Download Comparison of Local and Systemic Induction of Acquired Disease

Plant Cell Physiol. 40(4): 388-395 (1999)
JSPP © 1999
Comparison of Local and Systemic Induction of Acquired Disease
Resistance in Cucumber Plants Treated with Benzothiadiazoles or Salicylic
Acid
Yoshihiro Narusaka1, Mari Narusaka2, Takeshi Horio and Hideo Ishii
National Institute of Agro-Environmental Sciences (NIAES), 3-1-1 Kannondai, Tsukuba, Ibaraki, 305-8604 Japan
The accumulation of chitinase and its involvement in
systemic acquired disease resistance was analyzed using
acibenzolar-S-methyl and salicylic acid (SA). Resistance
against scab (pathogen: Cladosporium cucumerinum) and
the accumulation of chitinase were rapidly induced in cucumber plants after treatment with acibenzolar-S-methyl.
In contrast, SA protected the plants from C. cucumerinum and the accumulation of chitinase was induced
only on the treated leaves. The accumulation of chitinase in
response to inoculation with the pathogen was induced
more rapidly in cucumber plants previously treated with
acibenzolar-S-methyl than in plants pretreated with SA or
water. Thus, it appears that a prospective signal(s), that
induces systemic resistance, can be transferred from leaves
treated with acibenzolar-S-methyl to the untreated upper
and lower leaves where systemic resistance is elicited. In
contrast, exogenously applied SA is not likely to function
as a mobile, systemic resistance-inducing signal, because
SA only induces localized acquired resistance.
noninfected leaves (Uknes et al. 1992). This inducible
defense mechanism, which is known as systemic acquired
resistance (SAR) plays a central role in disease resistance
(Delaney et al. 1994, Ross 1961).
Acquired resistance is also induced by compounds
such as benzothiadiazoles, e.g., acibenzolar-S-methyl
(benzo[l,2,3]thiadiazole-7-carbothioic acid S-methyl ester), which is also referred to as BTH, 3-allyloxy-l,2benzisothiazole-1,1-dioxide (probenazole), 2,6-dichloroisonicotinic acid (INA), salicylic acid (SA), or certain other
benzoic acid derivatives, as well as inoculation with
necrotizing pathogens (Schurter et al. 1987, Watanabe et
al. 1977, Metraux et al. 1991, Uknes et al. 1992, Ward et
al. 1991). The SAR phenomenon suggests that a signal
originates at the site of infection or treatment with various compounds and then moves throughout the plant. It
has been shown that such a signal is produced in an infected or chemical-treated leaf and that detachment of
this leaf before the development of the hypersensitive response blocks the induction of SAR (Dean and Kuc 1986a,
b). Grafting and stem-girdling experiments with cucumber and tobacco have suggested that the SAR signal moves
in the phloem (Jenns and Kuc 1979, Guedes et al. 1980,
Tuzun and Kuc 1985). However, the nature of the signal is
as of unknown, although it has been shown that a mobile
signal leads to SA accumulation and SAR (Vernooij et al.
1994).
Key words: Acibenzolar-S-methyl — BTH — Chitinase —
Salicylic acid — Systemic acquired resistance.
The induction of local and systemic disease resistance by prior inoculation with necrotizing pathogens was
first reported by Chester (1933). Plants challenged by microbial pathogens are able to induce the expression of a set
of defense-related genes, such as the genes encoding pathogenesis-related proteins (PR proteins) (Uknes et al. 1992).
These genes are expressed locally as well as in distant,
The cucumber plant provides a good model system for
studying general disease resistance and induced disease resistance (Kuc 1982). Local and systemic increases in chitinase and/or peroxidase activity have been observed in
response to inoculation with necrotizing pathogens or to
treatment with SA, INA, and acibenzolar-S-methyl (Boiler
and Metraux 1988, Lawton et al. 1994, Dalisay and Kuc
1995, Ishii et al. in press, Narusaka et al. in press). In the
cucumber, SAR in response to inducers is correlated with
the systemic accumulation of extracellular peroxidase, /?1,3-glucanase, and chitinase (Hammerschmidt et al. 1982,
Boiler and Metraux 1988, Ji and Kuc 1995). The relationship between these enzymes and induced disease resistance in the cucumber has therefore been extensively investigated. Despite these investigations, however, the mode of
action of the disease-resistance inducers remains unclear.
We describe here the function of resistance inducers in
the cucumber. A time-course study has indicated that the
Abbreviations: CaMV, cauliflower mosaic virus; DIG, digoxigenin; INA, 2,6-dichloroisonicotinic acid; LAR, localized
acquired resistance; MDS, mean disease severity; PBS, phosphate-buffered saline; PR proteins, pathogenesis-related proteins; PVDF, polyvinylidene difluoride; SA, salicylic acid; SAR,
systemic acquired resistance; TNV, tobacco necrosis virus.
' Corresponding author present address: Biological Resources
Division, Japan International Research Center for Agricultural
Sciences (JIRCAS), 1-2 Ohwashi, Tsukuba, Ibaraki, 305-8686
Japan.
2
Present address: Laboratory of Plant Molecular Biology, The
Institute of Physical and Chemical Research (RIKEN), Tsukuba
Life Science Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074
Japan.
388
Acquired resistance by acibenzolar-S-methyl and SA
enhanced expression of the chitinase gene coincides with
the level of induced systemic disease resistance. Our results
show that acibenzolar-S-methyl activates the SAR signal
transduction pathway in cucumber plants, resulting in the
rapid induction of disease resistance in whole plants. In
contrast, SA induces resistance only in the treated leaf.
Materials and Methods
Growth and maintenance of plants and fungi—Cultures of
Cladosporium cucumerinum were maintained at 25°C on potato
dextrose agar (Difco). Cucumber plants (Cucumis sativus L.) cv.
Shin-Suyo Tsukemidori were grown at 25 °C in a growth chamber with a 14 h photoperiod at a photon flux density of 60-80/iE
m-2s-'.
Chemical application and inoculation of the pathogen—
The application of acibenzolar-S-methyl or SA at concentrations higher than 0.5 or 5 mM, respectively, caused phytotoxicity on the treated leaves, and the leaves subsequently died within
1 to 3 d (data not shown). In this study, therefore, the acibenzolar-S-methyl and SA were applied at concentrations of 0.5 mM
and 5 mM, respectively.
Distilled water, and 5 mM SA or 0.5 mM acibenzolar-Smethyl (50.%-water-dispersible granules, a gift from Novartis
Crop Protection AG) were applied by dipping for 5 s to the 1st
true leaf of cucumber plants (14 d postgermination), with the 1st
true leaf having fully expanded and the leaf above (2nd leaf)
having expanded from one third to one half, its full size. Twenty
drops (20 fA per drop) of inoculum (5 x 105 conidia ml" 1 water) of
C. cucumerinum were separately applied to the 1st true leaf and
incubated at 20°C in a dew chamber for 24 h. The treated plants
were grown under growth-chamber conditions until sampling of
the 1st leaf at various times or until challenged inoculation with a
conidial suspension of C. cucumerinum. The pathogen (1 x 105
conidia ml" 1 water containing 0.02% Tween 20) was applied as a
fine mist to the entire plant at approximately 2 to 3 ml per plant 7
d after treatment of the 1st leaf. Following the challenged inoculation, the plants were maintained at 20°C in a dew chamber for
24 h and then kept in a growth chamber. Seven days after inoculation, the plants were visually assessed for symptom development. To evaluate the control efficacy against disease, each leaf
was scored using the following scale according to disease development: 0, no visible lesion; 1, caused < 10 lesions; 2, caused > 10
lesions and/or deformity; 3, caused some lesions on the petiole
and < 10 lesions; 4, caused some lesions on the petiole and > 10
lesions; 5, petiole snapped off; 6, leaf died. Disease scores were
converted to mean disease severity (MDS) using the following
formula:
MDS = [(6A + 5B + 4C + 3D + 2E + F)/6G] x 100
where A, B, C, D, E, and F are the number of leaves corresponding to the scores 6, 5, 4, 3, 2, and 1, respectively. G is the
total number of leaves assessed. This experiment was performed
using 10-15 plants per treatment.
Expression of cucumber chitinase in Escherichia coli—The
genomic DNA encoding the mature chitinase of the cucumber
(CHI2, GenBank accession number M84214, Lawton et al. 1994)
was amplified by PCR using Pfu DNA polymerase and primers
with an in-frame Ndel site (sense), an in-frame stop codon, and a
BamHl site (antisense). The resulting fragment was purified by
phenol/chloroform extraction and ethanol precipitation, digested
389
overnight with Ndel/BamHl, and purified by agarose gel electrophoresis. The band of interest was excised from the gel and purified using a GENECLEAN II (Stratagene), it was then ligated
into the Ndel/BamHl site of the expression vector pET16b
(Novagen). The'5' splice site of the plasmid was sequenced to
confirm an in-frame insertion. The plasmid was then transformed into the host strain BL21DE3 of E. coli.
This strain was grown on LB medium with 200yUgmr' ampicillin at 37°C with vigorous aeration (220 rpm on a shaker) until
the absorbance at 600 nm reached 0.8, at this point expression was
induced by the addition of isopropyl-/?-D-thiogalactopyranoside
(final concentration 1 mM), and the culture was allowed to continue for an additional 3 h. The cells were pelleted by centrifugation and stored at — 80°C until use. The frozen cell pellet was
resuspended in phosphate-buffered saline (PBS), pH 7.4, — 2%
Triton X-100, and broken by an ultrasonic generator. The suspension was centrifuged at 15,000 rpm (18,000 xg) for 20min,
and the pellet (containing recombinant protein) was then collected. The pellet was washed with PBS-2% Triton X-100 three times.
Subsequently, the pellet was resuspended in washing buffer (50
mM Tris-HCl, pH 8.0, 10 mM EDTA, 1 M urea, 2% Triton X100) and then incubated at room temperature for 20 min. The
suspension was centrifuged at 15,000 rpm (18,000 xg) for 20 min.
This washing process was repeated three times. The pellet containing recombinant protein was dissolved in 50 mM Tris-HCl,
pH 8.0, plus 6M urea. This solution was centrifuged at 15,000
rpm (18,000 xg) for 20 min, and the supernatant was dialyzed at
4°C as follows: into 50 mM Tris-HCl, pH 8.0, plus 4 M urea for
1 h, 50 mM Tris-HCl, pH 8.0, plus 2 M urea for 1 h, 50 mM
Tris-HCl, pH 8.0, plus 100 mM NaCl for 1 h, and then 1/10 PBS
for 4 h. The last step was performed three times. The solution was
subsequently concentrated by freeze-drying and dissolved into the
PBS buffer. The recombinant protein was analyzed by SDS-PAGE
according to Laemmli's method (Laemmli 1970) to examine the
purity of the protein. The protein band with 23-25 kDa was considered to be the recombinant cucumber chitinase. To verify that
the recombinant protein was a chitinase, amino acid sequencing
was performed. Following electrophoresis, the protein was electroblotted onto an Immobilon-P polyvinylidene difluoride (PVDF)
membrane (Millipore) by the method of Matsudaira (1987) and
was then stained with Coomassie Brilliant Blue R-250 (Bio-Rad).
After the membrane was air-dried, the protein band was excised
and then sequenced using a Protein Sequencer, model LF3400DT
(BECKMAN).
Antiserum was raised against semi-purified chitinase by
Sawady Technology Co. (Tokyo, Japan).
Chitinase extraction—The extraction of chitinase from cucumber leaves was performed according to the method of Dalisay
and Kuc (1995).
Western blotting—The cucumber proteins (20 ^g) were separated by SDS-PAGE according to Laemmli's method (Laemmli
1970) and then electrophoretically transferred onto an Immobilon-P PVDF membrane (Millipore) according to the method
of Towbin et al. (1979). The chitinase on the membrane was detected with antiserum raised against chitinase, using an ECL
western blotting detection system (Amersham).
RNA extraction—The extraction of total RNA from the 1st
and 2nd leaves was performed with the TRIzol reagent (GibcoBRL) according to the manufacturer's protocol. Total RNA was
purified by lithium chloride precipitation, isopropyl alcohol precipitation, and then ethanol precipitation.
Northern blotting assay—The northern blotting was performed according to the manufacturer's protocol (Boehringer
Acquired resistance by acibenzolar-S-methyl and SA
390
Mannheim). Total RNA was hybridized with digoxigenin (DIG)labeled RNA probes and developed by immunochemiluminescence according to the method described previously (Penninckx et
al. 1996). DIG-labeled probes were made by run-off transcription, using the DIG RNA labeling kit of Boehringer Mannheim.
The probe for the chitinase gene was synthesized using T7 RNA
polymerase and the £coRI-linearized plasmid pCHI. The plasmid
pCHI was constructed by cloning the cucumber chitinase gene
(CHI2, GenBank accession number M84214, Lawton et al. 1994),
which was obtained by PCR with chitinase-specific primers, into
the EcoRl and Hindlll sites of plasmid pSPT19 (Boehringer
Mannheim).
1st leaf
2nd leaf
1
Acibenzolar-S-methyl
SA
Inoculation with
C.cucurmrinum
Results
Antisera to recombinant chitinase—the cucumber chitinase gene was isolated from the genomic DNA by PCR.
The gene isolated was the cucumber class III chitinase
gene (CHI2, GenBank accession number M84214, Lawton
et al. 1994), the expression of which is induced by the application of SA or INA (Lawton et al. 1994).
The N-terminal amino acids of the purified recombinant chitinase were sequenced. The N-terminal sequence
obtained was: MGHHHHHHHHHHSSGHIEGRHMAGIAIYWGQNGNE. This sequence was identical to the His-
Fig. 1 Accumulation of chitinase in response to treatment with
acibenzolar-S-methyl and SA as well as pre-inoculation with
C.cucumerinum. A suspension of each inducer was applied by
dipping the 1st true leaf for 5 s or by applying 20 drops (20//I per
drop) of the inoculum to the 1st leaf. The 1st and 2nd leaves were
collected 1, 3, 5, and 7 d after treatment. Total proteins were then
extracted from the cucumber leaves and separated by SDS-PAGE.
The chitinase was detected by anti-chitinase antibodies.
tag fusion cucumber class HI chitinase (Novagen Manufacturer's protocol, Lawton et al. 1994). Antiserum against
Table 1 Induction of systemic resistance against C. cucumerinum in cucumber plants in response to acibenzolar-Smethyl, SA, and pre-inoculation with the pathogen"
MDSC
Time of detaching
1st leaf after
treatment (day)
2nd leaf
3rd leaf
4th leaf
Water
0
72.9
72.9
79.2
0.5 mM Acibenzolar-S-methyl
0
1
70.8
333*rf
3
33.3*
33.3*
33.3*
68.8
31.3*
33.3*
29.2*
31.3*
75.0
33.3*
33.3*
29.2*
20.8*
64.6
70.8
62.5
70.8
70.8
68.8
70.8
70.8
70.8
72.9
72.9
70.8
83.3
83.3
79.2
70.8
66.7
70.8
50.0
41.7
70.8
79.2
75.0
50.0
35.4*
72.9
70.8
66.7
45.8
33.3*
Treatment *
.5
7
5mM SA
Q
l
3
3
7
Pre-inoculation with C. cucumerinum
0
1
3
5
7
" Four plants were used for each treatment. The experiment was conducted three times, and the reported values are the means from these
experiments.
* A solution of acibenzolar-S-methyl or SA was applied to the 1st leaf of the cucumber either by dipping for 5 s or by applying 20 drops
(20^1 per drop) of the inoculum to the 1st leaf. Seven days after induction, the entire plants were challenged with C. cucumerinum.
c
MDS: mean disease severity.
d
Means followed by an asterisk within a column are significantly different (P<0.05) from values obtained with water treatment according to the Dunn's multiple range test.
Acquired resistance by acibenzolar-S-methyl and SA
391
Table 2 Effect of a foliar spray of SA solution on local protection against C. cucumerinum in cucumber plants"
MDSC
Treatment *
1st leaf
2nd leaf
3rd leaf
4th leaf
Water
70.8
75.0
68.8
62.5
5mM SA
35.7*
33.3*
42.9*
35.7*
" Four plants were used for each treatment. The experiment was conducted three times, and the
reported values are the means from these experiments.
4
A solution of SA or distilled water was sprayed on the entire plants. After 7 d, treated plants were
challenge-inoculated with C.cucumerinum.
c
MDS: mean disease severity.
d
Means followed by an asterisk within a column are significantly different (.P<0.05) from values
obtained with water treatment according to the Dunn's multiple range test.
the recombinant chitinase that was diluted 25,000-fold
could detect the 23-25 kDa protein band in the proteins
extracted from the cucumber leaves treated with SA by
western blotting (data not shown). The protein band was
identical to the size of the cucumber class III chitinase
(Lawton et al. 1994).
Western blotting assay—Figure 1 illustrates the rapid
accumulation of a chitinase in the 2nd leaves within 3 d
after acibenzolar-S-methyl treatment. This accumulation
in the 2nd leaves was also detected at 5 d after preinoculation with C. cucumerinum (Fig. 1). In contrast, the accumulation of chitinase was observed only in the treated
leaves when the 1st leaves were treated with SA, indicating
that SA induces the accumulation of chitinase only locally. The level of disease suppression occurring in response to
each treatment is shown in Table 1.
Chemical application and disease suppression—The
acibenzolar-S-methyl treatment induced the systemic protection of cucumber plants from C. cucumerinum (Table
1). However, this systemic protective effect was not observed in response to the treatment with SA (Table 1).
To determine the timing for the induction of SAR, the
1st leaves, which had previously been treated with chemicals or C. cucumerinum, were detached at various times
Water
1st
Water
1st
SA
2nd 3rd 4th 1st 2nd 3rd 4th
after treatment and subsequently challenge-inoculated with
C. cucumerinum. The results indicated that for the induction of SAR, the 1st leaves must be attached to the plants
for 5 to 7 d after the pre-inoculation with C. cucumerinum (Table 1). In contrast, the 1st leaf needs to be attached for only 1 d or less after the acibenzolar-S-methyl
treatment. On cucumber plants treated with SA on the 1st
leaf, however, systemic protection was not observed in the
upper leaves even 7 d after the treatment (Table 1).
To analyze the acquired resistance induced by exogenously applied SA, the entire cucumber plants, on which
the 1st and 2nd leaves had expanded and the 3rd leaves had
just emerged from the bud and started to open while the
4th leaves were still unfolded, were sprayed with a 5 mM
SA solution (approx. 2 to 3 ml per plant). After 7 d of
treatment, the cucumber plants were inoculated with
C. cucumerinum. SA significantly induced a (P<0.05) local protective effect on the treated leaves and suppressed
disease development (Table 2). After 7 d of SA treatment, the accumulation of chitinase was detected in the 1st,
2nd, 3rd, and 4th leaves (Fig. 2).
To analyze the systemic acquired resistance induced by
Acibenzolar
-S-methyl
2nd 3rd 4th 1st 2nd 3rd 4th
leaf
position
leaf
position
Fig. 2 Accumulation of chitinase in response to SA treatment.
A solution of 5 mM SA or distilled water was applied as a fine mist
to the entire plant at approximately 2 to 3 ml per plant. The 1st,
2nd, 3rd, and 4th leaves were collected 7 d after treatment. Total
proteins were then extracted from the cucumber leaves and separated by SDS-PAGE. The chitinase was detected by anti-chitinase antibodies.
Fig. 3 Accumulation of chitinase in response to applying acibenzolar-S-methyl to the 3rd leaf of the cucumber plant. Twenty
drops (20 /A per drop) of a suspension of acibenzolar-S-methyl or
distilled water was applied on the upper surface of the 3rd leaf,
which has expanded to one third of its full size. The 1st, 2nd, 3rd,
and 4th leaves were collected 7 d after treatment. Total proteins
were then extracted from the cucumber leaves and separated by
SDS-PAGE. The chitinase was detected by anti-chitinase antibodies.
Acquired resistance by acibenzolar-S-methyl and SA
Table 3 Effect of acibenzolar-S-methyl application to the 3rd leaf of cucumber plants on
disease development of C. cucumerinum"
Treatment *
MDS'
2nd leaf
3rd leaf
1st leaf
4th leaf
Water
86.7
76.7
70.0
73.3
0.5 mM Acibenzolar-S-methyl
33.3 *d
29.2*
33.3*
29.2*
" Four plants were used for each treatment. The experiment was conducted three times, and the
reported values are the means from these experiments.
* Twenty drops (20 /A per drop) of acibenzolar-S-methyl solution or distilled water were applied to
the 3rd leaf. After 7 d, entire plants were challenge-inoculated with C. cucumerinum.
c
MDS: mean disease severity.
d
Means followed by an asterisk within a column are significantly different (P<0.05) from values
obtained with water treatment according to the Dunn's multiple range test.
acibenzolar-S-methyl, the 3rd leaves on cucumber plants
for which the 1st and 2nd leaves had expanded and the 3rd
leaves had expanded to one third, their full size were
treated with 20 drops (20 fA per drop) of 0.5 mM acibenzolar-S-methyl. After 7 d of treatment, the accumulation
of chitinase was detected in the 1st, 2nd, 3rd, and 4th
leaves (Fig. 3). At this time, the entire plants were inoculated with C. cucumerinum, and systemic protection against
C. cucumerinum was observed 7 d after the inoculation
(Table 3).
The first leaves on the cucumber plants were treated
with acibenzolar-S-methyl, SA, or water (dipping for 5 s).
After 1 h of treatment, the entire plants were inoculated
with C. cucumerinum and the subsequent accumulation of
the chitinase was examined at various times. The accumulation of chitinase was detected in the untreated 2nd leaves
more rapidly when plants were treated with acibenzolarS-methyl than with either SA or water (Fig.4A). In addition, the accumulation of chitinase in the 2nd leaves was
more rapidly induced in the plants inoculated with the
pathogen after acibenzolar-S-methyl treatment than in the
controls, i.e., the plants not inoculated with the patho-
Acibenzolar-S-methyl
Water
12
24
36
48
0
12
24
36
48
gen (Fig.4B).
Timing of mRNA accumulation—A chitinase mRNA
accumulated in the 2nd leaves 3 d after the treatment of the
1st leaves with acibenzolar-S-methyl (Fig. 5). In contrast,
the accumulation in the 2nd leaves was first detected 5 d
after the preinoculation with C. cucumerinum. For cucumber plants treated with SA in the 1st leaf, however, the
accumulation of the chitinase mRNA was observed only in
the 1st leaves even at 7 d after treatment. The disease suppression in response to each treatment is shown in Table 1.
The data show that induced resistance correlates with the
accumulation of a chitinase mRNA.
Discussion
The induction of chitinase activity occurs in different
plant species in response to infection, treatment with fungal cell wall preparations, or the stress-related hormone
ethylene, as well as to treatment with simple organic and
inorganic compounds. The relationship between chitinase
and disease resistance in the cucumber has been extensively investigated. It has been reported that the systemic pro-
B
SA
12
24
36
48 h
0
24
48
72
0
24
48
72
h
Fig. 4 Induction of chitinase in cucumber plants in response to inoculation with C. cucumerinum after treatment with acibenzolarS-methyl and SA (A) and the induction of chitinase in cucumber plants in response to water-treatment (a) or inoculation with
C. cucumerinum (b) after treatment with acibenzolar-S-methyl (B). The first leaves on cucumber plants were treated with acibenzolar-S-methyl, SA, or distilled water. One hour after treatment, the entire plants were inoculated with C. cucumerinum or were treated
with water, and the subsequent accumulation of the chitinase in 2nd leaves was detected at several time points after the inoculation.
Total proteins were then extracted from the cucumber leaves and separated by SDS-PAGE. The chitinase was detected by anti-chitinase antibodies.
Acquired resistance by acibenzolar-S-methyl and SA
393
sufficient to explain its accumulation in the upper leaves.
The export of SA from leaves inoculated with various
necrotizing pathogens and its appearance in the phloem
Acibenzolar-S-mettiyl
have been confirmed in tobacco and the cucumber (Metraux et al. 1990, Rasmussen et al. 1991, Yalpani et al.
1991). The physicochemical properties of SA make it well
SA
suited for long-distance phloem transport (Yalpani et al.
1991). Molders et al. (1996) have previously reported that
Inoculation with
[14C]SA is transported from cotyledons to 1st leaves after
C. cucumerinum
tobacco necrosis virus (TNV) inoculation in cucumber
plants. In addition, a large amount of SA synthesized in
Fig. 5 The accumulation of mRNA in cucumber plants follow- the TNV-inoculated leaf is translocated to the upper unining treatment with acibenzolar-S-methyl or SA, or following pre- oculated leaves in tobacco plants (Shulaev et al. 1995).
inoculation with C. cucumerinum. Cucumber tissues were harvested at the indicated time following treatment, and total RNA Moreover, exogenously applied SA leads to typical SAR
was extracted from these tissues. The northern blot was hybrid- responses such as increased resistance to viral infection
ized with DIG-labeled antisense RNA probes for chitinase.
(reviewed by Malamy and Klessig 1992, van Loon and
Antoniw 1982, White 1979, Ye et al. 1989). Direct eviduction of chitinase corresponds with the observed in- dence for the role of SA as a signalling molecule in the
duced resistance to cucumber anthracnose, but only a development of SAR has arisen from experiments with
transgenic tobacco plants that overexpress the salicylate
slight correlation with the activity of chitinase (Irving and
Kuc 1990, Dalisay and Kuc 1995). Metraux et al. (1988) and hydroxylase gene (nahG) from Pseudomonas putida
Smith-Becker et al. (1998) have reported that chitinase (Gaffney et al. 1993). This enzyme catalyzes the conversion
provides a useful molecular and biochemical marker for of SA to catechol, which has no SAR-inducing activity,
the induction of systemic acquired resistance in the cu- and thus reduces the concentration of active SA in plants.
cumber. More recently, direct evidence of the potential role Transgenic NahG plants do not accumulate SA in reof PR proteins containing chitinase in plant defense has sponse to pathogen infection, and they do not show an
been obtained by functional experiments demonstrating SAR response (Delaney et al. 1994). Nevertheless, recent
evidence suggests that SA may not be the induction signal
that the overexpression of pathogenesis-related genes can
lead to an enhanced resistance to certain pathogens. For for primary long-distance SAR and that the production of
example, a rice chitinase cDNA driven by the cauliflower this systemic signal is not dependent on SA accumulation
mosaic virus (CaMV) 35S promoter has been introduced (Vernooij et al. 1994). Instead, SA may be required in uninto the cucumber (Tabei et al. 1998), with the resulting infected tissues for transduction of the translocated signal
transgenic cucumber showing high resistance against into gene expression and resistance (Vernooij et al. 1994).
This study has demonstrated that acibenzolar-SBotrytis cinerea (Tabei et al. 1998). In the present study,
the accumulation of chitinase was detected in cucumber methyl induces the expression of the chitinase gene and
plants under disease-suppression conditions after treat- disease resistance both locally and systemically (Fig. 5,
ment with acibenzolar-S-methyl (Table 1, Fig. 1). Then, in Table 1,3). Furthermore, it has also been shown that exthe following analysis, chitinase was used as a marker for ogenously applied SA functions as an inducer of chitinase
and disease resistance only locally (Fig. 5, Table 1). These
SAR in the cucumber.
results suggest two hypotheses: (1) SA is transported
It has previously been reported that SAR is induced in
throughout the tissues and accumulates in these tissues
response to treatment with acibenzolar-S-methyl (Friend- systemically. In this study, however, exogenously applied
rich et al. 1996) and SA (Rasmussen et al. 1995) in plants. SA did not induce SAR. Therefore, SA does not appear to
It is known that treating 1st leaves with these compounds be a signalling molecule for SAR. (2) SA is not transinduces a systemic protection on the 2nd, 3rd, and 4th ported from treated leaves to untreated leaves. If SA is not
leaves without causing any stress signs or damage. SAR transported systemically, SA is not a signalling molecule
implies the existence of a signal molecule produced in in- for SAR. Therefore, exogenously applied SA stimulates
fected tissue that moves throughout the plant to activate signalling pathways for localized acquired resistance (LAR)
resistance (Ross 1966). Because the concentration of SA in treated leaves but does not induce those for SAR. In
rises dramatically after pathogen infection, it has been
contrast, acibenzolar-S-methyl rapidly induces SAR. More
proposed that SA may act as a signal molecule in the in- importantly, in cucumber plants treated with SAR-inducduction of SAR (Enyedi et al. 1992, Malamy et al. 1990, ing chemicals, a prospective mobile signal(s) for SAR,
Metraux et al. 1990, Rasmussen et al. 1991, Uknes et al. which might contain SAR-inducing chemicals and/or their
1993, Yalpani et al. 1991). To induce SAR in whole plants, metabolites, is systemically transferred to untreated leaves
SA must be exported from the treated leaf in amounts
1st leaf
1
0
1
3
If
2nd leaf
5
7 " 0
1
3
5
7 'day
Acquired resistance by acibenzolar-S-methyl and SA
394
within several hours to several days after treatment. Subsequently, the prospective mobile signal(s) induces disease
resistance systemically.
Although SAR is in general thought to be induced
several days post infection with necrotizing pathogens
(Dempsey and Klessig 1995), acibenzolar-S-methyl induced systemic disease resistance appeared within 24 h after the treatment (Table 1). In cucumber plants previously treated with acibenzolar-S-methyl, chitinase was rapidly
induced after attack of the pathogen (Fig.4A). The accumulation of chitinase occurred 12 h more rapidly than in
the control (Fig. 4A), and this may be a critical step in the
containment of the pathogen. This period immediately after the attack of the pathogen might be important in
determining whether protection will be successful in the
induced plants. During the 24 h following challenge inoculation with Colletotrichum lagenarium, electronopaque
epidermal walls develop in protected leaves (Xuei et al.
1988), and the rate of lignification also increases (Dean and
Kuc 1987, Hammerschmidt and Kuc 1982).
Interestingly, the induction of chitinase was detected
the 1st and 2nd leaves when the 3rd leaves were treated with
acibenzolar-S-methyl, i.e., a mobile signal was transported from treated leaves not only to upper but also to
lower leaves (Fig. 3). Although the action mechanism(s)
and SAR-signal(s) of acibenzolar-S-methyl and SA in restricting disease development are unclear as of yet, the systemic induction of chitinase suggests that these compounds
rapidly trigger the plant's immune system to induce and
activate these mechanisms. Further study should be undertaken to clearly determine the induction signal for acquired resistance in plant defense as well as the mechanism for the induction of systemic resistance. In addition, it
is necessary to characterize the LAR and SAR signal and
the signaling pathway(s).
This work was partly funded by Japan Science and Technology Corporation.
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(Received October 22, 1998; Accepted January 30, 1999)