Salicylate activity. 3. Structure relationship to systemic acquired resistance

Article abstract of DOI:10.1021/jf051383t

Salicylic acid (2-hydroxybenzoic acid; SA) is a primary signal inducing plant defenses against pathogens. This plant disease resistance, known as systemic acquired resistance (SAR), is an attractive target for the development of new plant protection agents. SAR induction is a multistep process that includes accumulation of pathogenesis-related (PR) proteins. The structure-activity profile of salicylates and related compounds has been evaluated using an inducible PR protein (PR-1a) and plant resistance to tobacco mosaic virus (TMV) as markers. Among the 47 selected monosubstituted and multiple-substituted salicylate derivatives tested, all 8 derivatives that induced more PR-1a protein than SA were fluorinated or chlorinated in the 3- and/or 5-position (3,5-difluorosalicylate > 3-chlorosalicylate > 5-chlorosalicylate > 3,5-dichlorosalicylate > 3-chloro-5-fluorosalicylate > 3-fluorosalicylate > 3-fluoro-5-chlorosalicylate > 3,5-dichloro-6-hydroxysalicylate > SA). In general, substitutions for or on the 2-hydroxyl group or at the 4-position of the ring reduced or eliminated PR-1a protein induction. In contrast, substitutions in positions ortho (3-position) or para (5-position) to the hydroxyl group with electron-withdrawing groups other than chlorine or fluorine decreased induction, and electron-donating groups in these positions also had a deleterious effect on PR-1a induction. PR-1a protein accumulation and reduction in TMV lesion diameter exhibited a log-linear relationship. The seven salicylate derivatives that were the most active TMV resistance inducers were all halogenated in the 3- and/or 5-position (3-chlorosalicylate > 3,5-difluorosalicylate > 3,5-dichloro-6-hydroxysalicylate > 3,5,6-trichlorosalicylate > 5-chlorosalicylate > 5-fluorosalicylate > 3,5-dichlorosalicylate > 4-fluorosalicylate > 3-fluorosalicylate > 3-chloro-5-fluorosalicylate > 4-chlorosalicylate > SA). The correlation between PR-1a protein induction and resistance to TMV confirms the value of using PR-1a induction as a screening tool for developing new plant disease control agents.

Full text of DOI:10.1021/jf051383t

J. Agric. Food Chem. 2005, 53, 9775−9780 9775
Salicylate Activity. 3. Structure Relationship to Systemic
Acquired Resistance
F. PAUL SILVERMAN,* PETER D. PETRACEK, DANIEL F. HEIMAN,
CHRISTINA M. FLEDDERMAN, AND PREM WARRIOR
Valent BioSciences Corporation, 6131 Oakwood Road, Long Grove, Illinois 60047
Salicylic acid (2-hydroxybenzoic acid; SA) is a primary signal inducing plant defenses against
pathogens. This plant disease resistance, known as systemic acquired resistance (SAR), is an
attractive target for the development of new plant protection agents. SAR induction is a multistep
process that includes accumulation of pathogenesis-related (PR) proteins. The structure-activity profile
of salicylates and related compounds has been evaluated using an inducible PR protein (PR-1a)
and plant resistance to tobacco mosaic virus (TMV) as markers. Among the 47 selected monosub-
stituted and multiple-substituted salicylate derivatives tested, all 8 derivatives that induced more PR-
1
>
3
a protein than SA were fluorinated or chlorinated in the 3- and/or 5-position (3,5-difluorosalicylate
3-chlorosalicylate > 5-chlorosalicylate > 3,5-dichlorosalicylate > 3-chloro-5-fluorosalicylate >
-fluorosalicylate > 3-fluoro-5-chlorosalicylate > 3,5-dichloro-6-hydroxysalicylate > SA). In general,
substitutions for or on the 2-hydroxyl group or at the 4-position of the ring reduced or eliminated
PR-1a protein induction. In contrast, substitutions in positions ortho (3-position) or para (5-position)
to the hydroxyl group with electron-withdrawing groups other than chlorine or fluorine decreased
induction, and electron-donating groups in these positions also had a deleterious effect on PR-1a
induction. PR-1a protein accumulation and reduction in TMV lesion diameter exhibited a log-linear
relationship. The seven salicylate derivatives that were the most active TMV resistance inducers
were all halogenated in the 3- and/or 5-position (3-chlorosalicylate > 3,5-difluorosalicylate > 3,5-
dichloro-6-hydroxysalicylate > 3,5,6-trichlorosalicylate > 5-chlorosalicylate > 5-fluorosalicylate > 3,5-
dichlorosalicylate > 4-fluorosalicylate > 3-fluorosalicylate > 3-chloro-5-fluorosalicylate > 4-chloro-
salicylate > SA). The correlation between PR-1a protein induction and resistance to TMV confirms
the value of using PR-1a induction as a screening tool for developing new plant disease control
agents.
KEYWORDS: Systemic acquired resistance; salicylic acid; tobacco mosaic virus; PR-1a protein;
halogenation
INTRODUCTION
plants. ISR, which is independent of SA, acts through jasmonate
and ethylene and may be either antagonized or complemented
by the SAR pathway (4).
Plants possess an inducible defense response known as
systemic acquired resistance (SAR). SAR is defined as an
increased resistance to disease in noninfected plant parts
following pathogen attack (1). Although SAR has been known
for more than 70 years (2), its basis is still being elucidated.
The trigger for SAR is the recognition of the invading pathogen,
and the result is the mounting of a resistance response. This
response is often characterized by a plant-mediated hypersensi-
tive response (HR) that may prevent the spread of a pathogen
Studies with SA have shown its importance in plant-
pathogen interactions. SA levels increased both locally and
systemically following the HR to invading pathogens (5, 6).
Furthermore, exogenous SA induces the accumulation of mRNA
for the pathogenesis-related (PR) proteins that are associated
with SAR (7). Two classes of PR proteins, chitinases and
glucanases, have antimicrobial properties in vitro or when
overexpressed in transgenic plants (8). Other PR proteins such
as PR-1 are of unknown function and may reduce pathogen
colonization through yet undetermined mechanisms (9). Whether
SA is the primary signal in tobacco SAR remains controversial
(3). SAR implies that a signal or signals move from pathogen-
exposed to pathogen-free parts of the plant. In addition to the
SAR pathway, which is salicylic acid (SA) dependent, an
induced systemic resistance (ISR) pathway is also active in
(10, 11). The necessity of SA for the expression of SAR in
tobacco was shown in experiments with the salicylate hydroxyl-
ase (nahG) transgene, which converts salicylate into catechol.
*
Author to whom correspondence should be addressed (e-mail
paul.silverman@valent.com).
1
0.1021/jf051383t CCC: $30.25
© 2005 American Chemical Society
Published on Web 11/15/2005

9776 J. Agric. Food Chem., Vol. 53, No. 25, 2005
Silverman et al.
Overexpression of the nahG gene in tobacco plants blocks SA
accumulation and prevents the development of SAR (12).
Although questions remain regarding the role and mode of action
of SA in SAR, most studies support the role of SA as a signal
in plant disease resistance (13).
was concentrated under a stream of argon until an abundant deposit of
crystals had formed, and a recrystallization was performed by heating
to redissolve them. Two crops of crystals were taken for a total yield
of 640 mg of colorless solid: mp 173-175 °C; GC-MS, after
+
methylation with boron trifluoride/methanol, m/z 204/206 [M for
methyl ester], 173/175, 172/174, 144/146, 117/119, 116/118 and 81;
An increased understanding of SAR has led to the develop-
ment of pesticides that induce plant defenses. For example,
acibenzolar-S-methyl, the active ingredient in Actigard 50WG,
was discovered and developed to induce disease resistance in
plants (3). In Japan, Oryzemate, with the active ingredient
probenazole, is used to control rice blast disease through SAR
induction (14). However, more effective crop protection agents
are needed. We have used a tiered approach to screen SA and
salicylate derivatives, in which the primary screen quantifies
the induction and accumulation of PR-1a protein, a prominent
acidic protein induced during SAR, and the second tier tests
the ability to increase resistance of tobacco to tobacco mosaic
virus (TMV).
1
H NMR (CD OD, 400 MHz) δ 7.40 (1H, dd, J ) 2.58, 10.48 Hz,
3
19
H-4), 7.62 (1H, dd, J ) 1.88, 2.50 Hz, H-6); F NMR (CD
MHz) δ -136.01 (1F, d, J ) 10.45 Hz, F-3).
3
OD, 376
3
-Methoxy-5-chlorosalicylic Acid. This compound was prepared
from 3-methoxysalicylic acid according to the method described above
for 3-fluoro-5-chlorosalicylic acid, except that the initial addition of
hydrogen peroxide was carried out in an ice-water bath. Total yield
for two crops of crystals was 65%: mp 189-192 °C; ESI-MS, m/z
-
2
01 (100%), 203 (31.3%) [M - H] .
3-Chloro-5-fluorosalicylic Acid. This compound was prepared from
5
3
-methoxysalicylic acid according to the method described above for
-fluoro-5-chlorosalicylic acid, except that 8 equiv of HCl and 9 equiv
of hydrogen peroxide were required to drive the reaction to comple-
tion: mp 216-218 °C (with sublimation) after recrystallization from
dichloroethane [lit., 220-221 °C (16)]; ESI-MS, m/z 189 (100%), 191
-
MATERIALS AND METHODS
(31.3%) [M - H] .
3
,5-Dichloro-6-hydroxysalicylate (2,6-Dihydroxy-3,5-dichloroben-
Chemicals. SA, most other salicylates, and other chemicals were
obtained from Sigma-Aldrich (St. Louis, MO). 3-Formylsalicylate was
obtained from Acros Organics (Fisher Scientific, Pittsburgh, PA).
zoic Acid). This compound was prepared from 2,6-dihydroxybenzoic
acid according to the method described above for 3-fluoro-5-chloro-
salicylic acid except that the initial addition of hydrogen peroxide was
carried out in an ice-water bath. Two crops of crystals provided a
total yield of 85%: mp 210-212 °C [lit., 210-212 °C (17)]; ESI-MS,
3
-Fluorosalicylic acid and 3′,5′-difluoro-2′-hydroxyacetophenone were
obtained from Matrix Scientific (Columbia, SC). Crop oil concentrate
COC) used in all foliar sprays comprised 83% Orchex 796 (Exxon
(
-
m/z 221 (100%), 223 (62%), 225 (11%) [M - H] .
Co., Houston, TX) and 17% AT Plus 300F (Uniqema, New Castle,
DE). The syntheses of 6-methylsalicylic acid, 3,5-difluorosalicylic acid,
2,6-Dichloroisonicotinic Acid. This compound was prepared by the
reaction of citrazinic acid with phosphoryl chloride at 125 °C under
pressure for 24 h (18): ESI-MS, m/z 190 (100%), 192 (58.3%), 193.9
10.0%) [M - H] .
Plant Material. Tobacco (Nicotiana tabacum cv. Xanthi-nc) seed
3
5
-fluoro-5-chlorosalicylic acid, 3-methoxy-5-chlorosalicylic acid, 3-chloro-
-fluorosalicylic acid, 3,5-dichloro-6-hydroxysalicylate, and 2,6-dichloro-
isonicotinic acid are described below.
-Methylsalicylic Acid. Ethyl 6-methylsalicylate was hydrolyzed
-
(
6
was obtained from Dr. Ilya Raskin (Rutgers University, New Brunswick,
NJ). The Xanthi-nc variety carries the N-gene for resistance to TMV;
thus, it responds hypersensitively to TMV. Tobacco plants were grown
in Promix PGX (25 °C; 16:8 h light/dark cycle). Plants were given 1
g/L of 20-20-20 fertilizer twice a week. Plants were treated with test
compounds or virus at 5-6 weeks after sowing, at which time 5-7
leaves were fully expanded.
by heating under reflux for 2 h with 3 equiv of dilute aqueous sodium
hydroxide solution. Addition of 0.5 M sulfuric acid precipitated the
product in 95% yield. The analytical sample was recrystallized from
dichloroethane: mp 165-167 °C with sublimation; GC-MS, m/z 152
+
-
[
M ], 134, 106, 105, 78; ESI-MS, m/z 151 [M - H] .
,5-Difluorosalicylic Acid. 3′,5′-Difluoro-2′-hydroxyacetophenone
1.033 g, 6 mmol, 1 equiv) was dissolved in 5 mL of dioxane and
3
(
PR-1a Protein Induction. Test compounds were dissolved in ethanol
and then diluted in water to the target concentration. Treatment solutions
contained 0.1% (v/v) ethanol. Treatment solutions were infused through
the stomata of the abaxial surface of the youngest fully expanded leaves.
The solutions were forced into the leaves by a disposable syringe
without a needle at multiple locations of the leaves until they were
saturated. In all studies, a positive control treatment of 1 mM SA was
included to allow determination of the relative PR-1a induction at 96
h.
converted to the sodium salt by the addition of 1.0 mL of 6 M NaOH
aq) and 5 mL of water. The resulting suspension was stirred in an
ice-water bath while a solution of 4.72 g of iodine (18.6 mmol, 3.1
equiv) and 3.08 g of potassium iodide (18.6 mmol, 3.1 equiv) in 30
mL of water was added in small portions, alternating with five additional
(
1.0 mL portions of 6 M NaOH (aq). The addition took 1 h, and stirring
was continued in the cold for an additional hour. Treatment with a
little sodium bisulfite removed excess iodine. The pH of the mixture
was adjusted to 9 with 5 N sulfuric acid, and the dioxane was removed
as an azeotrope on a rotary evaporator. The mixture was again cooled
to ice-bath temperature, and the precipitated iodoform was removed
by filtration. Further acidification to pH 1.5 with sulfuric acid
precipitated the product, which was collected by filtration, washed with
chilled water acidified by the addition of a small amount of trifluoro-
acetic acid, and air-dried. Yield was 582 mg, plus another 107 mg upon
concentration of the mother liquors (65% total): mp 182-184 °C with
Proteins were harvested according to the method of Yalpani et al.
(19). All harvests were performed 96 h after infiltration unless otherwise
indicated. Briefly, treated leaves were deveined, and the remaining tissue
was cut into 1 cm × 1 cm pieces and vacuum-infiltrated by immersion
in ice-cold extraction buffer. The extraction buffer contained 25 mM
2 2
Tris, pH 7.8, 25 mM MgCl , 10 mM CaCl , 0.5 M sucrose, 20 µM
phenylmethanesulfonyl fluoride, and 150 µM 2-mercaptoethanol (20).
Leaf tissue was blotted to remove excess buffer and placed in a spin
column assembly. The intercellular fluid containing the secreted PR-
+
sublimation [lit., 185-186 °C (15)]; GC-MS, m/z 174 [M ], 156, 128,
1
00.
1
a protein was collected by centrifugation at 1000g for 10 min at 4 °C
3
-Fluoro-5-chlorosalicylic Acid. 3-Fluorosalicylic acid (624 mg, 4
and stored at -80 °C prior to electrophoresis.
mmol, 1.0 equiv) was dissolved in 4 mL of dioxane to give a clear,
colorless solution. Hydrochloric acid (0.70 mL of 6.0 M, 4.2 mmol,
.05 equiv) was added, producing a two-phase mixture. Hydrogen
peroxide (0.50 mL of 30%, 4.4 mmol, 1.1 equiv) was added with rapid
stirring over a period of 1 min, and the reaction mixture was stirred
overnight at ambient temperature and protected from light. The next
day a further 0.70 mL of 6 M hydrochloric acid and 0.50 mL of 30%
hydrogen peroxide were added, and stirring was continued for another
Protein concentrations of intercellular fluids were determined with
Bradford dye reagent (Bio-Rad, Richmond, CA) using BSA as a
standard. Aliquots of 10 µg of total protein/sample were analyzed by
electrophoresis on precast 14% native Tris-glycine polyacrylamide gels
(Invitrogen, Carlsbad, CA). Gels were stained with colloidal blue
staining reagent (Invitrogen), and the band corresponding to PR-1a
protein was quantified using a model GS-710 densitometer (Bio-Rad).
The identity of the 14 kDa protein band that corresponds to PR-1a has
been confirmed using a polyclonal antibody (19, 21).
1
2
4 h. Finally, another 0.05 mL of 30% hydrogen peroxide was added,
and stirring was continued overnight once more. The reaction mixture

Salicylate Activity. 3
J. Agric. Food Chem., Vol. 53, No. 25, 2005 9777
rates (e.g., 2 mM SA) induced more PR-1a in a dose-dependent
manner, but also produced phytotoxicity. Because phytotoxicity
for 1 mM SA was limited only to the points of infiltration, this
rate was used as the standard in further studies. PR-1a induction
was initially measurable 24 h after infiltration with 1 mM SA
(Figure 1A) and increased with time through 192 h. However,
the most consistent response among studies was found at 96 h
after infiltration. In all studies, 1 mM SA was used as a treatment
and an internal standard against which all other compounds were
compared. At least two plants (one leaf per plant) were treated
with each compound, and the samples obtained were used to
obtain PR-1a induction data. The densities of the PR-1a bands
obtained with the 1 mM SA samples were averaged and
normalized to 1.0. The densities of the PR-1a bands for each
test compound were averaged, and their induction is expressed
relative to 1 mM SA. All test compounds were assayed in at
least two independent experiments, and the final induction
number is an average of these studies.
Among the 36 monosubstituted salicylates tested (Table 1),
3
had substantially greater PR-1a induction than SA (3-
chlorosalicylate, 3-fluorosalicylate, and 5-fluorosalicylate with
67, 113, and 60% greater induction, respectively). Within a
1
family of derivatives, 3- or 5-position substitutions were more
active than 4- or 6-position substitutions (e.g., 3-fluorosalicylate
>
5-fluorosalicylate > 4-fluorosalicylate > 6-fluorosalicylate).
With the exception of 3-aminosalicylate, 3-methoxysalicylate,
and 5-cyanosalicylate (relative PR-1a inductions of 0.54, 0.49,
and 0.85, respectively), the only 3- or 5-substituted salicylate
derivatives showing substantial PR-1a induction were the small
halogen-substituted salicylate derivatives. The 5-substituted
halosalicylates show decreasing activity with the increase in
atomic weight and steric bulk of the substituent group (i.e.,
activity of 5-fluorosalicylate > 5-chlorosalicylate > 5-bromo-
salicylate > 5-iodosalicylate). Substitution with other strong
electron-withdrawing groups with larger steric requirements,
such as nitro, cyano, or aldehyde, resulted in less activity than
with the smaller halogens at comparable positions.
Figure 1. Time course of salicylate (SA) induction of pathogenesis-related
protein (PR-1a) induction in Xanthi-nc tobacco (A) and effects of salicylate
(SA; 5-fluorosalicylate, and 3,5-difluorosalicylate) concentration (B) and
time after infiltration (C) on PR-1a induction. Relative induction of PR-1a
is the amount of PR-1a protein induced by test compound (1 mM) divided
by that induced by SA (1 mM) 96 h after infiltration. Means were separated
Among the 11 di- and trisubstituted salicylates tested (Table
2), 5 had substantially greater PR-1a induction than SA (3,5-
difluorosalicylate, 3,5-dichlorosalicylate, 3-chloro-5-fluorosali-
cylate, 3-fluoro-5-chlorosalicylate, and 3,5-dichloro-6-hydroxy-
salicylate with 283, 81, 70, 46, and 41% greater induction,
respectively). Whereas fluorination and/or chlorination in both
the 3- and 5-positions improved activity, PR-1a induction was
greatly reduced by the substitution of an additional halogen to
the 6-position (i.e., 3,5,6-trichlorosalicylate) or eliminated by
the substitution of bulkier groups, even if they are electron-
withdrawing, at the 3- and 5-positions (e.g., 3,5-dinitrosali-
cylate).
by Duncan’s new multiple-range test (p
) 0.05; n ) 3 replicate studies).
Means followed by the same letter are not significantly different.
TMV Resistance. Test compounds were dissolved in ethanol and
then diluted in water (treatment solutions contained 0.1% v/v ethanol).
Spray solutions were amended with 0.25% COC. Plants were sprayed
to drip and arrayed in a randomized complete block experimental design
in a greenhouse (25 °C; 16:8 h light/dark cycle). Unless otherwise stated,
one leaf per plant was inoculated 4 days after spray application with
1
.0 µg of TMV (U1 strain) (21). Six days after inoculation, inoculated
leaves were removed, and diameters of 20 necrotic lesions per leaf
were measured with the aid of a dissecting microscope.
Statistical Analysis. Data were subjected to analysis of variance,
and means were separated by Duncan’s new multiple-range test. The
relationship between relative induction of PR-1a protein and reduction
in TMV lesion diameter was determined by regression analysis using
PlotIT software (Scientific Programming Enterprises, Haslett, MI).
From the PR-1a-inducing activity of the monosubstituted and
multiply substituted salicylates, we can conclude that enhanced
activity is observed only when specific conditions are met: (1)
Enhanced activity is limited to substitution at the 3-
and 5-positions. Substitutions at positions other than 3 and 5 of
the ring result in decreased activity. (2) Substituents must be
electron-withdrawing to enhance activity. (3) Substituents may
contain no more than one atom and have a van der Waals radius
no greater than that of chlorine. Also, position 3 is more tolerant
of steric bulk than position 5.
RESULTS AND DISCUSSION
PR-1a Protein Induction. Induction of PR-1a protein by 1
mM SA increased with time (Figure 1A; optical density × 10/
2
mm is 0, 0.05, 0.30, 0.46, 0.83, and 0.98 for 0, 24, 48, 72, 96,
and 120 h, respectively). The optimal SA rate and time of PR-
The dose response of SA compared to two fluorinated
salicylates is shown in Figure 1B. At the lowest rate tested
(0.1 mM), 3,5-difluorosalicylate induced a measurable amount
of PR-1a, whereas neither SA nor 5-fluorosalicylate induced at
that rate. At either 0.5 or 1.0 mM, both 5-fluorosalicylate and
1
a collection were initially determined to establish a consistent
protocol for comparative studies. Although 0.1 mM SA did not
induce consistent induction of PR-1a in tobacco, induction
increased linearly with SA at 0.5 and 1 mM (Figure 1B). Higher

9778 J. Agric. Food Chem., Vol. 53, No. 25, 2005
Silverman et al.
Table 1. Induction of Pathogenesis-Related Protein (PR-1a) in
Xanthi-nc Tobacco by Salicylates with a Single Modification or
Substitution
Table 2. Induction of Pathogenesis-Related Protein (PR-1a) in
Xanthi-nc Tobacco by Salicylates with Multiple Substitutions
relative induction
compounda
of PR-1a
b
relative induction
compounda
control
substituent
of PR-1a
b
control
SA
0.00
1.00
0.00
1.00
SA
dihalogen substitutions
position 1 substitution
salicylamide
3,5-dibromosalicylate
3,5-dichlorosalicylate*
3,5-difluorosalicylate
0.25
1.81
3.83
CONH
2
0.00
position 2 substitution
acetylsalicylate*
benzoate
acetoxy
H
F
SH
1.10
0.00
0.06
0.59
mixed dihalogen substitutions
3-chloro-5-fluorosalicylate
3-fluoro-5-chlorosalicylate
other multiple substitutions
3,5-dichloro-6-hydroxysalicylate
2,6-difluorobenzoic acid
5-(2,4-difluorophenyl)salicylic acid
3,5-dinitrosalicylate
1.70
1.46
2-fluorobenzoate
thiosalicylate
position 3 substitution
1.41
0.00
0.00
0.00
0.15
0.25
0.02
3
3
3
3
3
3
3
3
3
3
-aminosalicylate
-chlorosalicylate
-fluorosalicylate
-formylsalicylate
-hydroxylsalicylate
-isopropylsalicylate
-methoxysalicylate
-methylsalicylate
-nitrosalicylate
NH
Cl
F
CHO
OH
2
0.54
2.67
2.13
0.06
0.00
0.00
0.49
0.22
0.27
0.00
3-methoxy-5-chlorosalicylate
3,5,6-trichlorosalicylate
2,3,6-trifluorobenzoic acid
isopropyl
OCH
CH
NO
phenyl
3
3
a
Asterisk denotes known SAR inducer [Conrath et al. (28)]. b Amount of PR-
a protein induced by test compound (1 mM) divided by that induced by salicylate
1 mM; SA) 96 h after infiltration.
2
1
(
-phenylsalicylate
position 4 substitution
4-aminosalicylate
4-chlorosalicylate*
4-fluorosalicylate
4-hydroxylsalicylate
4-methoxysalicylate
4-methylsalicylate
NH
Cl
F
OH
OCH
2
0.08
0.32
0.83
0.00
0.17
0.05
3
CH
3
position 5 substitution
5
5
5
5
5
5
5
5
5
5
5
-aminosalicylate
-bromosalicylate
-chlorosalicylate*
-cyanosalicylate
-fluorosalicylate
-formylsalicylate
-hydroxylsalicylate
-iodosalicylate
NH
Br
Cl
CN
F
CHO
OH
I
2
0.00
0.41
1.07
0.85
1.60
0.00
0.00
0.00
0.12
0.00
0.15
-methoxysalicylate
-methylsalicylate
-nitrosalicylate
OCH
CH
NO
3
3
3
2
position 6 substitution
6
6
6
6
-fluorosalicylate
F
OH
OCH
CH
0.29
0.86
0.31
0.16
-hydroxylsalicylate
-methoxysalicylate
-methylsalicylate
3
Figure 2. Relationship between relative induction of pathogenesis-related
protein (PR-1a) and percent reduction in tobacco mosaic virus (TMV)
lesion diameter in Xanthi-nc tobacco for methoxysalicylates (square),
chlorosalicylates (triangle), fluorosalicylates (inverted triangle), and salicylate
a
Asterisk denotes known SAR inducer [Conrath et al. (28)]. b Amount of PR-
a protein induced by test compound (1 mM) divided by that induced by salicylate
1 mM; SA) 96 h after infiltration.
1
(
(
SA; asterisk over circle) with multiple substitutions (diamond). Relative
3
,5-difluorosalicylate acid induced significantly more PR-1a
induction of PR-1a is the amount of PR-1a protein induced by test
compound (1 mM) divided by that induced by SA (1 mM) 96 h after
infiltration. TMV lesion diameter was determined for 1 mM of test
compound. Line represents TMV lesion diameter (percent reduction)
3
protein than SA at the same concentrations. This enhanced
accumulation of PR-1a protein extended through 192 h after
infiltration (Figure 1C).
TMV Resistance Induction. Among the 16 methoxy-,
chloro-, and fluorosalicylates tested, induction of resistance to
TMV generally paralleled induction of PR-1a (Figure 2). The
relationship between relative induction of PR-1a protein and
reduction in TMV lesion diameter was log-linear:
)
2
5.5
+
23.4
×
log(relative induction of PR-1a protein); r
)
0.54. Structure
of SA is in inset.
cylate > 5-fluorosalicylate > 3,5-dichlorosalicylate > 4-fluoro-
salicylate > 3-fluorosalicylate > 3-chloro-5-fluorosalicylate >
4-chlorosalicylate > SA). All four methoxylated salicylate
derivatives were less active than SA.
TMV lesion diameter (% reduction) )
3
5.5 + 23.4 × log(relative induction of PR-1a protein);
2
At 48 h after treatment, 1 mM 3,5-difluorosalicylate reduced
TMV lesion size slightly more than an equal molar concentration
of SA, and almost as well as 5 mM SA (Figure 3). At 96 h
after inoculation, 1 mM 3,5-difluorosalicylate was more effective
at reducing TMV lesions than 5 mM SA. This indicates that
3,5-difluorosalicylate, which is the best PR-1a inducer among
r ) 0.54
Except for 6-fluorosalicylate, all fluoro- and chlorosalicylates
tested were more active TMV resistance inducers than SA (3-
chlorosalicylate > 3,5-difluorosalicylate > 3,5-dichloro-6-
hydroxysalicylate > 3,5,6-trichlorosalicylate > 5-chlorosali-

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1
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(
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JF051383T