized to 32p-labelledA1MV cDNA
synthesized in vitro to detect viral plus-strand RNAs (e to h). The positions of A1MV RNAs 1, 2, 3 and 4
are indicated.
Table 1. Effect of salicylic acid and p-coumaric acid on A l M V replication
Host
Mode of treatment
with water, salicylic
or p-coumaric acids
A. Tobacco plants Sprayedon leaves
B. Bean plants
Sprayedon leaves
C. Cowpea plants Sprayedon leaves
before the isolation
of protoplasts
Added to protoplast
suspension
Combination of the above
r
Water
Effect* of treatment with
x
p-Coumaric acid
Salicylic acid
635
2037
1776
64 (10~)
504 (25~)
325 (18~)
2687 (420~)
1757 (86%)
NDt
1776
70 (4~)
1670 (94~)
1776
4 (0.2~)
1420 (80~)
* Effect was measured by inoculating homogenates on bean leaves and counting local lesions (A, C) or counting
lesions directly (B). The total number of lesions on seven half-leaves is given; figures in parentheses give the
relative production of lesions as compared to the water-treated control.
l" ND, Not determined.
obtained with this compound in any of the host systems (P < 5~). The fourfold stimulation of
A1MV production in tobacco leaves by p-coumaric acid was reproducibly observed in several
experiments.
Inhibition by salicylic acid of viral RNA and protein synthesis
Fig. 3 shows a Northern blot hybridization of R N A from healthy protoplasts (d, h) and
infected protoplasts which had been incubated for 24 h in standard medium (a, e), or m e d i u m
containing either I mM-salicylic acid (b,f) or 1 mM-p-coumaric acid (c, g). Strand-specific probes
were used to visualize A1MV minus-strand R N A s 1, 2 and 3 and plus-strand R N A s 1, 2, 3 and 4.
Although the plus-strand-specific probe detected R N A 4 poorly in this experiment, it was clear
that salicylic acid strongly blocked synthesis of viral minus-strand and plus-strand R N A ,
whereas p-coumaric acid had no inhibitory effect.
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R. A. M. H O O F T VAN H U I J S D U I J N E N
(a)
(b)
H
I
AND O T H E R S
(c)
H
I
H
I
-CP
......... i
......... . . . . . . . . . .
Fig. 4. Effect of salicylic acid and p-coumanc acid on AIMV coat protein synthesis. 3sS-labeUed
proteins from healthy (lanes H) and A1MV-infected (lanes I) cowpea protoplasts were electrophoresed
in 11 ~ polyacrylamide gel. The protoplasts were incubated for 24 h in standard medium (a), in medium
containing 1 raM-salicylic acid (b) or in medium containing 1 mM-p-coumaric acid (c). The position of
A1MV coat protein (CP) is indicated.
Fig. 4 shows the results of polyacrylamide gel electrophoresis of radiolabelled proteins from
healthy (H) and infected (I) protoplasts which had been incubated for 24 h in standard medium
(a), or medium containing either 1 raM-salicylic acid (b) or I mM-p-coumaric acid (e). Compared
to the control, p-coumaric acid did not inhibit AIMV coat protein synthesis. Salicylic acid
blocked the synthesis of detectable amounts of viral coat protein (CP) but did not qualitatively
affect the synthesis of host proteins, nor did it reduce the overall incorporation of radiolabel.
Under the conditions used, no salicylic acid-induced radiolabelled host proteins were detected in
a homogenate of the protoplasts (Fig. 4) or in the medium in which the protoplasts had been
incubated (result not shown).
DISCUSSION
Induction of PR proteins and 'acquired resistance' in plants by natural pathogens such as
viruses, fungi or bacteria is frequently associated with a necrotic reaction. It has been proposed
that ethylene production evoked by this reaction triggers the synthesis of one or more benzoic
acid derivatives which transmit the signal that eventually activates the transcription of PR
genes (Van Loon, 1983). In this model salicylic acid would mimic one of the phenolic
compounds. In agreement with this concept, our results show that systemic replication of A1MV
in tobacco does not induce PR proteins, as has been reported for the replication of TMV under
non-hypersensitive conditions (Van Loon & Van Kammen, 1970; Antoniw et al., 1985; Ohashi
& Matsuoka, 1985).
It is likely that the inhibitory effect of salicylic acid on the replication of AIMV in cells of
tobacco, bean and cowpea is due to the same underlying mechanism. The inhibition of the
systemic replication in tobacco shows that the induced resistance does not necessarily involve
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the formation of local lesions. The results with the cowpea protoplasts demonstrate that
inhibition does not occur at the level of cell-to-cell spread of the virus, but that salicylic acid is
acting on primarily infected cells. If this conclusion also holds for whole leaves, the reduction in
size and number of lesions on bean leaves by salicylic acid treatment might be due to a reduced
virus production in the primarily infected cells. The observation that viral R N A and coat
protein synthesis are inhibited indicates that salicylic acid treatment blocks an early step in the
A1MV replication cycle. Because salicylic acid is effective when applied after inoculation of the
protoplasts, an effect on the uptake or uncoating of the virus is not likely. This possibility is
being investigated by infecting protoplasts with viral RNA instead of nucleoprotein.
As salicylic acid does not inhibit host protein synthesis, the viral RNAs that enter the
protoplasts are probably translated into the encoded viral proteins. The observation that A1MV
RNAs 1 and 2 are able to replicate in protoplasts in the absence of R N A 3 indicates that the
126K and 90K proteins encoded by these genome segments, respectively, are involved in the
formation of a replicase activity (Nassuth & Bol, 1983). Apparently, salicylic acid treatment
blocks the synthesis of minus-strand and plus-strand RNAs by this replicase activity. Possibly,
the inhibition occurs at the level of the synthesis of double-stranded replicative intermediates.
The lack of coat protein synthesis may be a consequence of the block of viral R N A synthesis.
The observation that salicylic acid treatment activates transcription of PR genes, does not
inhibit host protein synthesis, does not affect the viability of cells and does not alter the
appearance of sprayed plants indicates that inhibition of virus multiplication by salicylic acid is
a specific process and is not due to a general block of host metabolism.
It is tempting to speculate that salicylic acid-induced PR proteins (or salicylic acid-induced
RNA molecules) are responsible for the inhibition of viral RNA synthesis. The parallel between
the induction of acquired resistance and the induction of PR proteins lends support to this
hypothesis. However, a role for PR proteins in a defence mechanism has been questioned, as in
several studies no quantitative or temporal relationship between the onset of resistance and PR
protein production was found, and some chemicals are known to cause resistance without
inducing PR proteins and vice versa (Fraser & Clay, 1983; Sherwood, 1985). Moreover, we
observed that addition of the intercellular fluid from salicylic acid-sprayed cowpea plants to
cowpea protoplasts did not inhibit AIMV replication (unpublished result). Recently, we have
cloned cDNAs of six TMV-induced tobacco mRNAs, three of which are also induced by
salicylic acid (Cornelissen et al., 1986b; Hooft van Huijsduijnen et al., 1986). The m R N A most
strongly induced by salicylic acid was found to encode a protein that did not react with an
antiserum to PR proteins. Thus, the possibility exists that a salicylic acid-induced non-PR
protein is involved in a resistance mechanism. As an alternative to the above hypothesis, the
possibility must be considered that salicylic acid inhibits viral R N A synthesis without the
involvement of the induction of host gene transcription. At a concentration of 1 mM, salicylic
acid did not interfere with A1MV RNA synthesis in vitro by a replicase complex isolated from
infected cells (C. J. Houwing & E. M. J. Jaspars, personal communication). Of 27 phenolic
compounds tested, salicylic acid was the most potent inhibitor of encephalomyocarditis virus
replication in mouse tissue cultures (Kochman et al., 1965). It would be interesting to see
whether salicylic acid treatment induces the synthesis of similar proteins in plant and animal
cells.
This work was sponsored in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial
aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).
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