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Plant Physiol. (1998) 116: 387-392
Endogenous Methyl Salicylate in Pathogen-Inoculated Tobacco
Plants1
Mirjana Seskar,
Vladimir Shulaev, and
Ilya Raskin*
AgBiotech Center, Cook College, Rutgers University, P.O. Box 231, New Brunswick, New Jersey 08903-0231
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ABSTRACT |
The tobacco (Nicotiana
tabacum) cultivar Xanthi-nc (genotype NN)
produces high levels of salicylic acid (SA) after inoculation with the
tobacco mosaic virus (TMV). Gaseous methyl salicylate (MeSA), a major
volatile produced in TMV-inoculated tobacco plants, was recently shown
to be an airborne defense signal. Using an assay developed to measure
the MeSA present in tissue, we have shown that in TMV-inoculated
tobacco plants the level of MeSA increases dramatically, paralleling
increases in SA. MeSA accumulation was also observed in upper,
noninoculated leaves. In TMV-inoculated tobacco shifted from 32 to
24°C, the MeSA concentration increased from nondetectable levels to
2318 ng/g fresh weight 12 h after the temperature shift, but
subsequently decreased with the onset of the hypersensitive response.
Similar results were observed in plants inoculated with
Pseudomonas syringae pathovar
phaseolicola, in which MeSA levels were highest just
before the hypersensitive response-induced tissue desiccation.
Transgenic NahG plants unable to accumulate SA also did not accumulate
MeSA after TMV inoculation, and did not show increased resistance to
TMV following MeSA treatment. Based on the spatial and temporal
kinetics of its accumulation, we conclude that tissue MeSA may play a
role similar to that of volatile MeSA in the pathogen-induced defense
response.
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INTRODUCTION |
Tobacco (Nicotiana tobaccum L. cv Xanthi-nc) plants
carrying the N gene produce high levels of SA after
inoculation with TMV. An increase in the tissue content of SA follows
the appearance of the virus-induced HR (Malamy et al., 1990 ). The
correlation between SA accumulation and pathogen resistance has also
been shown in cucumber (Metraux et al., 1990; Rasmussen et al., 1991), tomato (Hammond-Kosack et al., 1996), and Arabidopsis (Delaney et al.,
1995). Following the primary infection, the inoculated plant becomes
resistant to subsequent pathogen attack, both locally and systemically,
in pathogen-free leaves. This phenomenon, called SAR, has attracted
scientific interest for more than 60 years (Chester, 1933). The
dependence of SAR on SA accumulation was shown in transgenic NahG
tobacco plants expressing the salicylate hydroxylase, which accumulate
little or no SA and do not exhibit SAR (Gaffney et al., 1993).
While investigating the movement and distribution of SA in
TMV-inoculated tobacco, we discovered that the plants evolve large amounts of gaseous MeSA (Shulaev et al., 1997). In contrast, healthy or
mechanically wounded plants did not evolve any detectable amounts of
MeSA. Application of gaseous MeSA to healthy tobacco plants increased
the expression of the PR-1 gene and TMV resistance (measured as the size reduction of the TMV-induced lesions). The amount of MeSA
produced by TMV-inoculated tobacco plants was sufficient to induce
PR-1 gene expression and resistance in healthy plants receiving air from the headspace of enclosed TMV-inoculated plants.
The accumulation of SA in tissue exposed to MeSA gas and labeling
studies using [14C]SA and MeSA suggest that
MeSA is synthesized from SA and acts by being converted back to SA in
the target tissues. MeSA is not the only metabolite of SA found in
plants. Large amounts of glucosyl SA accumulate in and around the HR
lesions (Enyedi et al., 1992; Malamy et al., 1992). However, indirect
evidence suggests that glucosyl SA is neither mobile nor biologically
active and may function as a storage form of SA (Enyedi et al., 1992;
Malamy et al., 1992). In contrast, data indicate that MeSA may serve as
an airborne signal involved in inter- and intra-plant communication during the pathogen infection (Shulaev et al., 1997).
The fact that MeSA is naturally produced by a number of plants is well
known (Wilson et al., 1987; Hamilton-Kemp et al., 1988; Hayden and
Clough, 1990; Loughrin et al., 1993); however, the suggestion that
gaseous MeSA serves as an inducible signal for resistance in tobacco
prompted further investigation of this compound. MeSA, also known as
oil of wintergreen, is a volatile liquid at room temperature (boiling
point 220-224°C). Whereas previous work examined the effects of MeSA
vapor (Shulaev et al., 1997), the work presented here investigates the
production and role of nongaseous MeSA in tobacco plants using an
analytical method developed for this purpose. In addition, this study
provides further evidence that the conversion of MeSA to SA is required
for the biological activity of this compound.
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MATERIALS AND METHODS |
Plant Material and Inoculation with Pathogens
Wild-type and transgenic NahG (gift from J. Ryals, CIBA-Geigy, NC)
tobacco (Nicotiana tabacum L. cv Xanthi-nc, NN
genotype) seeds were germinated and grown as described previously
(Yalpani et al., 1991). Fully expanded leaves of 6- to 8-week-old
seedlings were inoculated with TMV strain U1 (5 µg per leaf) in 5 mm potassium phosphate buffer, pH 7.0, or mock inoculated
with buffer by gently rubbing the leaves with carborundum, as described
previously (Yalpani et al., 1991). Plants were kept under continuous
illumination provided by incandescent and cool-white fluorescent lamps
(200 µmol m 2 s1). HR
was induced by incubating TMV-inoculated plants at 32°C and then
shifting them to 24°C after 4 d, as described previously (Malamy
et al., 1992).
For bacterial inoculation, overnight cultures of Pseudomonas
syringae pv phaseolicola (NPS3121) (Lindgren et al.,
1986) (a gift from R. Mittler, Rutgers University, New Brunswick, NJ)
were infiltrated into the abaxial surface of tobacco leaves using a sterile plastic syringe. Following the inoculation, 18-mm leaf discs
immediately surrounding the infiltrated areas were sampled and
analyzed.
For experiments with gaseous MeSA, 5-week-old wild-type and transgenic
NahG tobacco plants were enclosed in 4-L gas-tight glass jars
containing an upright cotton swab. The jars were opened daily and
liquid MeSA was applied to the cotton swab to produce a concentration
of 250 µg L1 in the headspace after
evaporation. After 6 d of exposure to MeSA, leaves from one subset
of plants were excised for SA and PR-1 determination. Another subset of
plants was virus inoculated on all fully expanded leaves (see above),
and the diameter of TMV-induced lesions was measured 72 h
postinoculation with a low-power microscope.
Determination of SA
Total SA (the sum of free and glucosyl SA) was extracted and
quantified as described previously (Enyedi et al., 1992). Leaf tissue
samples (0.5 g fresh weight) were frozen in liquid nitrogen, ground to
a fine powder, and sequentially extracted with 90 and 100% methanol.
The combined methanolic extracts were vacuum dried and the pellets were
resuspended in 5 mm sodium acetate buffer, pH 5.5, containing 80 units/g fresh weight  -glucosidase (Sigma). Following
enzymatic hydrolysis (90 min at 37°C), the reaction was stopped with
the addition of 10% TCA. The supernatant was partitioned with ethyl
acetate:cyclopentane:isopropanol (100:99:1, v/v). SA was determined by
fluorescence (excitation 301 nm, emission 412 nm) after separation on a
C18 reverse-phase HPLC column (Waters). The
column was maintained at 40°C and equilibrated in 0.5% glacial acetic acid:methanol (75:25, v/v) with a flow rate of 1.5 mL
min1. Three minutes after injection, a methanol
gradient (25-60%) was applied over 7 min, after which the methanol
concentration was returned to 25%. All data were corrected for
recovery using spiked samples.
Determination of MeSA
Leaf tissue samples (0.5 g fresh weight) were frozen and ground in
liquid nitrogen. The resulting powder was resuspended in 2.5 mL of 100 mm phosphate buffer, pH 7.0, containing 0.125 µg mL1 vanillin as an internal standard. After 10 min of sonication and centrifugation at 3000g, the
supernatant was loaded onto a 3-mL C18 column
(Bakerbond SPE, J.T. Baker) preconditioned with 3 mL of 100%
methanol and 3 mL of phosphate buffer added sequentially. MeSA was
eluted with 2.5 mL of 80% methanol. The methanolic fraction was
further diluted with water to 40% methanol (approximately 5 mL total
volume), and loaded onto a second 3-mL C18 column
preconditioned with 3 mL of 100% methanol and 3 mL of 40% methanol
added sequentially. MeSA was eluted with 2.5 mL of 100% methanol. The
volume of column eluent was reduced to 0.5 mL by partial drying under
vacuum (SpeedVac, Savant Instruments, Holbrook, NY). The recovery of
MeSA was 55 to 75%.
Vanillin and MeSA were detected and quantified by online UV absorption
at 280 nm (model 486 tunable absorbance detector, Waters) and
fluorescence detection at an excitation of 292 nm and an emission of
360 nm (model 474 scanning fluorescence detector, Waters), respectively. The samples (30-µL volume) were injected (model 717-Plus autosampler, Waters), and the compounds were separated on a
C8 reverse-phase HPLC column (Symmetry Shield
RP8, Waters) maintained at 40°C. MeSA was
separated during a 35-min acetonitrile (10-90%):1.2% acetic acid
(90-10%) gradient (flow rate of 1 mL min1)
maintained by HPLC pumps (model 510, Waters). Under these conditions the retention time for MeSA was approximately 22.04 min (Fig. 1,
A and B). This procedure gave the
detection limit of 200 ng/mL of MeSA (6 ng per injection). An
absorption spectrum of an authentic MeSA standard, obtained using a
photodiode array detector (model 996, Waters), was almost identical to
that of a compound that coeluted with authentic MeSA. GC-MS analysis of
TMV-inoculated tobacco leaf extracts demonstrated directly the presence
of a compound with the retention time and fragmentation pattern of authentic MeSA (data not shown).
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| Figure 1.
Identification of MeSA in leaf extracts of
TMV-inoculated tobacco. HPLC-elution profile (fluorescence at 360 nm
upon excitation at 292 nm) of authentic MeSA standard (A) and of leaf
extract of TMV-inoculated tobacco obtained 12 h after temperature
shift (B). Insets, UV-absorption spectra of the corresponding MeSA
peaks from A and B.
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RNA Isolation and Analysis
Total RNA was isolated using the guanidine thiocyanate and
phenol-chloroform method (Chomczynski and Sacchi, 1987). RNA (10 µg)
was separated by electrophoresis, transferred to nylon membrane (Zeta-Probe, Bio-Rad), and hybridized according to the manufacturer's protocols. PR-1 transcripts were detected with a corresponding 32P-labeled tobacco cDNA probe (a gift from R. Smith, Rutgers University, New Brunswick, NJ) prepared by specific
priming. As a control for loading and transfer efficiency, blots were
stripped and rehybridized with an 18S gene probe.
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RESULTS |
MeSA Production in TMV-Inoculated Tobacco Leaves
Approximately 48 h after tobacco leaves were inoculated with
TMV, HR lesions developed around the site of inoculation. At that time
the SA concentration increased 8-fold and continued to accumulate in
the inoculated leaf (Fig. 2A). MeSA
accumulation was also first detected at the time of HR appearance and
reached a maximum of 386 ng g1 fresh weight.
MeSA levels remained relatively steady from 48 to 96 h
postinoculation.
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| Figure 2.
Accumulation of MeSA and SA in tobacco plants
after TMV inoculation of a single leaf. Eight-week-old tobacco plants
were inoculated on the third leaf from the bottom. MeSA and SA
accumulation was analyzed in the inoculated leaf (A) and in the leaf
located directly above it (leaf 8) (B). Each point is the mean ± se of three replicates. The experiment was repeated twice
with similar results. FW, Fresh weight.
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SA content increases systemically after TMV inoculation (Malamy et al.,
1990), which at least partially explains its signaling role in SAR.
This systemic SA increase is particularly pronounced in the leaf
located directly above the inoculated leaf (Shulaev et al., 1995). To
investigate the possible systemic mobility of MeSA, we measured the
concentration of SA and MeSA in the leaf directly above the
TMV-inoculated leaf. As expected, the SA concentration in the
uninoculated leaf gradually increased, reaching a maximum of 318 ng
g1 fresh weight at 144 h postinoculation
(Fig. 2B). The uninoculated leaf also showed a significant but
transient increase in nonvolatile MeSA content. Immediately following
inoculation, MeSA concentration was below detection levels but reached
36 ng g1 fresh weight 96 h after the leaf
below was inoculated with TMV. MeSA concentration returned to below
detection levels at 144 h postinoculation. In most other
experiments the MeSA concentration in the systemic leaf did not show
such a rapid decline and remained elevated for a longer period of time
(data not shown).
Xanthi-nc tobacco plants inoculated with TMV do not develop HR lesions
and do not accumulate SA when they are placed at 32°C following TMV
inoculation (Kassanis, 1952; Malamy et al., 1992). Within 24 h of
being returned to 24°C, inoculated plants respond with massive tissue
necrosis and a dramatic accumulation of SA in the tissues to which the
virus has spread. We used the temperature-shift experiment to amplify
the biochemical changes associated with the HR response and MeSA
production. MeSA concentration increased significantly as early as
8 h after temperature shift, and reached a maximum of 5858 ng
g1 fresh weight after 12 h, just before
the tissue started to collapse (Fig. 3).
From 12 to 24 h the concentration of MeSA decreased in parallel
with tissue desiccation during massive necrosis. However, tissue SA
continued to accumulate throughout the 24-h period.
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| Figure 3.
Accumulation of MeSA and SA in TMV-inoculated
tobacco leaves after temperature shift. Tobacco plants were inoculated
with TMV on a fully expanded leaf and kept at 32°C for 96 h. At
time 0, the temperature was lowered to 24°C. Each point is the
mean ± se of three replicates. The experiment was
repeated twice with similar results. FW, Fresh weight.
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MeSA Production in P. syringae-Inoculated Tobacco Leaves
To establish whether the production of MeSA in tobacco is specific
for TMV, we measured MeSA accumulation in tobacco leaves
inoculated with the bacterial pathogen P. syringae pv phaseolicola, which induces strong HR
lesions around the site of infiltration 12 to 18 h
postinoculation. Total SA concentration in the vicinity of the lesions
reached 13,466 ng g1 fresh weight after 24 h (Fig. 4). Following the increase in
tissue SA, a large increase in MeSA concentration of 1,126 ng
g1 fresh weight was detectable 12 h
postinoculation. However, by 18 h, when necrotic HR lesions had
developed, the concentration of MeSA decreased to 504 ng
g1 fresh weight. In contrast, tissue
desiccation did not substantially reduce SA content.
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| Figure 4.
Accumulation of MeSA and SA in tobacco leaves
inoculated with P. syringae pv.
phaseolicola. One fully expanded leaf per plant was inoculated
with a strain that induces the HR. The infiltrated tissue was harvested
with a cork borer at the times indicated. Each point is the mean ± se of three replicates. The experiment was repeated with
similar results. FW, Fresh weight.
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Effect of MeSA on Transgenic NahG Tobacco Plants
In the previous experiments we observed that MeSA accumulation
paralleled or followed an increase in tissue SA (Figs. 2, 3, and 4). To
investigate whether SA accumulation is required for MeSA production, we
used transgenic tobacco plants expressing the salicylate hydroxylase
gene (nahG) from Pseudomonas putida, which
converts SA to catechol (Gaffney et al., 1993). NahG tobacco plants,
which are unable to accumulate SA, and wild-type plants were inoculated
with TMV and kept at 32°C for 4 d. In contrast to the wild type,
TMV inoculation of NahG tobacco plants did not cause SA or MeSA
accumulation (Table I). Even though the
leaf tissue of both wild-type and NahG plants became necrotic
approximately 12 h after the temperature shift, the amounts of SA
in NahG plants remained close to basal level, whereas MeSA was below
the limit of detection.
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Table I.
SA and MeSA concentration in TMV-inoculated
wild-type and NahG tobacco plants after temperature shift
Tobacco plants were inoculated with TMV and incubated at 32°C for
96 h. At time 0, the temperature was lowered to 24°C. Tissue was
sampled 0 and 12 h after temperature shift. Each value is the mean
of three replicates ± se. The experiment was repeated with similar results.
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Gaseous MeSA was shown to induce PR-1 transcripts and to
reduce the size of TMV-induced lesions in tobacco, presumably through conversion to SA (Shulaev et al., 1997). To further test this hypothesis, we investigated whether MeSA can induce PR-1
transcripts and TMV resistance at low SA levels by exposing NahG plants
for 6 d to 250 µg L1 of gaseous MeSA.
MeSA treatment of NahG plants resulted in only a 1.9-fold increase in
leaf SA levels compared with a 193-fold increase in the wild type
(Table II). In addition, the diameter of
the TMV lesions on NahG leaves exposed to MeSA was 2.1 times greater
than those on wild-type tobacco leaves, an indication of decreased TMV
resistance. Moreover, MeSA treatment did not induce PR-1
transcripts in NahG plants, in contrast to its dramatic effect in the
wild-type plants (Fig. 5).
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Table II.
Effect of MeSA on SA concentration and TMV-lesion
size in wild-type and NahG tobacco plants
Tobacco plants were enclosed in gas-tight glass chambers to which 250 µg L1 MeSA was supplied daily. After 6 d of
incubation the plants were sampled and analyzed for SA content. Each
value is the mean ± se of three replicates. Plants were
then inoculated with TMV, and the diameter of the TMV-induced lesions
was measured 72 h postinoculation. Data represent the mean
diameter ± se of at least 30 lesions per plant. The
experiment was repeated with similar results. The diameters of TMV
lesions in untreated wild-type and NahG plants were 1.328 ± 0.09 and 1.800 ± 0.11 mm, respectively.
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| Figure 5.
PR-1 gene expression in wild-type (WT) and
transgenic NahG tobacco plants exposed to gaseous MeSA. Five-week-old
tobacco plants were enclosed in gas-tight glass chambers containing 250 µg L1 MeSA. After the 6-d incubation period, fully
expanded leaves were removed from plants and analyzed for
PR-1 gene transcription. Total RNA was isolated and the
PR-1 transcript was detected by hybridization with a
cDNA probe (top). A probe for rRNA (18S) was used to demonstrate equal
RNA loading (bottom).
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DISCUSSION |
We previously demonstrated that gaseous MeSA produced in
TMV-inoculated tobacco leaves acts as an airborne defense signal involved in the communication between infected and healthy plants (Shulaev et al., 1997). The amounts of gaseous MeSA produced after the
infection were sufficient to induce expression of PR-1 proteins and TMV
resistance in nearby healthy plants.
SA glucoside, which is believed to be biologically inactive and
immobile, was previously identified as a major metabolite of SA in
TMV-infected tobacco tissue (Enyedi et al., 1992; Hennig et al., 1993;
Edwards, 1994; Lee et al., 1995). Since MeSA is a volatile liquid at
room temperature, earlier steps (such as drying) used in tissue
extraction prevented researchers from detecting MeSA as another
important metabolite of SA. Measurements of MeSA in the air around
virus-inoculated plants provided the first assessment, but
underestimated the total MeSA production in the inoculated tissue.
Thus, we have developed a method for measuring MeSA in plant tissues
(Fig. 1). Our results demonstrate that large amounts of nonvolatile
MeSA accumulate in the TMV-inoculated tissue around the time of its
appearance in the gas phase (Shulaev et al., 1997).
The amounts of endogenous MeSA in pathogen-inoculated tobacco leaves
represent about 10 to 20% of the total SA detected in nontemperature-shifted plants (Figs. 2 and 4), and approximately 30%
in temperature-shifted tobacco. Since a substantial portion of MeSA
escapes as a vapor, the total amounts of MeSA produced in inoculated
leaves should represent an even larger fraction of total SA production.
It is important to mention that the induction of MeSA production is not
specific to TMV. P. syringae pv phaseolicola, a
bean pathogen that is incompatible in tobacco (Lindgren at al., 1986),
also caused dramatic MeSA accumulation, indicating that MeSA production
is not pathogen specific.
It was shown earlier that MeSA is made from SA and acts by being
converted back to SA (Shulaev et al., 1997). Transgenic tobacco plants
expressing the salicylate hydroxylase gene nahG are unable to accumulate SA (Gaffney et al., 1993; Friedrich et al., 1995) and
provided a useful tool to test the biochemical mechanisms of SA
production and action. Since NahG plants inoculated with TMV were
unable to accumulate detectable amounts of MeSA (Table I), it is likely
that MeSA is synthesized from SA. In addition, the observation that
MeSA could not induce TMV resistance (measured as smaller HR lesions)
(Table II) and PR-1 gene expression in NahG plants (Fig. 5)
suggests that MeSA acts by being converted to SA. However, these
results can also be explained by MeSA serving as a substrate for the
salicylate hydroxylase. Another indication that SA is a precursor of
MeSA comes from Nicotiana glutinosa × debneyi, a tobacco hybrid that contains constitutively high levels of SA (Yalpani et al., 1993). The MeSA concentration in healthy leaves (n = 9) of this hybrid is 480 ± 248 ng g1 fresh weight, suggesting that
the high levels of SA give rise to high levels of MeSA.
In inoculated leaves MeSA accumulation always paralleled the
accumulation of endogenous SA (Figs. 2-4), consistent with the proposed synthesis of MeSA from SA. However, in the postinoculation period SA concentration increased continually and MeSA concentration decreased as HR lesions developed (Figs. 2-4). This observation agrees
with our previously published observation that the highest production
of gaseous MeSA is reached at the onset of tissue desiccation (Shulaev
et al., 1997). Here we suggest that the differences in the kinetics of
MeSA and SA accumulation can be at least partially explained by the
increased volatilization of MeSA facilitated by tissue desiccation
during HR-associated necrosis.
It has been previously suggested that SA may not be the only signal
involved in SAR (Rasmussen et al., 1991; Vernooij et al., 1994; Shulaev
et al., 1995), although it was shown to be required for the
establishment of SAR (Gaffney et al., 1993). The observation that MeSA
accumulation was detected in the healthy tobacco leaves located above
the inoculated leaf (Fig. 2B) suggests that MeSA may function as a
translocatable form of SA, into which it can be readily converted in
the target tissue. Therefore, like the SA precursor benzoic acid
(Shulaev et al., 1995), MeSA may play at least an accessory role in SAR
signaling. We have detected MeSA in phloem exudates of TMV-inoculated
leaves after a temperature shift, using a previously established
exudate-collection technique (Yalpani et al., 1991; data not shown).
Therefore, phloem translocation of nongaseous MeSA may complement its
aerial spread and provide an early warning defense system during SAR.
However, relatively low levels of MeSA compared with SA argue for a
supplemental rather than an exclusive role for MeSA in SAR signaling.
On the other hand, MeSA vapor may be the sole signal inducing defense
responses in an adjacent plant. Further elucidation of MeSA
biosynthesis and the cloning of MeSA biosynthetic enzymes may
enable us to manipulate MeSA and SA levels in plants, thereby
enhancing inter- and intra-plant resistance to pathogens.
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FOOTNOTES |
1
This research was funded by grant no.
96-35304-3874 from the U.S. Department of Agriculture. Additional
support was provided by the New Jersey Agricultural Experiment Station
and the New Jersey Commission for Science and Technology.
*
Corresponding author; e-mail raskin{at}aesop.rutgers.edu; fax
1-908-932-6535.
Received July 30, 1997;
accepted October 13, 1997.
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ABBREVIATIONS |
Abbreviations:
HR, hypersensitive response.
MeSA, methyl
salicylate.
PR protein, pathogenesis-related protein.
SA, salicylic
acid.
SAR, systemic acquired resistance.
TMV, tobacco mosaic
virus.
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Top
Abstract
Introduction