The Plant Cell, Vol. 11, 1393–1404, August 1999, www.plantcell.org © 1999 American Society of Plant Physiologists

Salicylic Acid Induction–Deficient Mutants of Arabidopsis

Express

PR-2

and

PR-5

and Accumulate High Levels of Camalexin after Pathogen Inoculation

Christiane Nawrath

1

and Jean-Pierre Métraux

Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland

In Arabidopsis, systemic acquired resistance against pathogens has been associated with the accumulation of salicylicacid (SA) and the expression of the pathogenesis-related proteins PR-1, PR-2, and PR-5. We report here the isolation oftwo nonallelic mutants impaired in the pathway leading to SA biosynthesis. These SA induction–deficient (

sid

) mutantsdo not accumulate SA after pathogen inoculation and are more susceptible to both virulent and avirulent forms of

Pseudomonas syringae

and

Peronospora parasitica.

However,

sid

mutants are not as susceptible to these pathogensas are transgenic plants expressing the

nahG

gene encoding an SA hydroxylase that degrades SA to catechol. In con-trast to NahG plants, only the expression of

PR-1

is strongly reduced in

sid

mutants, whereas

PR-2

and

PR-5

are stillexpressed after pathogen attack. Furthermore, the accumulation of the phytoalexin camalexin is normal. These resultsindicate that SA-independent compensation pathways that do not operate in NahG plants are active in

sid

mutants. Oneof the mutants is allelic to

eds5

(for enhanced disease susceptibility), whereas the other mutant has not been describedpreviously.

INTRODUCTION

Infection with necrotizing pathogens often leads to the sub-sequent systemic induction of resistance. This is referred toas systemic acquired resistance (SAR), which is character-ized by a long-lasting defense response against a broadspectrum of pathogens (Kuc, 1982; Ryals et al., 1994;Sticher et al., 1997). After the initial observation that treat-ment of plants with salicylic acid (SA) leads to resistance(White, 1979), several lines of evidence have shown that SAis associated with SAR. The expression of a number ofpathogenesis-related (PR) proteins is highly correlated withacquired resistance (Ward et al., 1991; Uknes et al., 1992).The synthesis of PR proteins can be induced by treatmentwith SA and also by the application of functional analogs ofSA, such as 2,6-dichloroisonicotinic acid (INA; Métrauxet al., 1991) or benzo(1,2,3)thiadiazole-7-carbothioic acid

S

-methyl ester (BTH; Görlach et al., 1996).The accumulation of SA in plant tissues correlates with

the presence of PR proteins and resistance (Malamy et al.,1990; Métraux et al., 1990; Yalpani et al., 1991). In addition,transgenic tobacco (Gaffney et al., 1993) and Arabidopsis(Delaney et al., 1994) plants that express the

Pseudomonasputida nahG

gene, encoding an SA hydroxylase, which de-

grades SA to catechol, are hypersusceptible to infectionwith virulent pathogens and cannot develop SAR. Experi-ments with NahG plants also showed that SA is involved ingene-for-gene defense responses (Delaney et al., 1994).These findings provide strong evidence for the involvementof SA in SAR.

SA also has been proposed to be the systemic signal forSAR (Malamy et al., 1990; Métraux et al., 1990). However,although SA synthesized in infected leaves can be shown tobe transported to uninfected leaves in tobacco and cucum-ber (Shulaev et al., 1995; Mölders et al., 1996), grafting ex-periments with tobacco plants expressing NahG, choleratoxin, or reduced levels of phenylalanine ammonia–lyaseshow that other primary systemic signals are involved in theonset of SAR (Vernooij et al., 1994; Beffa et al., 1995; Pallaset al., 1996). Recently, several lines of evidence indicate thatone mode of action of SA in pathogen defense is to potenti-ate defense reactions (Mur et al., 1996; Shirasu et al., 1997).

The dissection of the signal transduction pathway con-necting SA to resistance has been studied primarily in to-bacco and Arabidopsis. In tobacco, a protein has beenfound that binds SA in vitro with a very high affinity (Du andKlessig, 1997). Furthermore, an SA-activated mitogen-acti-vated protein (MAP) kinase has been identified, suggestingthat a MAP kinase cascade participates in SA-dependentdefense signaling (Zhang and Klessig, 1997). In Arabidopsis,the signal transduction pathway downstream of SA hasbeen characterized by analyzing the

npr1

,

nim1

, and

sai1

1

To whom correspondence should be addressed. E-mail christiane.nawrath@unifr.ch; fax 41-26-300-9740.

1394 The Plant Cell

mutants that have been isolated by several groups (for no

PR

gene expression [Cao et al., 1994], noninducible immu-nity [Delaney et al., 1995], and SA insensitive [Shah et al.,1997], respectively).

PR

genes are not induced in the

npr1

/

nim1

/

sai1

mutants after the application of typical inducers ofSAR, and they are highly susceptible to infection by patho-gens. The

NPR1

/

NIM1

gene has been cloned recently, and itencodes a protein containing ankyrin repeats (Cao et al.,1997; Ryals et al., 1997). Its postulated role as a transcrip-tional regulator could be substantiated by its localization inthe nucleus upon treatment with SA (X. Dong, personalcommunication).

Other mutants affected in the signal transduction pathwaylinking pathogen infection with plant resistance could be po-sitioned upstream of the synthesis or action of SA.

lsd

(forlesions simulating disease resistance response; Dietrich etal., 1994) and

acd2

(for accelerated cell death; Greenberg etal., 1994) mutants have high SA levels when expressing le-sions, which is reminiscent of a hypersensitive reaction (HR).Interestingly, in most

lsd

mutants, lesion formation is inde-pendent of SA; however, for

lsd6

and

lsd7

mutants, lesionformation depends on SA (Weymann et al., 1995). Severalmutants were identified by constitutive expression of PRproteins (

cpr

). These mutants have constitutively high levelsof SA and therefore act upstream of SA.

cpr

mutants also may be affected in other disease re-sponse pathways. Whereas

cpr1

seems to be specificallyaffected in the SA pathway (Bowling et al., 1994),

cpr5

formsspontaneous lesions (Bowling et al., 1997) and shows con-stitutive expression of the SA-independent regulated plantdefensin

PDF1.2.

(Penninckx et al., 1996). The

cpr6

mutanthas high constitutive SA and

PDF1.2

levels and provides ev-idence for an SA-dependent expression of

PR-1

,

PR-2

, and

PR-5

that is independent of NPR1/NIM1/SAI1 (Clarke et al.,1998). The

dnd1

(for defense no cell death) mutant also ex-hibits high constitutive levels of SA,

PR-1

, and

PR-2

and abroad spectrum of resistance (Yu et al., 1998). The

dnd1

mutant, however, does not undergo an HR reaction in agene-for-gene resistance response. Enhanced disease sus-ceptibility (

eds

) mutants are compromised in resistance(Glazebrook et al., 1996; Parker et al., 1996; Rogers andAusubel, 1997; Volko et al., 1998); however, their SA levelshave not been reported. Interestingly,

eds5-1

is only defec-tive in the expression of

PR-1

, but not in that of

PR-2

or

PR-5

(Rogers and Ausubel, 1997).In addition to being important for SAR, SA has been found

to be necessary but not sufficient for the accumulation ofthe phytoalexin camalexin (Zhao and Last, 1996; Zhou et al.,1998). In Arabidopsis plants that express NahG, the tryp-tophan pathway and accumulation of camalexin are down-regulated coordinately (Zhao and Last, 1996). However, the

cpr1

mutant shows no alterations in the pathway leading tothe formation of tryptophan or camalexin, despite havinghigh levels of SA (Zhao and Last, 1996). The

pad4

mutant isimpaired in the accumulation of SA and camalexin as well asin the expression of

PR-1

after infection with virulent bacte-

ria. Application of SA to

pad4

plants restores camalexin pro-duction and

PR-1

expression in the presence of the bacteria(Zhou et al., 1998).

In this study, we describe two Arabidopsis mutants thatdo not accumulate SA after pathogen infection. These SAinduction–deficient (

sid

) mutants are more susceptible to

P.syringae

and

Peronospora parasitica

and show a reducedSAR response. After pathogen inoculation, only

PR-1

ex-pression was strongly reduced in

sid

mutants, whereas

PR-2

and

PR-5

were still normally expressed. Furthermore,

sid

mutants had normal levels of camalexin after bacterial infec-tion. A model of pathogen response pathways in

sid

mu-tants is presented.

RESULTS

sid1

and

sid2

Do Not Accumulate SA afterPathogen Inoculation

Approximately 4500 Arabidopsis plants (accession Colum-bia [M

2

]) mutagenized by using ethyl methanesulfonate werescreened for altered levels of total (free and conjugated) SAafter inoculation with the avirulent strain

P. syringae

pv

to-mato

DC3000 carrying the avirulence gene

avrRpm1

. Fourmutant lines that accumulate much less SA in comparison towild-type plants were isolated; they were called

sid

becausethey were deficient in the induction of SA accumulation.

Genetic complementation tests indicated that the fourmutant lines fall into two complementation groups, onegroup consisting of

sid1

and the other of three alleles of

sid2

(

sid2-1

,

sid2-2

, and

sid2-3

; data not shown). Therefore, twoloci,

SID1

and

SID2

, were identified by mutation. Leaf ex-tracts from each mutant were analyzed by using HPLC toquantify free and conjugated SA after inoculation with theavirulent strain

P. s. tomato

DC3000 carrying

avrRpt2

. Fig-ure 1 shows an

z

10- to 20-fold reduction in the amounts offree and conjugated SA in

sid1

and

sid2

mutants when com-pared with the wild type. The amounts of free SA in

sid

mu-tants were similar to that in NahG plants, whereas slightlyhigher amounts of conjugated SA were observed for the

sid1

mutant (Figure 1). Similar results were obtained after in-fection with the virulent strain

P. s. tomato

DC3000 and withavirulent strains carrying the

avrRpm1

or

avrRps4

gene (datanot shown).

Genetic Characterization

Measurements of the amount of SA in pathogen-inoculatedF

1

hybrids from crosses between wild-type plants and

sid

mutants indicated that both

sid1

and

sid2

mutants are re-cessive (total SA content:

SID1

/

sid1

, 2.45

6

0.15

m

g/g freshweight,

n

5

3;

SID2

/

sid2

, 2.37

6

0.378

m

g/g fresh weight,

SA Induction–Deficient Mutants 1395

n

5

3; Columbia [Col-0], 3.27

6

0.535

m

g/g fresh weight,

n

5

3). Both

sid1

and

sid2

segregated in the F

2

progeny assingle Mendelian loci (

SID1:sid1

, 45:9 [0.5

, P , 1.0, x2 test]and SID2:sid2, 43:9 [0.5 , P , 1.0, x2 test]). To map thegenes on the Arabidopsis genome, we outcrossed sid1 andsid2 to Arabidopsis accession Landsberg erecta (Ler), andF2 plants were scored for cosegregation of low SA levelswith simple sequence length polymorphisms (Bell andEcker, 1994) and cleaved amplified polymorphic sequencemarkers (Konieczny and Ausubel, 1993). Analysis of 48 sid1plants selected from a Ler 3 sid1 F2 population mappedsid1 to the lower arm of chromosome 4, at a distance of z2centimorgans from the marker nga1107. Analysis of 48 sid2plants from a Ler 3 sid2 F2 population mapped sid2 be-tween AthATPase and g17311 on the lower arm of chromo-some 1.

sid1 and sid2 Have a Blockage in SA Biosynthesis

Accumulation of SA has been found to be triggered by vari-ous abiotic stresses that also lead to pathogen resistance

(Yalpani et al., 1994). sid mutants were analyzed for theirability to accumulate SA after callus formation or after sub-jecting plants to abiotic stresses, such as UV-C or ozone.Ozone fumigation and callus formation gave rise to substan-tial amounts of SA in wild-type plants, as illustrated in Fig-ures 2A and 2B, whereas UV-C only weakly induced SAsynthesis (data not shown). However, none of thesestresses could trigger accumulation of SA in sid mutants(Figures 2A and 2B). Because lack of SA accumulation couldbe explained by a higher rate of SA degradation, the endog-enous SA content was analyzed in sid mutants after expo-sure to high amounts of SA. Figure 2C shows that after asoil-drench treatment with 1 mM SA, the amounts of totalSA were as high in sid1 and sid2 mutants as in wild-typeplants, whereas NahG plants had a very low content of SA.Thus, the low levels of SA in sid mutants do not result fromenhanced degradation of SA but rather from a blockage in

Figure 1. Endogenous Levels of Free and Conjugated SA in Leavesof Arabidopsis Col-0 Plants, NahG Plants, and sid Mutants after In-oculation with P. s. tomato DC3000 Carrying avrRpt2.

Leaf material was analyzed 2 days after inoculation with a bacterialsuspension of OD600 of 0.2. The values represented are the averagefor three treated plants (6SE). The experiment was repeated twicewith similar results. Conj, conjugated SA; FW, fresh weight.

Figure 2. Total Amounts of SA in Leaves of Arabidopsis Col-0Plants, NahG Plants, and sid Mutants.

(A) Leaf tissue 24 hr after ozone treatment.(B) Callus.(C) Leaf tissue 1 day after drenching the soil with 1 mM SA was ana-lyzed. Each histogram represents the average of two samples con-taining callus material or leaf material of treated plants (6SE).The experiment was repeated once for (A) and (B) or twice for (C)with similar results. FW, fresh weight.

1396 The Plant Cell

the steps leading to the biosynthesis of SA after pathogeninfection.

sid1 and sid2 Have Enhanced Susceptibility toP. parasitica

SA has been shown to play an important role in resistanceto the obligate biotroph P. parasitica (Mauch-Mani andSlusarenko, 1996). To investigate whether sid1 and sid2 mu-tants were more susceptible to virulent P. parasitica isolates,we inoculated 4-week-old plants with the P. parasitica iso-late NOCO (105 conidiospores per mL), and the number ofadult leaves showing conidiospores was scored 7 dayslater. Whereas sporulation only occurred on z40 to 50% ofthe wild-type leaves, sporulation was observed on 75 to85% of the leaves of sid1 and sid2 mutants. In comparison,nearly 100% of the leaves of NahG plants showed sporula-tion under the same conditions.

When NahG plants were inoculated with isolates of P. par-asitica that are avirulent on Arabidopsis accession Col-0, noHR reaction was observed, and the microorganism couldcomplete its reproductive cycle, although growth of the hy-phae was surrounded by a trailing necrosis indicating an in-termediate resistance (Delaney et al., 1994). The resistancephenotype of sid mutants to the incompatible P. parasiticaisolates EMWA and WELA (105 conidiospores per mL) there-fore was determined. Microscopic investigation of lactophe-nol–trypan blue—stained leaves at 1, 3, 5, 7, and 9 daysafter infection showed that both sid mutants have a similarlydecreased resistance to P. parasitica WELA and EMWA. Theoomycete could complete its reproductive cycle, formingboth oospores and conidiospores, although the growing hy-phae were surrounded by a trailing necrosis similar to that inNahG plants. However, conidiospore formation was in gen-eral somewhat lower than in NahG plants. Typical reactionsof wild-type plants, NahG plants, and sid mutants are pre-sented in Figures 3A to 3D.

sid Mutants Are More Susceptible toP. s. tomato DC3000

Resistance to pathogens was investigated further using in-oculation with P. syringae. After inoculations with a low titer(z105 colony-forming units per mL) of the virulent P. s. to-mato DC3000 strain, sid mutants developed enhanced dis-ease symptoms (data not shown) and supported strongerbacterial growth in comparison to wild type, as shown inFigure 4A. Although sid1 and sid2 mutants react with an HRin ,24 hr after a high-titer inoculation (z108 colony-formingunits per mL) with avirulent strains of P. s. tomato DC3000carrying the avrRpm1, avrRpt2, or avrRps4 gene, reducedresistance could be observed after a low titer inoculation(z105 colony-forming units per mL), as indicated by en-hanced growth of P. s. tomato DC3000 carrying avrRpt2

Figure 3. Resistance Response of Arabidopsis Col-0 Plants, NahGPlants, and sid Mutants to Incompatible P. parasitica Isolates.

Nine days after spray inoculation of 2-week-old plants with 105 con-idiospores per mL of P. parasitica WELA ([A], [B], and [D]) or EMWA(C), respectively, leaves were stained with lactophenol–trypan blueand viewed under the microscope.(A) Col-0 with blue staining of typical HR reactions.(B) NahG plants with blue staining of hyphae, oospores, and necro-sis following a hypha.(C) sid1 mutant with blue staining of conidiospores.(D) sid2 mutant with blue staining of hyphae with trailing necrosis.Bars 5 25 mm.

SA Induction–Deficient Mutants 1397

spots and inducing SA-independent defense pathways thatlead to the accumulation of PDF1.2 (Penninckx et al., 1996,1998). The effect of A. brassicicola therefore was tested insid mutants to determine whether the PDF1.2 pathway isoperative in these plants. sid1 and sid2 mutants as well as

shown in Figure 4B. However, bacteria grew to a lower titerin sid mutants compared with NahG plants, as seen best 3days after inoculation (Figures 4A and 4B).

sid1 and sid2 Mutants Have an Impaired SAR Response

SAR was tested in sid mutants by giving the lower leaves ahigh-titer inoculation of P. s. tomato DC3000 carryingavrRpt2. The result was that these leaves became necrotic.Five days after the primary inoculation, plants were sprayedwith a spore suspension of P. parasitica isolate NOCO.Seven days after the secondary infection, the disease wasevaluated on the upper leaves of the rosette by using a five-step rating of sporulation. In wild-type plants, SAR occurred,as evidenced by a strong reduction of symptoms measuredon the basis of either the total number of leaves showingsporulation or the degree of sporulation (Table 1). By con-trast, NahG plants showed severe disease symptoms, withalmost 100% sporulation and collapse of the infectedleaves. sid1 and sid2 mutants were much more susceptibleto P. parasitica NOCO than were wild-type plants, but not assusceptible as their NahG counterparts.

PR Expression in sid1 and sid2 Mutants

Leaves were infected with the avirulent strain P. s. tomatoDC3000 carrying avrRpt2 to examine the expression of PRgenes in infected and systemic leaves. RNA gel blots of leafsamples taken 2, 3, 4, and 5 days after inoculation wereprobed for PR-1 (unknown function), PR-2 (b-1,3-gluca-nase), and PR-5 (thaumatin-like protein; Uknes et al., 1992).Figure 5 shows the expression of these PR genes in unin-fected, infected, and uninfected systemic leaves of infectedplants 4 days after pathogen inoculation. PR-1 expressionwas reduced z10-fold in infected and systemic leaves of sidmutants to a level similar to NahG plants. However, the ex-pression of PR-2 and PR-5 in both infected and systemicleaves of sid mutants remained similar to that in controls inmost experiments. In some experiments, a slight reductionin the expression of either PR-2 or PR-5 could be seen. Sim-ilar results were obtained from plants 2 and 3 days after in-oculation with P. s. tomato DC3000 carrying avrRpt2, and 2and 4 days after infection with P. s. tomato DC3000 (datanot shown). As shown in Figure 6, PR-1 expression could berestored to the wild-type level in both sid mutants by the ap-plication of the SAR inducers INA and BTH, indicating thatthe signal transduction pathway downstream of SA is not af-fected in sid1 and sid2 mutants.

PDF1.2 Is Induced Normally in sid Mutants

Arabidopsis reacts to inoculation with the necrotizing fungusAlternaria brassicicola by developing small dark necrotic

Figure 4. Growth of the Virulent Strain P. s. tomato DC3000 and ofthe Isogenic Strain Carrying the Avirulence Gene avrRpt2 in Leavesof Arabidopsis Col-0 Plants, NahG Plants, and sid Mutants.

(A) P. s. tomato DC3000.(B) P. s. tomato DC3000 carrying avrRpt2.Leaves were analyzed for bacterial density at different time pointsafter inoculation with 0.5 3 103 bacteria per cm2. Data points repre-sent the means of three pools derived from eight replicate samples(6SE; see Methods). By using the Student-Newman-Keuls analysisperformed for the 3-day values, we placed sid mutants in a separategroup compared with NahG plants and Col-0 with a significancelevel of 0.01. The experiments were repeated twice with similar re-sults. cfu, colony-forming unit; diamonds, Col-0; squares, NahG; tri-angles, sid1; circles, sid2.

1398 The Plant Cell

NahG and wild-type plants developed similar necrotic spotsafter inoculation with A. brassicicola (data not shown). InFigure 7, the results of a RNA gel blot analysis of leaves 2days after inoculation with A. brassicicola is presented.Shown is a strong induction of PDF1.2 in wild-type andNahG plants as well as in sid1 and sid2 mutants. Therefore,the SA-independent defense pathway leading to the induc-tion of PDF1.2 seems to operate normally in sid1 and sid2mutants.

sid Mutants Accumulate Camalexin Normally

Accumulation of camalexin has been shown to be affectedby the amount of SA in leaves (Zhao and Last, 1996; Zhou etal., 1998). After inoculation with P. s. tomato DC3000 carry-ing avrRpt2, the camalexin levels in sid1 and sid2 werehigher in comparison to wild-type plants and much higher incomparison to NahG plants, as shown in Figure 8. After in-fection with P. s. tomato DC3000, a similar tendency was

Table 1. SAR Analysis of Arabidopsis Col-0 Plants, NahG Plants, and sid Mutants Infected with P. parasitica Isolate NOCO after Preinoculation of Lower Leaves with P. s. tomato DC3000 Carrying avrRpt2

Disease Ratingb

Genotype Treatmenta 0 1 2 3 4No. of LeavesAnalyzed

No. of Leaveswith Sporulation

Leaves withSporulation (%)c

Col-0 Mock 35 3 12 9 0 59 24 41SAR 112 23 9 2 0 146 34 23

NahG Mock 3 0 0 0 45 48 45 94SAR 5 0 0 0 103 108 103 95

sid1 Mock 8 3 18 24 0 53 45 85SAR 35 7 32 66 0 140 105 75

sid2 Mock 11 4 15 24 0 54 43 80SAR 34 18 28 30 0 110 76 69

a Five days after inoculation of five leaves with a high-titer inoculation of a bacterial suspension (SAR) or H2O (mock).b A rating was given according to the intensity of the sporulation: 0, no sporulation; 1, few sporangia; 2, ,50% of the leaf area covered by spo-rangia; 3, .50% of the leaf area covered by sporangia; 4, heavily covered with sporangia, with additional chlorosis and leaf collapse.c The experiment was repeated twice with similar results.

Figure 5. Expression of PR-1, PR-2, and PR-5 in Leaves of Arabidopsis Col-0 Plants, NahG Plants, and sid Mutants after High-Titer Inoculationwith P. s. tomato DC3000 Carrying avrRpt2.

Four days after inoculation, infected, uninfected systemic leaves of infected plants, and leaves of control plants were harvested. Two indepen-dent samples containing RNA (10 mg) of two treated plants were analyzed together with one control sample (C, control; I, infected leaves; S, un-infected systemic leaves of infected plants). Membranes were probed with PR-1, PR-2, and PR-5, respectively, and with the 18S rDNA asinternal standard. The experiment was repeated twice with similar results.

SA Induction–Deficient Mutants 1399

observed (data not shown). The enhancement of camalexinaccumulation in sid mutants might reflect a larger proportionof the leaf area reacting to pathogen infection in sid mutantsin comparison to wild-type plants. However, it is striking thatsid mutants are able to accumulate high levels of camalexinindependently of SA.

sid1 Is Allelic to eds5

Similar to sid mutants, the eds5 mutant exhibits enhanceddisease susceptibility toward virulent strains of P. syringae(Rogers and Ausubel, 1997; Volko et al., 1998). The eds5-1mutant is characterized by reduced expression of PR-1, butunaltered expression of PR-2 and PR-5 and unaltered accu-mulation of camalexin in response to infection with virulentbacteria (Rogers and Ausubel, 1997). sid1 and eds5 map tosimilar positions on the Arabidopsis genome. Therefore, theamounts of SA after pathogen infection were measured ineds5-1 (Rogers and Ausubel, 1997) and eds5-2 (Volko et al.,1998). Four days after infection with P. s. tomato DC3000carrying avrRpt2, SA had accumulated to similar levels insid1 and eds5 mutants (sid1, 38.3 6 2.4 ng/g fresh weight;eds5-1, 30.5 6 1.7 ng/g fresh weight; eds5-2, 34.0 6 3.3 ng/gfresh weight). Complementation analyses were performedbetween sid1 and eds5-1 and eds5-2, respectively. The F1

generations of the crosses eds5-1 3 sid1 and eds5-2 3 sid1were analyzed for accumulation of SA 3 days after inocula-tion of 3-to-4-week-old plants with P. s. tomato DC3000carrying avrRpt2. The amounts of SA in the F1 plants eds5-1/sid1 and eds5-2/sid1 were 403 6 49 (n 5 4) and 460 6

110 ng/g fresh weight (n 5 4), respectively. These values aresimilar to those of the parents (eds5-1, 394 6 55 ng/g freshweight [n 5 3]; eds5-2, 423 6 87 ng/g fresh weight [n 5 3];sid1, 658 6 138 ng/g fresh weight [n 5 3]; as a comparisonCol-0, 5340 6 545 ng/g fresh weight [n 5 3]), indicating that

sid1 belongs to the same complementation group as eds5.sid1 will therefore be referred to as eds5-3 in subsequentpublications. The small differences in the amounts of SA in-dicate that eds5-1 is a tighter allele than is sid1.

DISCUSSION

Both sid1 and sid2 mutants have very low levels of SA afterinfection by virulent or avirulent bacterial and fungal patho-gens. SA levels in these mutants do not increase in re-sponse to a variety of abiotic stresses, and the capacity ofthe mutants to degrade SA is not enhanced. Therefore, bothsid mutants are very probably affected in the pathway lead-ing to the biosynthesis of SA. sid mutants are more suscep-tible to local infection by a virulent strain of P. syringae aswell as a virulent isolate of P. parasitica and are also moresusceptible to pathogens that are normally avirulent on Ara-bidopsis accession Col-0. Although sid mutants exhibit anormal HR reaction ,1 day after high-titer inoculation withavirulent bacteria, P. parasitica isolates that normally triggeran HR response can grow and sporulate in sid mutants.Avirulent bacteria grow to a higher titer in sid mutants thanthey do in wild-type plants when inoculated at low titers.SAR is reduced but not totally suppressed in sid mutants,indicating that both local and systemic resistance dependon normal SA levels.

Similar results have been observed for transgenic Arabi-dopsis plants that express NahG, an SA hydroxylase from P.putida that converts SA to catechol (Delaney et al., 1994).However, sid mutants exhibit less severe phenotype alter-ations after most pathogen infections in comparison toNahG plants. Interestingly, the different levels of resistanceare reflected in the pattern of PR gene expression. Whereas

Figure 6. PR-1 Expression in Leaves of Arabidopsis Col-0 Plants,NahG Plants, and sid Mutants after the Application of SAR Inducers.

Soil was drenched with wetting agents for 2 days (C), 5 ppm INA (I)for 2 days, or 330 ppm BTH (B) for 1 day before harvesting the leafmaterial. Ten micrograms of total RNA was probed with PR-1 andwith the 18S rDNA as internal standard. The experiment was re-peated once with similar results.

Figure 7. Expression of PDF1.2 in Arabidopsis Col-0 Plants, NahGPlants, and sid Mutants after Inoculation with A. brassicicola.

Two days after inoculation with A. brassicicola (I) or H2O (C), leavesof two plants were harvested together for each treatment. Ten mi-crograms of RNA was probed with PDF1.2 and with the 18S rDNAas an internal standard. The experiment was repeated once withsimilar results.

1400 The Plant Cell

expression of PR-1 was abolished to a similar extent in sidmutants and NahG plants, PR-2 and PR-5 were expressedto wild-type levels in sid mutants, but their expression wasstrongly reduced in NahG plants.

The absence of coordinate regulation between PR-1 andPR-2/PR-5 observed in sid mutants stands in contrast withother results showing that PR-1, PR-2, and PR-5 are regu-lated coordinately. For example, application of SA or SARinducers, such as INA or BTH, induces the expression ofPR-1, PR-2, and PR-5 coordinately (Uknes et al., 1992;Görlach et al., 1996). In addition, the npr1/nim1 mutant (Caoet al., 1994; Delaney et al., 1995; Shah et al., 1997) affectsexpression of PR-1, PR-2, and PR-5, whereas cpr mutantsconstitutively express all three PR genes (Bowling et al.,1994, 1997; Clarke et al., 1998). However, different lines ofevidence hinted at the existence of an SA-independent PR-2/PR-5 expression after inoculation with P. syringae. For ex-ample, after pathogen infection, npr1 plants are deficient inPR-1 expression but still express PR-2 and PR-5 (Glazebrooket al., 1996). However, from these results, it could not be de-termined whether this expression of PR-2/PR-5 is SA de-pendent or independent. Furthermore, the pad4 mutant,which has normal expression of PR-2/PR-5, but diminishedexpression of PR-1 and a decreased accumulation of cama-lexin after infection with virulent pathogens, has been foundto be affected in its ability to accumulate SA (Zhou et al.,1998). In addition, eds5-1 has been reported to be specifi-cally affected in its expression of PR-1, but not in its expres-sion of PR-2/PR-5 (Rogers and Ausubel, 1997).

Our findings that SA does not accumulate in eds5-1 and

eds5-2 after pathogen inoculation and that sid1 is allelic toeds5 confirm the indications that inoculation with P. syrin-gae leads to SA-independent expression of PR-2/PR-5. Whythis pathway for the expression of PR-2/PR-5 is not active inNahG plants remains an open question.

Because eds5-1 does not accumulate SA after infectionwith pathogens, the observation that eds5-1 npr1 doublemutants are more susceptible to Erysiphe orontii and have alower expression of PR-1 than do both parental lines cannotbe explained by EDS5 acting downstream of SA (Reuber etal., 1998). However, it is more likely that the eds5 mutationinterrupts the pathway upstream of SA. SA might activateseveral proteins that act in synergy to induce the expressionof PR-1. In addition, both mutants may be slightly leaky;double mutants thus could give a stronger phenotype, al-though being part of the same pathway (Reuber et al., 1998).

Another major difference between NahG plants and sidmutants is the different level of accumulation of camalexin.In both sid mutants, camalexin was found to accumulate tohigh levels in contrast to NahG plants. The low amount ofcamalexin in NahG plants has been interpreted to indicatethat SA is essential for the normal expression of the camal-exin pathway (Zhao and Last, 1996). One possible explana-tion for the differences in the pathogen response pathwaysleading to accumulation of camalexin between sid mutantsand NahG plants might lie in fundamental changes of theirrespective metabolisms. Because SA is permanently de-graded in NahG plants, the metabolic drain from the phenyl-propanoid pathway may cause a downregulation of thecamalexin pathway, which branches off further upstream inthe phenylpropanoid pathway. However, the pad4 mutantshows reduced accumulation of camalexin that could berescued by the application of SA when the pathogen waspresent (Zhou et al., 1998).

Figure 8. Accumulation of Camalexin in Leaves of ArabidopsisCol-0 Plants, NahG Plants, and sid Mutants after Inoculation withP. s. tomato DC3000 Carrying avrRpt2.

Two days after inoculation with a bacterial suspension, leaves wereanalyzed for accumulation of camalexin. The data represent meansof three replicate samples (6SE). sid mutants were placed in a sepa-rate group compared with Col-0 and NahG plants by using theStudent-Newman-Keuls analysis with a significance level of 0.01.The experiment was repeated twice with similar results.

Figure 9. Model for the Role of sid Mutants in Pathogen ResponsePathways.

CA, camalexin; JA, jasmonic acid; question mark, unknown signal-ing molecule; double lines crossing an arrow, blocked pathway; Tbar crossed by double lines, blocked inhibitor function.

SA Induction–Deficient Mutants 1401

This observation seems to be in contradiction with the resultsreported in this study. Normal accumulation of camalexinalso has been reported for eds5-1 mutants (Rogers andAusubel, 1997). The normal accumulation of camalexin insid1/eds5 and sid2 mutants may be explained by an SA-independent compensation pathway that is only active insid1/eds5 and sid2 mutants. Such a pathway might be spe-cifically activated by the presence of metabolic precursorsthat accumulate if enzymes leading to the synthesis of SAare affected in sid mutants. It is also possible that SID pro-teins have a dual regulatory function in activating the path-ways that depend on SA and inactivating pathways that donot depend on SA. In the differentiation pathway of yeast,MAP kinases have been found to have such dual regulatoryfunctions (Madhani et al., 1997). However, it also might bepossible that SA regulates several pathways with differentaffinity.

A working model for the signal transduction pathways insid mutants is proposed in Figure 9. sid mutants are placedupstream of SA and downstream of HR and of the branchpoint of the SA-independent pathways, leading either to ex-pression of PDF1.2 or PR-2/PR-5 or to the SA-independentpathway for accumulation of camalexin. A closer geneticpositioning of the eds5/sid1 and sid2 mutants in the patho-gen response pathways might be obtained in epistasis ex-periments between different mutants affected in pathogendefense, such as the cpr1, cpr5, and cpr6 mutants (Bowlinget al., 1994, 1997; Clarke et al., 1998).

It is also noteworthy that no significant difference ingrowth of bacteria after infection with P. syringae pv maculi-cula ES4326 carrying avrRpt2 was observed among eds5-1,eds5-2, and wild-type plants (Rogers and Ausubel, 1997;Volko et al., 1998), whereas the resistance was strongly di-minished in the eds5 mutants after infection with theisogenic virulent strain. Because eds5/sid1 is defective inaccumulation of SA after inoculation with avirulent patho-gens (see above), it can be concluded that eds5 can estab-lish a race-specific incompatible reaction in the absence ofSA after inoculation with certain pathovars of P. syringae.The bacterial growth experiments presented in this studyshow a small but significant growth enhancement of P. s. to-mato DC3000 carrying avrRpt2 when inoculated onto sid1/eds5 plants. In addition, experiments with incompatible iso-lates of P. parasitica showed that resistance to incompatiblepathogens is decreased (see above). SA can therefore affectresistance to both virulent and avirulent pathogens, as hasbeen shown with NahG plants.

In conclusion, the identification of sid mutants providesnew information on the relationship between SA and SAR,on the induction pathways for PR proteins, and the accumu-lation of camalexin. The finding that eds5 and sid1 fall intothe same complementation group adds evidence to thegeneral notion that a defense network is present in plantsand that different branches of the network may contributedifferently to resistance against different pathogens. Char-acterization of the eds5/sid1 and sid2 genes will provide fur-

ther information on the elements involved in the induction ofSA accumulation.

METHODS

Growth Conditions for Plants, Fungi, and Bacteria

Ethyl methanesulfonate–mutagenized M2 seeds of Arabidopsisthaliana accession Columbia (Col-0; a gift from C.R. Somerville, Car-negie Institution, Stanford, CA) and ethyl methanesulfonate–muta-genized Col-0 M2 seeds homozygous for a mutation in ferulic acid5-hydroxylase (fah1-2) (Chapple et al., 1992; Glazebrook et al., 1996;a gift from E. Rogers and F. Ausubel, Massachusetts General Hospi-tal, Boston, MA) were used for the mutant screen. Salicylic acid (SA)induction–deficient mutants sid1 and sid2-1 were isolated from Col-0/fah1-2 M2 seeds, whereas sid2-2 and sid2-3 were isolated from Col-0M2 seeds. For physiological analyses, plant lines of sid1 and sid2-1backcrossed at least twice, but usually four or five times were used.Because of linkage between sid1 and fah1, sid1 still carries the fah1-2background mutation that has been shown not to influence plant–pathogen interactions (Glazebrook et al., 1996).

Plants were grown on a pasteurized soil mix of humus/perlite (3:1)under a 16-hr-light and 8-hr-dark cycle for genetic analysis and a12-hr-light and 12-hr-dark cycle for physiological experiments, witha night temperature of 168C and a day temperature of 20 to 228C (60to 70% humidity), when not otherwise indicated.

Arabidopsis accessions Col-0, Landsberg erecta (Ler), andWassilewskija were obtained from the Arabidopsis Biological Re-search Center (Columbus, OH). NahG plants were obtained from J.Ryals, Novartis Corp, Basel, Switzerland.

Peronospora parasitica isolate NOCO was transferred every weekon 2-to-3-week-old Arabidopsis accession Col-0 plants by spray in-oculation with a spore suspension. P. parasitica isolate WELA wascultivated in a similar manner every 10 days on Arabidopsis acces-sion Ler, and P. parasitica isolate EMWA was cultivated every weekon accession Wassilewskija (Holub et al., 1994; isolates obtainedfrom B. Mauch-Mani, University of Fribourg, Fribourg, Switzerland).Plants inoculated with P. parasitica were kept in a 12-hr-light and12-hr-dark cycle with 198C day temperature and 168C night temper-ature and, for the first day and the last day of the growth cycle, under100% humidity to ensure infection and sporulation, respectively(Dangl et al., 1992).

Alternaria brassicicola was cultivated on half-strength potato dex-trose agar at 208C (Penninckx et al., 1996).

Strain DC3000 of Pseudomonas syringae pv tomato and theisogenic strains carrying the avirulence genes avrRpt2, avrRpm1, oravrRps4 (Whalen et al., 1991) were cultured at 288C and 220 rpm inLuria-Bertani or Kings B medium (pH 7.0, 10 mg/mL protease pep-tone, 15 mg/mL glycerol, 1.5 mg/mL K2HPO4, and 4 mM MgSO4)containing the appropriate antibiotic.

Mutant Screen

Five to 6-week-old M2 plants were syringe-inoculated with P. s. to-mato carrying avrRpm1 at a titer of z108 colony-forming units per mL(OD600 nm 0.2) on four to five leaves by infiltrating z20 to 25% of the

1402 The Plant Cell

leaf surface. Four days after inoculation, 0.05 to 0.20 g of leaf mate-rial was analyzed for total SA content.

Measurements of SA and Camalexin

For the analysis of total SA and camalexin, 5-to-6-week-old plantswere inoculated on approximately one-quarter to one-fifth of the leafsurface and analyzed individually, when not described otherwise.When 3-to-4-week-old plants were analyzed, z30 to 50% of the leafwas inoculated and the leaves of several plants were pooled. TotalSA and camalexin were determined by extracting 0.1 to 0.2 g ofleaves with 90% methanol at 658C for 4 hr in the presence of 250 ngof o-anisic acid as internal standard (Meuwly and Métraux, 1993). Af-ter evaporation of the methanol in a vortex evaporator, each samplewas incubated with 2 mg of b-glucosidase (Fluka, Buchs, Switzer-land) in 150 mL of 0.1 M Na acetate, pH 5.5, for 1.5 hr at 308C. Afterprecipitation with 0.5 mL of 5% trichloroacetic acid, the phenol com-pounds, including camalexin, were partitioned against cyclohexane/ethyl acetate (1:1).

For the analysis of free and conjugated SA and camalexin, 0.1 to0.3 g of leaves (with 250 or 1000 ng of o-anisic acid) were extractedonce with 2 mL of 70% methanol and once with 2 mL of 90% meth-anol by using a homogenizer (Polytron; Kinematica, Littau, Switzer-land). After evaporation of the methanol from the combined extractsand trichloroacetic acid precipitation as described above, free phe-nols and camalexin were extracted into cyclohexane/ethyl acetate(1:1). The remaining aqueous phase was submitted to acid hydrolysisin the presence of 4 N HCl at 808C for 1 hr, and the liberated phenolswere extracted into cyclohexane/ethyl acetate, as described above.

For HPLC, the organic phase was evaporated, and the sampleswere resuspended in 85% phosphate buffer/15% acetonitrile. Chro-matography was performed on a reverse phase HPLC column(ABZ1, 25 cm 3 4.6 mm; Supelco, Buchs, Switzerland) (Meuwly andMétraux, 1993). The detection limit of SA was 2 ng.

RNA Gel Blot Analysis

RNA (1 g) was extracted with 3 mL of 2 M Tris-HCl, pH 8.0, 0.5 MEDTA, pH 8.0, and 20% SDS (1:2:1) (Chirjwin et al., 1979). The aque-ous phase was extracted once with 1 volume of phenol–chloroform–isoamyl alcohol (50:49:1) and twice with 1 volume of chloroform andprecipitated with 1 volume of 6 M LiCl overnight at 48C. The RNA wasresuspended in H2O and reprecipitated with one-tenth of a volume of3 M NaAc, pH 5.5, and 3 volumes of ethanol. Ten micrograms of RNAwas separated on a formaldehyde/agarose gel, visualized under UVlight, and transferred to a Nylon membrane (Hybond-N; AmershamPharmacia Biotech, Little Chalfont, UK). The membrane was probedwith the cDNA of pathogenesis-related proteins PR-1, PR-2, PR-5(Uknes et al., 1992), and PDF1.2 (Penninckx et al., 1996). As internalstandard, the membranes were probed with a gene encoding the18S RNA of tomato (Kiss et al., 1988).

Treatments with Chemicals, UV Light, and Ozone

Two-to-three-week-old plants were soil-drenched with 1 mM SA forSA uptake experiments. To induce PR proteins, we soil-drenched2-to-3-week-old plants with 5 ppm of 2,6-dichloroisonicotinic acid(INA; Novartis Corp, Basel, Switzerland) or 330 mM benzo(1,2,3)thia-

diazole-7-carbothioic acid S-methyl ester (BTH; Novartis Corp,Basel, Switzerland). Two-to-three-week-old plants were illuminatedfor 20 min with 254 nm UV-C light at a 30-cm distance. Four-week-old plants were subjected twice to 300 ppm of ozone for 6 hr at 24-hrintervals. Samples were taken after 3, 6, 12, 18, 24, and 48 hr fromthe beginning of the fumigation.

Callus Induction

Leaves were surface-sterilized with a 5% sodium hypochlorite and0.01% Triton X-100 solution and incubated on callus-inducingmedium (Axelos et al., 1992). Emerging calli were maintained onMurashige and Skoog medium (Sigma, Buchs, Switzerland) contain-ing 0.2 g/L glutamine, 1 mg/L 2,4-D, and 0.3 mg/L kinetin.

Inoculation with P. syringae

The hypersensitive response (HR) was induced by inoculation with abacterial suspension of z108 colony-forming units per mL in 10 mMMgCl2. To examine the growth of the bacteria, we inoculated 5-to-6-week-old plants with a bacterial suspension of z105 colony-formingunits per mL. The bacterial titer was determined from three leaves(0.5 cm2 discs per leaf) per plant from six to eight replicates at differ-ent time points after inoculation. To obtain the mean values and stan-dard error, we analyzed either three leaves of each plant together,and each replicate plant was taken separately, or we pooled one in-fected leaf disc per plant from the different replicates, resulting inthree pools. Both methods gave similar results.

Inoculation with P. parasitica

To examine local resistance to P. parasitica isolate WELA or EMWA,2-to-3-week-old plants were sprayed with a suspension of 105 con-idiospores per mL and incubated as described above. Either plantswere examined for sporulation or leaf samples were stained with lac-tophenol–trypan blue every 2 days and examined under the micro-scope (Koch and Slusarenko, 1990). To examine virulence or thesystemic acquired resistance response, we challenge-inoculated4-to-5-week-old plants 5 days after treatment with H2O (mock) or high-titer inoculation with P. s. tomato DC3000 carrying avrRpt2 (z108

colony-forming units per mL) with 105 conidiospores per mL of P.parasitica isolate NOCO, and sporulation was examined 7 days later.

Inoculation with A. brassicicola

Four-to-five-week-old plants were inoculated with droplets (6 3 5mL) of a spore suspension (106 spores per mL) on five leaves perplant. After 2 days at 100% humidity, plants were visually examined,and inoculated leaves were harvested for RNA gel blot analysis.

ACKNOWLEDGMENTS

We gratefully acknowledge the financial support of the Swiss Na-tional Science Foundation (Grant Nos. 31-34098 and 31-55662.98 toJ.-P.M.) and the EMBO and Novartis Foundations (fellowships to

SA Induction–Deficient Mutants 1403

C.N.). We thank Drs. Antony Buchala and Ghislaine Rigoli for helpwith HPLC; Drs. Roger Innes, Jeff Dangl, Brigitte Mauch-Mani, andWilliam Broekaert for providing pathogens and for helpful discus-sions; and Drs. Fred Ausubel and Chris Somerville for supplying uswith mutagenized seed and seed of eds5-1 and eds5-2 mutants. Weare grateful to Dr. Jürg Fuhrer for performing the ozone fumigationsand to Drs. Antony Buchala, Thierry Genoud, Brigitte Mauch-Mani,Philippe Reymond, and Yves Poirier for critical reading of themanuscript.

Received April 12, 1999; accepted May 20, 1999.

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DOI 10.1105/tpc.11.8.1393 1999;11;1393-1404Plant Cell

Christiane Nawrath and Jean-Pierre MétrauxAccumulate High Levels of Camalexin after Pathogen Inoculation

andPR-5 and PR-2Deficient Mutants of Arabidopsis Express −Salicylic Acid Induction

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