Proc. Natl. Acad. Sci. USAVol. 92, pp. 7143-7147, August 1995Plant Biology

Two inducers of plant defense responses, 2,6-dichloroisonicotinicacid and salicylic acid, inhibit catalase activity in tobacco

(reactive oxygen species/enhanced disease resistance/pathogenesis-related proteins/plant signal transduction/salicylicacid-binding protein)

UWE CONRATH*, ZHIXLANG CHEN, JOSEPH R. RICIGLIANOt, AND DANIEL F. KLESSIGtWaksman Institute and Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, P.O. Box 759, Piscataway, NJ 08855

Communicated by Clarence A. Ryan, Washington State University, Pullman, WA, May 9, 1995 (received for review August 21, 1994)

ABSTRACT 2,6-Dichloroisonicotinic acid (INA) and sal-icylic acid (SA) are potent inducers of plant defense responsesincluding the synthesis of pathogenesis-related (PR) proteinsand the development ofenhanced disease resistance. A solubleSA-binding protein has been purified from tobacco with anaffinity and specificity of binding that suggest it is a SAreceptor. Recently, this protein has been shown to be acatalase whose enzymatic activity is inhibited by SA binding.We have proposed that the resulting increase in intracellularlevels of reactive oxygen species plays a role in the inductionof defense responses such as PR protein gene expression. Herewe report that INA, like SA, -binds the SA-binding pro-tein/catalase and inhibits its enzymatic activity. In fact, thedose-response curves for inhibition of catalase by these twocompounds are similar. Furthermore, the ability of both INAanalogues and SA derivatives to bind and inhibit tobaccocatalase correlates with their biological activity to inducePR-1 gene expression and enhance resistance to tobaccomosaic virus. Comparison of the structures of INA, SA, andtheir analogues reveals several common features that appearto be important for biological activity. Thus, these results notonly suggest that INA and SA share the same mechanism ofaction that involves binding and inhibition ofcatalase but alsofurther indicate an important role for reactive oxygen speciesin the induction of certain plant defense responses. This issupported by the demonstration that INA-mediated PR-1 geneactivation is suppressed by antioxidants.

Plant disease resistance is often manifested as a restriction ofpathogen growth and spread to a small zone around theinfection site. In many cases, this restriction is accompanied bylocalized cell death of host tissue. This local cell death resultsin the formation of visible necrotic lesions (hypersensitiveresponse) (1) and resembles programmed cell death (apopto-sis) (2). After lesion formation, many plants develop enhanced(acquired) resistance to a broad spectrum of pathogens nearthe area of primary infection and often throughout the plant(3, 4). In the resistant tissue, there is the expression of acomplex set of defense-related genes (4) whose products intobacco include five or more families of pathogenesis-related(PR) proteins (5, 6). The enzymatic activities of several PRproteins have been identified and include 1,3-03-glucanases andchitinases, which can hydrolyze microbial cell wall compo-nents. Moreover, overexpression of a variety of these PR genesin transgenic plants results in enhanced resistance to differentfungal pathogens (3, 4, 7). Thus, their synthesis serves as amarker for disease resistance.A growing body of evidence suggests that salicylic acid (SA)

is an endogenous signal for the activation of certain plantdefense responses including PR gene expression and estab-lishment of enhanced disease resistance (3, 4, 7). In the past

few years, considerable progress has been made in the eluci-dation of the SA signal transduction mechanism. In tobacco,SA binds a soluble 280-kDa SA-binding protein (SABP) whosebinding specificity and affinity are consistent with a receptorfunction (8, 9). The SABP readily converts 1J202 to H20 and02 and the sequence of a cDNA encoding SABP shows highsimilarity to that of catalases (H202:H202 oxidoreductase, EC1.11.1.6) (10). A variety of experiments strongly argue that oneof SA's modes of action in tobacco involves binding to andinhibition of SABP/catalase's enzymatic activity. The result-ing increase in the levels of H202 or related reactive oxygenspecies (ROS) may act as a signal for the expression ofdefense-related genes and, possibly, the development of en-hanced disease resistance. Indeed, injection of tobacco leaveswith H202 or compounds that increase H202 levels in vivoinduces expression of PR-1 genes (10).The identification ofSA as a natural inducer ofplant defense

responses has prompted characterization of synthetic chemi-cals that are able to mimic SA. 2,6-Dichloroisonicotinic acid(INA) and its methyl ester, which induce disease resistance invarious host-pathogen systems, are the best studied of thesechemicals (11). In tobacco, SA and INA induce the same nineclasses of genes (including the PR genes) that are expressedsystemically after tobacco mosaic virus (TMV) infection (5).However, INA's mechanism of action is unclear. In contrast tomany other abiotic inducers of PR gene expression andenhanced disease resistance such as polyacrylic acid andthiamine, INA does not stimulate accumulation of SA or itsglucoside in treated tobacco plants (J. Malamy, P. Sanchez-Casas, J. Hennig, A. Guo, and D.F.K, unpublished results).This result indicates that INA does not act through SA butenters the signal transduction pathway after SA biosynthesis oracts via a different pathway. Since mammalian catalases havebeen shown to bind NAD(P)H (12), which shares with INA thenicotinic acid moiety, it seemed'probable that INA might alsobind catalase. However, in contrast to the binding ofNAD(P)H, which does not decrease the enzyme's catalyticactivity (12), we suspected that INA binding, like that of SA,could inhibit catalase activity. Here we report that INA indeedbinds and inhibits catalase.

MATERIALS AND METHODSMaterials. [7-14C]SA (55 Ci/mol; 1 Ci = 37 GBq) was

obtained from New England Nuclear; unlabeled SA and its

Abbreviations: INA, 2,6-dichloroisonicotinic acid; ROS, reactive ox-ygen species; SA, salicylic acid; PR, pathogenesis related; SABP,SA-binding protein; SAR, systemic acquired resistance; TMV, tobaccomosaic virus; CSA, chlorinated derivative of SA; HBA, hydroxyben-zoic acid.*Present address: Universitat Kaiserslautern, Fachbereich Biologie,Abteilung Pflanzenphysiologie, Postfach 3049, 67653 Kaiserslautern,Germany.

tPresent address: Paradigm Biosciences, 2401 Foothill Boulevard, SaltLake City, UT 84109.lTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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analogues were purchased from Sigma or Aldrich; H202 wasfrom Sigma. INA and its analogues were synthesized byCIBA-Geigy. INA, SA, and their analogues were dissolved inwater as 10 mM stock solutions and adjusted to pH 5.8 withKOH.

Plant Material. Tobacco (Nicotiana tabacum cv. Xanthi nc)plants were grown at 22°C in a 14-hr light cycle and used forexperimentation at 6-8 weeks. The cell suspension culture wasderived from callus tissue originating from leaf tissue of N.tabacum cv. Xanthi and grown as described (13).

Determination of Catalase Activity in Vivo. For the deter-mination of catalase activity in vivo, 5 ml of a cell suspensionobtained 3 days after subculturing was agitated (100 rpm) atroom temperature in the absence or presence of INA, SA, ortheir analogues. After 1 hr, 1 ml of the treated cell suspensionwas diluted 1:8 with fresh MS medium without inhibitors (-6mg of cells per ml). After establishment of a constant baseline,the in vivo catalase activity was measured by continuouslyrecording for 2-3 min the rate of H202-dependent 02 pro-duction with a commercial oxygen electrode probe (model5739; YSI, Yellow Springs, OH) after addition of H202 to 10mM. The rates of 02 evolution obtained with cell suspensionstreated with SA, INA, or their analogues were compared tothat of water-treated control cells and given as percent inhi-bition.

Determination of Intracellular SA Levels. After varioustimes of incubation of tobacco cells in suspension culture ineither 250 AM or 1000 ,uM SA, 3 ml of the cell suspension wascentrifuged for 5 min in a tabletop centrifuge (Dynac, Parsip-pany, NJ) at 1400 x g, washed three times with 10 ml of MSmedium, and frozen in liquid nitrogen. Cells from each samplewere extracted and quantitated for free SA as described (14).Intracellular SA levels were based on the assumption thatintracellular volume was "50% of packed cell volume. Therecovery rate of SA is '50%; numbers in Table 1 have beencorrected accordingly.

Analysis ofPR-1 Induction. To determine the ability of INA,SA, and their analogues to induce PR-1 gene expression, twoleaf discs (1 cm in diameter) of tobacco were floated in Petridishes (3.5 cm in diameter) containing water (control) oraqueous solutions of 1 mM INA, 1 mM SA, or their analoguesat 1 mM in a total volume of 3 ml. After vacuum infiltrationfor 10 min, the discs were agitated (50 rpm) at room temper-ature for 24 hr in the presence of light and then homogenizedin 50 mM Tris HCl, pH 8.0/1 mM EDTA/12 mM 2-mercap-toethanol/phenylmethylsulfQnyl fluoride (10 ,ug/ml). Afterclarification by centrifugation (12,000 x g), the homogenatewas fractionated by SDS/PAGE and the separated proteinswere electrophoretically transferred to a nitrocellulose filter.Immunoblot analysis was performed as described with a 1:1000dilution of mouse monoclonal antibody 33G1, which specifi-cally recognizes PR-1 proteins (9).

0

C

U

3iaCa2

i

100-

80 -

60-

40-

20-

u

10° 101 102 103 104

Concentration (pM)105

FIG. 1. Dose-response curves for the inhibition by INA or SA ofcatalase activity in tobacco suspension cells. Cells were treated withincreasing concentrations of INA (solid line) or SA (broken line) for1 hr after which in vivo catalase activity was determined. Repeatexperiments for both INA and SA gave similar results.

Influence of Antioxidants on PR-1 Induction. Leaves of 6-to 8-week-old tobacco plants were injected with 1 mM INA inthe absence or presence of either 20 mM N-acetylcysteine or1 mM L-ascorbate by using a 10-ml syringe. After 14.5 hr, twoleaf discs were harvested from the treated tissue and analyzedfor PR-1 induction as described above.

Determination of Enhanced Disease Resistance. Leaves of 6-to 8-week-old tobacco plants were injected with 1 mM INA, SA,or their analogues. After 6 days, treated leaves were infected witha suspension of the Ul strain of TMV (0.1 ,ug/ml) in 50 mMsodium phosphate (pH 7.0). After an additional 7 days, the size(average ± SD) of 20 lesions was determined and compared tothe size of lesions on water-treated control plants.

Purification of SABP/Catalase and Binding Assay. Prep-aration of highly purified SABP/catalase and determination of[14C]SA binding to SABP/catalase was performed as de-scribed (8, 9).

RESULTS

INA- and SA-Mediated Inhibition of Catalase Activity inTobacco Cells in Suspension Culture. To avoid potentialartifacts that might result from the use of purified SABP/catalase, we used an in vivo assay to investigate the effect ofINA and SA on catalase activity in tobacco cells. Treatment oftobacco cells in suspension culture for 1 hr with increasing

Table 1. Inhibition by SA of catalase activity in tobacco cells in suspension culture

250 AM SA 1000 ,uM SA

Incubation Intracellular % Intracellular %time, hr SA, ,uM inhibition SA, ,uM inhibition

0 1 0 1 01 197 43 291 712 110 86 102 814 53 90 88 916 11 80 21 71

Tobacco cells in suspension culture were treated with 250 AM or 1000 ,uM SA and analyzed at theindicated times for catalase activity and intracellular levels of free SA. At the beginning of the experiment,just before addition of SA, two aliquots of the cell suspension were removed (0-hr time points; one aliquotwas used for determination of intracellular SA concentration and the other was employed for determi-nation of catalase activity in vivo). At the end of the experiment (6 hr), catalase activity of untreatedcontrol suspension cells was again determined and found to be within a few percent of that determinedat the 0-hr point.

SA

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Cle )0

0 zC.) L) g = =L)*-e r cn4 Lh ciFAPR-1 __

FIG. 2. Induction by INA, SA, and their analogues of PR-1 proteinaccumulation in tobacco leaf tissue. Excised leaf discs were assayed foraccumulation of PR-1 proteins. PR-1 induction was also tested intobacco cells in suspension culture with similar results, except that thelevels of induction by the active compounds were not as high as in leafdiscs (data not shown). 3,5-DCSA,3,5-dichloro-SA.

concentrations of INA or SA had no effect on the viability ofthe cells (data not shown) but caused a dose-dependentinhibition of cellular catalase activity (Fig. 1). Additionally, thedose-response curves for both INA- and SA-mediated inhi-bition were similar. Half-maximal inhibition was reached at"500 ,uM for INA and "300 pLM for SA. In the experimentshown, almost total inhibition was obtained at 10 mM INA orSA. However, in many other experiments (e.g., see Table 2)inhibition was nearly complete at 1 mM.To determine how the levels of free SA within the cells

affected catalase activity, the intracellular SA concentrations,as well as catalase activity, were monitored over 6 hr at twoextracellular SA concentrations (Table 1). After a 1-hr incu-bation with 250 ,uM or 1000 AM SA, catalase activity within thecells in suspension culture was inhibited "40% or "70%, whileintracellular SA levels were '200 ,iM or -300 ,uM, respec-tively. With time, the intracellular SA levels dropped dramat-ically, as shown (13). In contrast, inhibition of catalase in-creased to 4 hr, at which time catalase activity was inhibited":90% and intracellular levels of SA ranged from 50 ,uM to 100,uLM.

Biological Activity of INA, SA, and Their Analogues. As afirst step toward assessing the functional relevance of catalaseinhibition by INA and SA, the biological activity of severalanalogues for each of these two compounds was determined.These analogues, whose structures are shown in Table 2, weretested for their ability to induce PR-1 protein accumulation(Fig. 2) and enhanced resistance to TMV infection (Table 2)in tobacco. Two halogenated analogues of INA (analogues 3and 8) and the aldehyde form of INA (compound 7) werebiologically active in both assays, whereas three other INAanalogues (compounds 1, 6, and 9) were not active at theconcentration used (1 mM).Three chlorinated derivatives of SA (CSAs) (4-CSA, 5-CSA,

and 3,5-dichloro-SA) were also effective at inducing PR-1protein accumulation (Fig. 2) and enhanced resistance toTMV(Table 2). In contrast, the SA derivatives 3-hydroxybenzoicacid (3-HBA) and 4-HBA at 1 mM were inactive in both assays.Comparison of the results from both assays shows a good

correlation between the ability of an analogue to induce PR-1protein accumulation and enhanced resistance. However, thiscorrelation was not always quantitative. While the fluorinatedanalogue of INA (compound 8) was somewhat less effective

INA + + + - -

NAC - + - + -

ASC - -+ -+=| _ -PR-1

FIG. 3. Inhibition by antioxidants of INA-induced PR-1 proteinaccumulation in tobacco leaf tissue. Healthy leaves of 6-week-oldtobacco plants were injected with INA (1 mM) in the absence (-) orpresence (+) of N-acetylcysteine (NAC, 20 mM) or L-ascorbate (ASC,1 mM) or with the antioxidant alone. After 14.5 hr, two leaf discs wereharvested and analyzed for PR-1 protein accumulation as in Fig. 2.

than INA at activating PR-1 protein accumulation, it wasslightly more active than INA in inducing enhanced resistance.In contrast, the chlorinated analogues of SA were somewhatless effective than SA in both assays at the concentrationtested.

Correlation Between Biological Activity and Catalase Bind-ing and Inhibition. To assess whether the biological activity ofINA, SA, and their analogues is mediated through bindi'ng andinhibition of catalase, the various biologically active, as well asinactive, analogues of INA and SA were tested for their abilityto bind highly purified SABP/catalase from tobacco leaves invitro and also to inhibit catalase activity in tobacco suspensioncells. As shown in Table' 2, there was a good correlationbetween the biological activity of the compounds and theirability to bind and inhibit catalase. The only exception to thiscorrelation was INA analogue 7, which was biologically activeand inhibited catalase activity in vivo but competed poorly with[14C]SA for binding to catalase in vitro. This inconsistency maybe due to oxidation of the aldehyde form (compound 7) to theacid form (INA) in vivo, a process that may not readily occurin vitro at 4°C during the binding assay.

Inhibition of PR-i Induction by Antioxidants. The aboveresults suggest that INA, like SA, induces certain defenseresponses such as PR-1 gene activation by inhibition of catalaseactivity and elevation of ROS levels. If this is correct, thenINA-mediated PR-1 gene induction should be inhibited by thepresence of antioxidants that scavenge ROS. As anticipated,simultaneous injection of INA and the antioxidant N-acetyl-cysteine or L-ascorbate repressed INA-induced PR-1 geneexpression (Fig. 3).

DISCUSSIONINA and SA are potent inducers of plant defense responsesincluding the accumulatiQn of PR proteins and the develop-ment of enhanced disease resistance (refs. 3-7, 1I; Fig. 2 andTable 2). INA, like SA, bound to tobacco catalase (Table 2)and inhibited its enzymatic activity in vivo (Fig. 1) and in vitro(Table 2), suggesting that the biological activity of bothcompounds is mediated through decreased catalase activityand the resulting elevation of ROS levels. In support of thismodel is the finding that the concentrations of exogenous SAor INA (200 ,zM to 1 mM) necessary for substantial inhibitionof catalase activity in tobacco cells in suspension culture weresimilar to the concentrations of exogenous SA or INA (300 ,uMto 2 mM) required for good induction of PR gene expressionand enhanced disease resistance (3-7, 11). Moreover, aspredicted by this model, the antioxidants N-acetylcysteine andL-ascorbate, which scavenge ROS, repressed INA induction ofPR-1 gene expression (Fig. 3).

Interestingly, we have found that treatment of tobacco cellswith exogenous SA for prolonged times results in a decline inintracellular SA levels, whereas catalase inhibition increases.This finding may indicate that within peroxisomes, which is theprimary location of catalases, the level of SA first increases andthen decreases relatively slowly compared to the surroundingcytoplasm or adjoining vacuole. Thus, determination of theoverall intracellular levels ofSA may be a poor indicator of theeffective concentrations of SA in the immediate vicinity of thetarget enzyme.

In TMV-infected tobacco leaves, SA levels increase 10- to100-fold and range from 7 ,uM to 150 ,uM (15-18). Theseconcentrations should be quite adequate to effectively inhibitcatalases and thereby induce certain local defense responsessince they are similar to those seen in tobacco suspensioncultures (10-100 ,M SA) several hours after incubation inexogenous SA, where catalase activity was inhibited 70-90%(Table 1). In contrast, the role of SA-mediated inhibition ofcatalase in systemic acquired resistance (SAR) is less clearsince overall levels of SA in the systemic tissue are much lower

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Table 2. Comparison of INA, SA, and their analogues with respect to biological activity, inhibitionof catalase activity, and inhibition of [14C]SA binding by SABP/catalase

Biological activity Inhibition, %

INA, SA, and Reduction in PR-i Catalase activity [14C]SA bindinganalogues lesion size, % induction in vivo in vitro

a>° (INA) 80±7 + 98±6 58±3

Cl OBr

> (#3) 72±3 + 100±9 60±5~OH

BrCi

>j (#7) 65 ± 14 + 79 ± 7 20 ± 4

aF

0 (#8) 86 ± 7 + 48 ± 6 58 ± 2F

\ ° (#1) 1_±18 4 ±_3 7_1HOHQ

(#6) 4±26 - 25±4 18 2HO

° 0 (#9) 7±16 - 15±6 12±2

OH

OH0 (4-CSA) 76± 11 + 95 ± 5 94 ± 2

OHa>° (5-CSA) 66±9 + 100±5 92±2

OH

COH>° (3,5-DCSA) 70 ± 16 + 100 ± 0 93 ± 4

Cl OH

pM (3-HBA) 18±28 - 0±7 3±2HO

HO-Q--/( (4-HBA) 10±24 - 0±9 1 ±2

Reduction in lesion size was used as an indicator for enhanced disease resistance. PR-i induction wasbased on the ability to induce accumulation of PR-i proteins in leaf discs (Fig. 2). To measure catalaseactivity and [14C]SA binding, tobacco cells in suspension culture were treated with 1 mM INA, 1 mM SA,or their analogues at 1 mM. After 1 hr, catalase activity was assayed in vivo. SA and INAwere less effectiveat inhibiting catalase activity in extracts prepared from the tobacco cells in suspension culture or leaves.At 1 mM SA, inhibition of catalase activity ranged from 70% to 100% in vivo and 40% to 60% in extractsfrom leaves or cells in suspension culture. Inhibition by INA ranged from 70% to 100% in vivo and 20%to 35% in extracts from leaves or cells in suspension culture. Note that with the luminol assay and a lowerconcentration of H202 (1 mM) catalase activity in leaf extracts was inhibited '70% by 1 mM SA (10).This is consistent with our observation that inhibition by SA is inversely proportional to substrate (H202)concentration (Z.C. and D.F.K, unpublished results). [14C]SA (20 ,uM) binding was assayed in vitro withhighly purified SABP/catalase from tobacco leaves (9). Assays were done in the absence or presence of1 mM unlabeled INA, 1 mM SA, or their analogues at 1 mM. In the absence of competitor, there was-200,000 cpm of bound [14C]SA per 100 ,ul of excluded volume after spin-column exclusion chroma-tography of the binding reaction. In the absence of SABP/catalase, the background was 120-150 cpm/100Al. Two additional compounds, polyacrylic acid and thiamine, were used as controls. These twocompounds repress TMV lesion size (70-100% reduction) and induce PR-i gene expression, probably dueto their stimulation of production of SA and its glucoside (J. Malamy, P. Sanchez-Casas, J. Hennig, A.Guo, and D.F.K., unpublished results). Both compounds were poor (2-9%) inhibitors of [14C]SA bindingto SABP/catalase in vitro and had no inhibitory effect on catalase activity in vivo after 1 hr of treatment.This is so because SA levels were probably not yet elevated within this short time period as SA levels donot increase until 6 hr (thiamine) to 10 hr (polyacrylic acid) after their injection into tobacco leaves. Incontrast, PR-i gene expression and enhanced resistance were measured one or more days after treatment.3,5-DCSA, 3,5-dichloro-SA.

than the Kd (14 ,uM) for its binding to catalase. For example,this laboratory (ref. 15 and A. Guo, J. Malamy, and D.F.K.,unpublished results) has observed a 2- to 5-fold increase of SAlevels systemically to 1-3 ,M SA after TMV infection oftobacco, and Vernooij et al. (18) reported that systemic SAconcentrations increased to 0.5-1 ,uM after infection. In the

latter study a scion derived from a nontransgenic Xanthi nctobacco plant was grafted onto a rootstock derived fromnahG-expressing transgenic tobacco. [NahG plants producesalicylate hydroxylase that converts SA to catechol; as a resultthey fail to accumulate SA or develop SAR after TMVinfection (19).] After infection of the rootstock with TMV,

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only a 10% increase of SA was detected in the uninfectedleaves of the scion. They argued that this slight increase in SAlevels was responsible for induction ofPR gene expression andestablishment of SAR but was unlikely to be sufficient forinhibition of catalase.However, other results suggest that in the uninfected tissue

additional factors besides SA may be involved in developmentof SAR. In the same grafting experiments (18), TMV inocu-lation of rootstock leaves derived from a nontransgenic to-bacco plant also produced a 10% elevation of SA levels in thetransgenic nahG scion, yet neither PR gene expression norSAR were induced. In addition, treatment of transgenic nahGplants with 1 mM SA has been found to increase the levels ofboth free and glucosylated SA at least severalfold without acorresponding induction of PR-1 gene expression or enhancedresistance to TMV (J. Malamy, P. Sanchez-Casas, and D.F.K.,unpublished results). Since marked increases in SA levels arenot always correlated with the induction of defense responses,additional factors such as SA compartmentation, the intracel-lular redox state, or the presence of other defense-inducingcompounds may be involved.

Analysis of INA analogues and SA derivatives furthersupports the model that INA and SA induce defense responsesthrough the inhibition of catalase activity. The three deriva-tives of INA (compounds 3, 7, and 8), which effectivelyinhibited catalase activity (Table 2), induced PR-1 gene ex-pression (Fig. 2) and enhanced resistance to TMV infection(Table 2). These three analogues also activated expression ofa chimeric PR-1-uid4 reporter gene in transgenic tobacco andelevated resistance to Colletotrichum lagenarium in cucumber(H. Kessmann, personal communication). Similarly, threeCSAs were potent inhibitors of catalase and strong activatorsof PR-1 gene expression and enhanced disease resistance intobacco. These CSAs have also been shown to enhanceresistance of barley to the fungal pathogen Erisyphe graminis(20) and increase the sensitivity of parsley cells to a fungalelicitor that stimulates phytoalexin secretion by these cells(21). In contrast, the three INA analogues (compounds 1, 6,and 9), which were inactive for inducing PR gene expressionand enhanced resistance, were also poor inhibitors of catalase.And similarly, the biologically inactive SA analogues failed toblock catalase activity. INA, SA, and their analogues weretested in all four assays at 1 mM, the concentration mostfrequently used in many different laboratories. Since dose-response curves were done only for INA's and SA's inhibitionof catalase activity, the results need to be interpreted withcaution.Comparison of the structures of INA and its active ana-

logues with those of SA and its active derivatives suggests thatthese compounds share several features that may allow orfacilitate binding by SABP/catalase and inhibition of itsenzymatic activity (Table 2). All active compounds contain asix-membered conjugated ring and a carboxyl group. Substi-tution of hydrogen atoms with halide groups at the 3, 4, and 5positions of the ring (relative to the carboxyl group) appearsto enhance binding by catalase in vitro, whereas the presenceof hydroxyl groups at the same positions blocks binding (Table2). Addition of a hydroxyl group at position 2 of benzoic acidappears to facilitate binding while substitution of a chloro,ethoxyl, or thiol group at this position inhibits binding (9).

The strong correlation between biological activity and abil-ity to bind and inhibit catalase suggests that the physiologicaleffects of INA, SA, and their active analogues are mediated bythe same mechanism of action, namely, inhibition of catalase'sability to degrade H202. These results thus not only elucidatea likely mode of action of INA but also support the proposedrole of ROS in the induction of plant defense responses (10).

We thank members of the laboratory, particularly D'Maris Demp-sey, Sudam Pathirana, and Jyoti Shah, for their critical reading of themanuscript. Helmut Kessmann, Theo Staub, and John Ryals areacknowledged for generously providing INA and its analogues. Thiswork was supported, in part, by Grant MCB-9310371 from theNational Science Foundation and Grant 92-37301-7599 from the U.S.Department of Agriculture.

1. Matthews, R. E. F. (1991) Plant Virology (Harcourt Brace Jo-vanovich, San Diego), 3rd Ed.

2. Greenberg, J. T., Guo, A., Klessig, D. F. & Ausubel, F. M. (1994)Cell 77, 551-563.

3. Ryals, J., Uknes, S. & Ward, E. (1994) Plant Physiol. 104,1109-1112.

4. Klessig, D. F. & Malamy, J. (1994) Plant Mol. Bio. 26, 1439-1458.

5. Ward, E. R., Uknes, S. J., Williams, S. C., Dincher, S. S., Wie-derhold, D. L., Alexander, D. C., Ahl-Goy, P., Metraux, J. P. &Ryals, J. A. (1991) Plant Cell 3, 1085-1094.

6. Cutt, J. R. & Klessig, D. F. (1992) in Plant Gene Research: GenesInvolved in Plant Defense, eds. Boller, T. & Meins, F. (Springer,New York), pp. 209-243.

7. Dempsey, D. A. & Klessig, D. F. (1994) Trends Cell Biol. 4,334-338.

8. Chen, Z. & Klessig, D. F. (1991) Proc. Natl. Acad. Sci. USA 88,8179-8183.

9. Chen, Z., Ricigliano, J. W. & Klessig, D. F. (1993) Proc. Natl.Acad. Sci. USA 90, 9533-9537.

10. Chen, Z., Silva, H. & Klessig, D. F. (1993) Science 262, 1883-1886.

11. Kessman, H., Staub, T., Hofmann, C., Maetzke, T., Herzog, J.,Ward, E., Uknes, S. & Ryals, J. A. (1994)Annu. Rev. Phytopathol.32, 439-459.

12. Kirkman, H. N. & Gaetani, G. F. (1984) Proc. Natl. Acad. Sci.USA 81, 4343-4347.

13. Hennig, J., Malamy, J., Grynkiewicz, G., Indulski, J. & Klessig,D. F. (1993) Plant J. 4, 593-600.

14. Bowling, S. A., Guo, A., Cao, H., Gordon, S., Klessig, D. F. &Dong, X. (1994) Plant Cell 6, 1845-1857.

15. Malamy, J., Carr, J. P., Klessig, D. F. & Raskin, I. (1990) Science250, 1002-1004.

16. Malamy, J., Hennig, J. & Klessig, D. F. (1992) Plant Cell 4,359-366.

17. Enyedi, A., Yalpani, N., Silverman, P. & Raskin, I. (1992) Proc.Natl. Acad. Sci. USA 89, 2480-2484.

18. Vernooij, B., Friedrich, L., Morse, A., Reist, R., Kolditz-Jawhar,R., Ward, E., Uknes, S., Kessmann, H. & Ryals, J. (1994) PlantCell 6, 959-965.

19. Gaffney, T., Friedrich, L., Vernooij, B., Negrotto, D., Nye, G.,Uknes, S., Ward, E. R., Kessmann, H. & Ryals, J. A. (1993)Science 261, 754-756.

20. Buchenauer, H. & Fleischmann, C. (1992) in 48th DeutschePflanzenschutz-Tagung, ed. Biologische Bundesanstalt fiir Land-und Forstwirtschaft (Kommissionsverlag Paul Parey, Berlin), p.240.

21. Kauss,H., Theisinger-Hinkel, E., Mindermann, R. & Conrath, U.(1992) Plant J. 2, 655-660.

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uest

on

July

6, 2

020