A recessive mutation in the Arabidopsis SSI2 gene confers SA- and NPR1-independent expression of PR...

A recessive mutation in the Arabidopsis SSI2 gene confersSA- and NPR1-independent expression of PR genes andresistance against bacterial and oomycete pathogens

Jyoti Shah1,2,*, Pradeep Kachroo1,², Ashis Nandi2 and Daniel F. Klessig1,²

1Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers, The State University of New

Jersey, 190 Frelinghuysen Road, Piscataway, NJ 08855-8020, USA and2Molecular, Cellular and Developmental Biology Program, Division of Biology, Kansas State University, 303 Ackert Hall,

Manhattan, KS 66506-4901, USA

Received 6 December 2000; accepted 21 December 2000.*For correspondence (fax +1 785 532 6653; e-mail [email protected]).²Present address: Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853, USA.

Summary

The Arabidopsis thaliana NPR1 gene is required for salicylic acid (SA)-induced expression of

pathogenesis-related (PR) genes and systemic acquired resistance. However, loss-of-function mutations

in NPR1 do not confer complete loss of PR gene expression or disease resistance. Thus these responses

also can be activated via an NPR1-independent pathway that currently remain to be elucidated. The ssi2-

1 mutant, identi®ed in a genetic screen for suppressors of npr1-5, affects signaling through the NPR1-

independent defense pathway(s). In comparison with the wild-type (SSI2 NPR1) plants and the npr1-5

mutant (SSI2 npr1-5), the ssi2-1 npr1-5 double mutant and the ssi2-1 NPR1 single mutant constitutively

express PR genes [PR-1, BGL2 (PR-2) and PR-5]; accumulate elevated levels of SA; spontaneously develop

lesions; and possess enhanced resistance to a virulent strain of Peronospora parasitica. The ssi2-1

mutation also confers enhanced resistance to Pseudomonas syringae pv. tomato (Pst); however, this is

accomplished primarily via an NPR1-dependent pathway. Analysis of ssi2-1 NPR1 nahG and ssi2-1 npr1-5

nahG plants revealed that elevated SA levels were not essential for the ssi2-1-conferred phenotypes.

However, expression of the nahG transgene did reduce the intensity of some ssi2-1-conferred

phenotypes, including PR-1 expression, and disease resistance. Based on these results, SSI2 or an SSI2-

generated signal appears to modulate signaling of an SA-dependent, NPR1-independent defense

pathway, or an SA- and NPR1-independent defense pathway.

Keywords: systemic acquired resistance, salicylic acid-independent, pathogenesis-related, NPR1-independent.

Introduction

The hypersensitive response (HR) and systemic acquired

resistance (SAR) are important components of a plant's

defense arsenal against pathogens. The HR is a rapid

defense response characterized by localized programmed

host cell death and restriction of pathogen to the site of

pathogen entry (Dempsey et al., 1999; Hammond-Kosack

and Jones, 1996; Matthews, 1991; Staskawicz et al., 1995).

Concurrent with the HR, a systemic signal is released that

induces SAR in uninfected plant tissues. SAR is long-

lasting and confers resistance against a broad spectrum of

pathogens. Tightly correlated with the appearance of HR

and SAR is the accumulation of salicylic acid (SA) and the

expression of a subset of the pathogenesis-related (PR)

genes, some of which encode proteins with antimicrobial

activities. Hence the expression of these genes serves as

an excellent molecular marker for a resistance response

(Hunt and Ryals, 1996; Malamy and Klessig, 1992).

Salicylic acid has emerged as a key signal molecule in

the activation of SAR (Dempsey et al., 1999; Durner et al.,

1997; Hammond-Kosack and Jones, 1996; Ryals et al.,

1996). Exogenously applied SA induces resistance in

many plant species (Malamy and Klessig, 1992); it acti-

vates expression of the same classes of PR genes [PR-1,

BGL2 (PR-2) and PR-5] as those induced during SAR

The Plant Journal (2001) 25(5), 563±574

ã 2001 Blackwell Science Ltd 563

(Uknes et al., 1992; Ward et al., 1991). In addition, the in vivo

increases in endogenous SA levels correlate with PR gene

induction and the development of resistance in pathogen-

infected plants (Dempsey et al., 1997; Malamy et al., 1990;

MeÂ traux et al., 1990; Summermatter et al., 1995; Uknes

et al., 1993). Moreover, plants unable to accumulate SA

due to constitutive expression of the Pseudomonas putida

nahG gene, which encodes the SA-degrading enzyme

salicylate hydroxylase, fail to develop SAR and are

hypersusceptible to pathogen infection (Delaney et al.,

1994; Gaffney et al., 1993). Preventing SA accumulation

by application of SA biosynthesis inhibitors likewise

makes otherwise resistant Arabidopsis plants susceptible

to P. parasitica (Mauch-Mani and Slusarenko, 1996).

Conversely, the elevated levels of SA present in the

following Arabidopsis mutants lead to constitutive expres-

sion of PR genes and SAR: acd (accelerated cell death;

Greenberg et al., 1994; Rate et al., 1999); lsd (lesions

simulating disease; Dietrich et al., 1994; Weymann et al.,

1995); cpr (constitutive expressor of PR genes; Bowling

et al., 1994; Bowling et al., 1997; Clarke et al., 1998; Silva

et al., 1999); ssi1 (suppressor of salicylate insensitivity of

npr1-5; Shah et al., 1999); cim (constitutive immunity;

Ryals et al., 1996); and dnd1 (defense with no HR cell

death; Yu et al., 1998). In addition to PR expression and

SAR, recent studies have suggested that SA also regulates

the activation of cell death and the restriction of pathogen

spread (Dempsey et al., 1999).

The Arabidopsis NPR1/NIM1 gene is an important

component of the SA signal transduction pathway(s).

Plants carrying npr1/nim1 loss-of-function mutations are

insensitive to SA; following treatment with SA or its

functional analogs 2,6-dichloroisonicotinic acid (INA) and

benzothiadiazole (BTH), they fail to express PR genes or to

develop SAR (Cao et al., 1994; Delaney et al., 1995;

Glazebrook et al., 1996; Shah et al., 1997). In contrast,

overexpression of NPR1 in Arabidopsis leads to increased

resistance against bacterial and oomycete pathogens (Cao

et al., 1998). However, NPR1 overexpression does not

cause constitutive activation of defense responses. Hence

NPR1 or possibly a co-inducer, may require activation by

pathogen attack before SAR can be activated. NPR1

encodes a novel, 65 kDa protein containing ankyrin

repeats (Cao et al., 1997; Ryals et al., 1997). These repeats

are involved in the speci®c interaction between NPR1 and

certain members of the TGA family of bZIP DNA binding

proteins (Despres et al., 2000; Zhang et al., 1999; Zhou

et al., 2000).

Genetic screens for suppressors of npr1 have identi®ed

additional components of the SA signaling pathway(s).

The ssi1 and sni1 mutations restore SA responsiveness

and resistance in npr1 plants (Li et al., 1999; Shah et al.,

1999). The SNI1 protein, which shows no signi®cant

homology to any known protein, was proposed to be a

Figure 1. PR and PDF1.2 expression in ssi2-1 plants.(a) Expression of the PR-1 and BGL2 genes in water-treated (W) and SA-treated (S) wt (SSI2 NPR1); npr1-5 (SSI2 npr1-5); and ssi2-1 (ssi2-1 npr1-5and ssi2-1 NPR1) plants. RNA was extracted from leaves of 4-week-old,soil-grown plants 24 h after treatment.(b) Comparison of constitutive PR-1, BGL2 and PR-5 expression in ssi2-1plants homozygous for the npr1-5 (ssi2-1 npr1-5) or nim1-1 (ssi2-1 nim1-1) alleles. SSI2 plants homozygous for npr1-5 (SSI2 npr1-5) or nim1-1(SSI2 nim1-1) served as negative controls. RNA was extracted fromleaves of 4-week-old, soil-grown plants.(c) Comparison of constitutive expression of PDF1.2 in wt (SSI1 SSI2NPR1); npr1-5 (SSI1 SSI2 npr1-5); ssi2-1 npr1-5 double mutant (SSI1 ssi2-1 npr1-5); and ssi1 npr1-5 double mutant (ssi1 SSI2 npr1-5). RNA wasextracted from leaves of 4-week-old, soil-grown plants.Gel loading was monitored by photographing the ethidium bromide-stained gel (EtBr) before transferring the RNA to a Nytran Plusmembrane. Blots were sequentially probed for the indicated genes.

564 Jyoti Shah et al.

ã Blackwell Science Ltd, The Plant Journal, (2001), 25, 563±574

negative regulator of PR gene expression and SAR. By

contrast, the SSI1 protein may function as a switch that

regulates cross-talk between SA-dependent, and JA- and

ethylene-dependent, defense responses. The ability of the

ssi1 and sni1 alleles to restore SA responsiveness in

various npr1 mutant allele backgrounds argues against

SSI1 and SNI1 functioning as NPR1-interacting proteins.

Rather, the ssi1 and sni1 mutations may suppress the npr1

mutant phenotype, either by restoring function to the SA/

NPR1 pathway, or by activating an NPR1-independent but

SA-responsive pathway.

While NPR1 is critical for transducing the SA signal that

activates resistance to some pathogens, there is growing

evidence that an SA-dependent, NPR1-independent path-

way(s) also exists. For example, the level of disease

susceptibility exhibited by npr1 mutants is signi®cantly

less severe than that displayed by SA-de®cient NahG plant

(Delaney et al., 1994, Delaney et al., 1995; Zhao and Last,

1996). Likewise, the pad4-1 mutant, which does not accu-

mulate elevated levels of SA, is more susceptible than npr1

to Erysiphe orontii (Reuber et al., 1998). PR gene expression

is also mediated via an NPR1-independent pathway in

addition to the NPR1-dependent pathway; in contrast to

NahG plants, PR expression is elevated in pathogen

infected npr1 plants (Delaney et al., 1995; Glazebrook

et al., 1996; Shah et al., 1997; Zhao and Last, 1996).

Moreover, the ability of Arabidopsis to resist infection by

certain viral, oomycete and bacterial pathogens has been

shown to be mediated by an SA-dependent, NPR1-inde-

pendent pathway(s) (Bowling et al., 1997; Clarke et al., 1998;

Kachroo et al., 2000).

Mutant screens in Arabidopsis have made signi®cant

contributions in identifying various components of the SA

signaling pathway. However, very few of these mutants

have been shown to affect the NPR1-independent signaling

pathway. The paucity of mutants in genes affecting the

NPR1-independent pathway may, in part, be due to the

NPR1 pathway masking the role of the NPR1-independent

pathway. To identify genes in the NPR1-independent

pathway, we screened the progeny of ethyl methylsulfo-

nate (EMS)-mutagenized npr1-5 plants for individuals that

exhibit constitutive PR gene expression. Through this

process, several mutants, one designated ssi2-1, were

isolated. Unlike wild-type (wt) plants and the npr1-5 mutant,

the ssi2-1 npr1-5 double mutant constitutively expressed

several defense-associated responses and exhibited

enhanced resistance to P. parasitica. The ssi2-1 mutation

also conferred enhanced resistance to Pst, although via an

NPR1-dependent pathway. Interestingly, elevated SA levels

enhanced, but were not essential for, the manifestation of

several ssi2-1-associated phenotypes. Taken together, SSI2

appears to affect the activation of several defense

responses by modulating an NPR1-independent defense

pathway.

Results

A mutation in the SSI2 gene confers constitutive PR gene

expression and spontaneous development of HR-like

lesions in npr1-5 plants

The ssi2-1 mutant was isolated by screening 3- to 4-week-

old M2 progeny of EMS-mutagenized npr1-5 plants (NoÈ

ecotype) for mutants that constitutively accumulated tran-

scripts of the PR-1, BGL2 and PR-5 genes. As shown in

Figure 1(a,b), transcripts for all these genes were detected

in ssi2-1 npr1-5 double mutant plants, but not in water-

treated wt (SSI2 NPR1) and water- or SA-treated npr1-5

Figure 2. Comparison of morphological phenotypes of ssi2-1 and SSI2plants.(a,b) Comparison of the morphological phenotype of lesion± SSI2 npr1-5(a) and lesion+ ssi2-1 npr1-5 (b) plants. The two white arrows in (b) pointto areas showing lesions. The plant in (b) is at a 23 higher magni®cationthan that in (a).(c,d) UV microscopy of a leaf from an ssi2-1 npr1-5 plant (d) revealedincreased levels of auto¯uorescent material (white arrow) around thecells associated with a spontaneous lesion. By contrast, the control SSI2npr1-5 (c) plant did not exhibit any areas of increased auto¯uorescenceabove background.(e,f) Trypan blue staining of a lesion-bearing ssi2-1 npr1-5 leaf (f)revealed intensely blue-stained regions (white arrow) which areindicative of dead cells. The control SSI2 npr1-5 plant (e) did not revealany intensely stained regions.All plants were grown in soil and photographed when 4 weeks old.

Defense signaling in plants 565


(SSI2 npr1-5) plants. Even greater levels of transcripts for

PR-1, and occasionally BGL2 and PR-5, were detected in

ssi2-1 NPR1 plants. However, exogenously applied SA did

not further increase the accumulation of PR gene tran-

scripts in the ssi2-1 npr1-5 or ssi2-1 NPR1 mutants (Figure

1a). We also monitored constitutive expression of the

defensin gene PDF1.2 in the ssi2-1 mutant. As shown in

Figure 1(c), unlike the previously characterized ssi1 (ssi1

SSI2 npr1-5) mutant, which constitutively expresses PDF1.2

(Shah et al., 1999), the wt (SSI1 SSI2 NPR1), npr1-5 (SSI1

SSI2 npr1-5) and ssi2-1 npr1-5 double mutant (SSI1 ssi2-1

npr1-5) plants do not constitutively express the PDF1.2

gene.

In addition to constitutively expressing PR genes, as

shown in Figure 2(b,d,f), ssi2-1 npr1-5 plants exhibited

several unusual phenotypes not observed in wt or npr1-5

plants (Figure 2a,c,e). These include a small rosette, curled

leaves, and the appearance of spontaneous necrotic

lesions (Figure 2b). Analysis of these lesions revealed

that they were associated with the accumulation of

auto¯uorescent material (Figure 2d) and the presence of

dead cells (Figure 2f) ± features associated with a patho-

gen-induced HR. All the morphological phenotypes asso-

ciated with ssi2-1 also were prevalent in ssi2-1 NPR1 plants

(data not shown).

Genetic characterization of ssi2-1

F1 plants derived from a backcross of the ssi2-1 npr1-5

double mutant to the SSI2 npr1-5 parent lacked all ssi2-1-

conferred phenotypes, suggesting that ssi2-1 was reces-

sive to the wt SSI2 allele. Analysis of F2 progeny con®rmed

that the ssi2-1-conferred phenotypes are due to a recessive

mutation in a single gene; segregation of the ssi2-1

phenotype was consistent with a 3 : 1 constitutive PR-1±

lesion± : constitutive PR-1+ lesion+ Mendelian ratio (81

constitutive PR-1± lesion± plants to 26 constitutive PR-1+

lesion+ plants; c2 = 0.028; 0.9 > P > 0.5; P95% c2 = 3.84;

df = 1). It is possible that a second site mutation within

the npr1-5 allele results in suppression of the npr1-5

mutant phenotype. However, the recessive nature of the

ssi2-1 mutation argues that it is not an intragenic suppres-

sor of the npr1-5 allele. This conclusion was con®rmed by

demonstrating that the ssi2-1 mutant phenotypes segre-

gate independently from the npr1-5 allele. In the F2

progeny of a cross between ssi2-1 npr1-5 and a wt (SSI2

NPR1) plant, plants were identi®ed that were homozygous

for the NPR1 allele (as determined by Cleared Ampli®ed

Polymorphic Sequences (CAPS) analysis; Shah et al.,

1999), but that exhibited spontaneous lesion formation

and constitutive PR-1 expression. Moreover, while NPR1

maps on chromosome 1, SSI2 maps on chromosome 2

(0.2 cM from AthB102 and 3.7 cM from GBF). Thus ssi2-1 is

not an intragenic suppressor of npr1-5.

If the npr1-5 allele is leaky, then a mutation in a gene

functioning upstream of NPR1, which constitutively acti-

vates signaling through NPR1, could activate expression of

the PR genes in npr1-5 containing plants. We therefore

analyzed ssi2-1-conferred phenotypes in nim1-1 (allelic

with npr1) plants. A single base pair insertion in nim1-1 is

expected to cause premature termination of NPR1, result-

ing in a truncated protein that lacks the C-terminal 349

amino acids (Ryals et al., 1997). ssi2-1 nim1-1 plants

constitutively expressed the PR genes at levels compar-

able to those observed in ssi2-1 npr1-5 plants (Figure 1b).

Furthermore, like the ssi2-1 npr1-5 plant, the ssi2-1 nim1-1

plant also developed HR-like lesions (data not shown).

These results strongly suggest that the ssi2-1 mutant

phenotypes do not require the NPR1 protein.

ssi2-1 confers enhanced resistance to pathogen infection

As the ssi2-1 mutant constitutively expresses PR genes,

and enhanced expression of these genes often correlates

with enhanced resistance, we tested whether ssi2-1 plants

also show enhanced resistance to pathogen infection. The

Table 1. Disease ratings of SSI2 and ssi2-1 plants after inoculation with P. parasitica biotype Emco5

GenotypeaTotal number ofplants inoculated Diseasedb Healthy

Range of number ofsporangiophores/cotyledon

SSI2 NPR1 (NoÈ ) 120 113 7 30±40SSI2 NPR1 (Ler) 200 0 200 0SSI2 npr1-5 146 142 4 30±40ssi2-1 NPR1 160 10 150 10±20ssi2-1 npr1-5 173 13 160 10±20SSI2 NPR1 nahG 109 109 0 >50ssi2-1 NPR1 nahG 145 70 75 10±20

aAll plants were homozygous for the designated alleles.bPlants were scored as diseased if the inoculated cotyledons exhibited ®ve or more sporangiophores.



ssi2-1 mutant is in the Arabidopsis ecotype NoÈ . The

oomycete pathogen P. parasitica isolate Emco5 is virulent

on Arabidopsis ecotype NoÈ , causing extensive growth and

sporulation. As shown in Table 1, both wt NoÈ (SSI2 NPR1)

and npr1-5 (SSI2 npr1-5) plants exhibit profuse growth and

sporulation of P. parasitica. In contrast, plants from the

Arabidopsis ecotype Landsberg (Ler), which are resistant,

did not show any fungal growth. The majority of the ssi2-1

NPR1 (150 out of 160) and ssi2-1 npr1-5 plants (160 out of

173) also did not support fungal growth (Table 1). In the

few cotyledons of ssi2-1 NPR1 and ssi2-1 npr1-5 plants

where growth and sporulation were observed, the number

of sporangiophores per cotyledon were two- to fourfold

lower than observed in the cotyledons of infected SSI2

NPR1 and SSI2 npr1-5 plants.

Previously, we had demonstrated that the npr1-5 mutant

exhibits enhanced susceptibility to the bacterial pathogen

P. syringae pv. tomato DC3000 (Pst) carrying the aviru-

lence gene avrRpt2 (Shah et al., 1997, Shah et al., 1999). In

addition, the ssi1 mutation, which restores SA responsive-

ness in npr1-5 plants, was also shown to restore resistance

against avirulent Pst. To test if ssi2-1 also could restore

resistance to Pst in the npr1-5 plants, we compared the

growth of Pst carrying the avrRpt2 avirulence gene in ssi2-

1 NPR1, ssi2-1 npr1-5 and SSI2 npr1-5 plants, and in wt

SSI2 NPR1 plants as control. As shown in Figure 3, while

the mutation in ssi2-1 enhanced resistance approximately

®vefold in plants containing the wt NPR1 allele, it did not

enhance resistance in the plants containing the npr1-5

allele, suggesting that NPR1 is required for the ssi2-1-

conferred enhanced resistance against Pst.

The ssi2-1 mutant constitutively accumulates high levels

of SA and SAG

Several previously described mutants that constitutively

express PR genes and exhibit enhanced resistance also

constitutively accumulate elevated levels of SA (reviewed

by Dempsey et al., 1999; Durner et al., 1997; Ryals et al.,

1996). We therefore tested the levels of SA and its glucoside

(SAG) in SSI2 and ssi2-1 plants. As shown in Figure 4(a),

ssi2-1 NPR1 and ssi2-1 npr1-5 contained seven- and 15-fold

higher levels of SA than SSI2 NPR1 and SSI2 npr1-5 plants,

respectively. Likewise, SAG levels were >100-fold higher in

the ssi2-1 plants as compared with SSI2 plants.

SA is not essential for the ssi2-1-conferred phenotypes

As elevated SA levels are required for the phenotypes

exhibited by other constitutive PR-expressing mutants, we

assessed whether they are also required for the pheno-

types associated with the ssi2-1 mutation. An ssi2-1 plant

was therefore crossed with an NahG transgenic plant

expressing the SA-degrading enzyme salicylate hydroxy-

lase. F2 progeny of a cross between ssi2-1 npr1-5 and an

SSI2 NPR1 nahG plant showed a near 3 : 1 segregation of

lesion± : lesion+ plants. Constitutive PR expression and the

ssi2-1 small rosette phenotype also co-segregated with the

lesion+ phenotype. Nearly 75% of these ssi2-1-like plants

also expressed the nahG transcript, suggesting that high

levels of SA and SAG are not required for these ssi2-1-

conferred phenotypes. Analysis of the F3 generation

con®rmed this hypothesis, as spontaneous lesion forma-

tion and constitutive PR expression were observed in

plants containing the nahG transgene and homozygous for

ssi2-1 (Figure 5a,b; data not shown). In contrast, spontan-

eous lesion formation and PR gene expression were not

detected in SSI2 NPR1, SSI2 npr1-5, SSI2 NPR1 nahG and

SSI2 npr1-5 nahG plants. SA and SAG levels in these ssi2-1

npr1-5 nahG and ssi2-1 NPR1 nahG plants were down to

the basal levels found in uninfected npr1-5 and wt plants

(Figure 4b; data not shown).

While constitutively elevated SA levels were not essen-

tial for ssi2-1-conferred PR expression, the presence of

nahG transgene was observed to repeatedly lower the

absolute levels of PR-1 transcript accumulation in ssi2-1

NPR1 nahG and ssi2-1 npr1-5 nahG plants, as compared to

ssi2-1 NPR1 and ssi2-1 npr1-5 plants. By contrast, the effect

of nahG expression on ssi2-1-conferred BGL2 and PR-5

expression was more variable; decreases in transcript

levels were detected only in two out of four experiments.

To assess whether SA is required for ssi2-1-conferred

resistance to pathogen infection, the level of resistance to

P. parasitica Emco5 and Pst carrying the avirulence gene

avrRpt2 was compared among SSI2 NPR1, ssi2-1 NPR1,

SSI2 NPR1 nahG, and ssi2-1 NPR1 nahG plants. As shown

Figure 3. Growth of Pst in ssi2-1 plants.Pst DC3000 containing the avrRpt2 avirulence gene (OD600 = 0.001 in10 mM MgCl2) was in®ltrated into the abaxial surface of leaves from wt(SSI2 NPR1), npr1-5 (SSI2 npr1-5) and ssi2-1 (ssi2-1 npr1-5 and ssi2-1NPR1) plants with a syringe. Four leaf discs were harvested from infectedleaves at 3 days post-inoculation; they were then weighed, ground in10 mM MgCl2, and the bacterial numbers titered. The bacterial numbers6SD, presented as colony-forming units (cfu) per mg leaf tissue,represent the average of three samples. The experiment wasindependently performed twice with similar results.



in Table 1, SSI2 NPR1 nahG plants are hypersusceptible to

P. parasitica Emco5 as compared to SSI2 NPR1 plants. Not

only did all the SSI2 NPR1 nahG plants show disease

symptoms, they also supported higher levels of sporula-

tion. Furthermore, the newly emerging leaves showed the

presence of sporangiophores (data not shown).

Interestingly, the ssi2-1 allele partially restored resistance

in the ssi2-1 NPR1 nahG plants. Approximately 50% of

ssi2-1 NPR1 nahG plants showed little or no sign of

infection. Furthermore, the ssi2-1 NPR1 nahG plants that

did show infection had three- to ®vefold fewer sporangio-

phores compared to the SSI2 NPR1 nahG plants.

The SSI2 NPR1 nahG plants also were hypersusceptible

to Pst expressing avrRpt2; they supported »250-fold more

bacterial growth than the SSI2 NPR1 plants (Figure 6).

However, the presence of the ssi2-1 allele partially restored

resistance, evidenced as a 20-fold decrease in bacterial

titer in these ssi2-1 NPR1 nahG plants.

Discussion

The observation that most of the ssi2-1-associated pheno-

types are displayed by ssi2-1 plants carrying the npr1-5 or

nim1-1 alleles indicates that they are induced via an NPR1-

independent signaling pathway. However, ssi2-1 plants

containing the NPR1 allele accumulated greater levels of

PR-1 transcripts than ssi2-1 npr1-5 plants (Figure 1a). Thus

an NPR1-dependent signaling pathway appears to function

additively with the ssi2-1-induced NPR1-independent sig-

naling pathway to enhance PR-1 gene expression. By

contrast, the NPR1-dependent signaling pathway had little

to no additive effect on ssi2-1-conferred expression of

BGL2 or PR-5 (Figure 1a; Figure 5b; data not shown). A

similar difference in the requirement for NPR1 for activat-

ing PR-1 versus BGL2 and PR-5 expression was previously

noted in pathogen-infected npr1 plants (Glazebrook et al.,

1996; Reuber et al., 1998).

Figure 4. SA and SAG levels in ssi2-1 mutant.(a) SA and SAG levels in leaves of wt (SSI2 NPR1), npr1-5 (SSI2 npr1-5) and ssi2-1 (ssi2-1 NPR1 and ssi2-1 npr1-5) plants.(b) SA and SAG levels in leaves of npr1-5 (SSI2 npr1-5), ssi2-1 (ssi2-1 npr1-5) and two independent ssi2-1 nahG (ssi2-1 npr1-5 nahG) lines.Leaves from 4-week-old soil-grown plants were harvested, extracted and analyzed by HPLC, as described by Bowling et al. (1994). The SA and SAG values6SD, presented as mg SA g±1 FW tissue, are the averages of three to six sets of samples.



Like ssi2-1, constitutive PR gene expression in other

mutants, including ssi1, cpr6 and acd6, is mediated

independently of NPR1. However, elevated levels of

endogenous SA are required for the constitutive PR

expression exhibited by these mutants (Clarke et al.,

1998; Rate et al., 1999; Shah et al., 1999). By contrast,

expression of the nahG transgene did not abolish PR

expression in either ssi2-1 NPR1 nahG or ssi2-1 npr1-5

nahG plants (Figure 5b). It is unlikely that the PR expres-

sion detected in the ssi2-1 NPR1 nahG and ssi2-1 npr1-5

nahG plants was due to highly elevated levels of SA that

were not degraded by salicylate hydroxylase. The levels of

SA and SAG detected in ssi2-1 NPR1 nahG and ssi2-1 npr1-

5 nahG plants were comparable to those observed in

uninfected wt and npr1-5 plants (Figure 4b; data not

shown). In addition, the NahG transgenic line used in these

experiments was used previously to suppress constitutive

SA accumulation and PR gene expression in ssi1 mutant

plants (Shah et al., 1999), and ssi1 and ssi2-1 mutants

contain comparable levels of SA and SAG. Rather, our

results suggest that the ssi2-1-conferred constitutive PR

expression and enhanced resistance are due to altered

signaling through an SA-dependent, NPR1-independent

pathway (Figure 7a) or an SA- and NPR1-independent

pathway (Figure 7b).

In addition to constitutive PR-1 expression, SA-de®cient

ssi2-1 NPR1 nahG plants also exhibited enhanced resist-

ance to P. parasitica and Pst. Interestingly, ssi2-1-conferred

resistance to P. parasitica Emco5 was mediated by an

NPR1-independent pathway, while enhanced resistance to

Pst was NPR1-dependent. These seemingly contradictory

results can be explained if resistance to Pst is dependent

primarily on the NPR1-dependent SA signal transduction

pathway, while resistance to P. parasitica is conferred

primarily by an NPR1-independent signaling pathway that

is activated by the combined presence of SA and an

unidenti®ed second factor `X' (Figure 7a). SSI2 or an SSI2-

generated signal could inhibit this pathway in the absence

of pathogen infection. The reduction or absence of SSI2

activity in ssi2-1 plants would allow SA-independent

Figure 5. SA-independent expression of ssi2-1-conferred phenotypes.(a) Microscopy of trypan blue-stained lesion-bearing leaves from an ssi2-1 npr1-5 and an ssi2-1 npr1-5 nahG plant showing intensely stained deadcells.(b) Comparison of constitutive PR-1 and BGL2 expression in SSI2 andssi2-1 plants with or without the nahG transgene. All lines werehomozygous either for the wt NPR1 or for the npr1-5 mutant allele. Thewild-type (wt) or mutant (m) genotype at the NPR1 and SSI2 loci isindicated at the top. The presence of the nahG transgene wasdetermined based on detection of a signal for the nahG transcript. Gelloading was monitored by photographing the ethidium bromide-stainedgel (EtBr) before transferring the RNA to a Nytran Plus membrane. Theblot was sequentially probed for the indicated genes.All plants were grown in soil and sampled when 4 weeks old.

Figure 6. Growth of Pst in SA-de®cient SSI2 and ssi2-1 plants.Pst DC3000 containing the avrRpt2 avirulence gene (OD600 = 0.001 in10 mM MgCl2) was in®ltrated into the abaxial surface of leaves from wt(SSI2 NPR1), ssi2-1 (ssi2-1 NPR1), SSI2 nahG (SSI2 NPR1 nahG) and ssi2-1 nahG (ssi2-1 NPR1 nahG) plants with a syringe. At 3 days post-inoculation, four leaf discs were harvested from infected leaves, weighed,ground in 10 mM MgCl2, and the bacterial numbers titered. The bacterialnumbers 6SD are presented as colony-forming units (cfu) per mg leaftissue; they represent the average of three samples. The experiment wasindependently performed twice with similar results.



constitutive activation of the NPR1-independent pathway;

this would confer strong resistance to P. parasitica even in

ssi2-1 npr1-5 plants. Activation of this pathway (either by

the ssi2-1 mutation or pathogen infection) would also

confer the partial resistance to Pst observed in infected

ssi2-1 npr1-5 and SSI2 npr1-5 plants, as compared with the

lack of resistance displayed by infected SSI2 NPR1 nahG

and SSI2 npr1-5 nahG plants (Figures 3; Figure 6; data not

shown). In addition, the elevated SA levels that accumulate

in the ssi2-1 NPR1 plant would activate the NPR1-depend-

ent defense response pathway. Combined activation of the

NPR1-dependent and -independent signaling pathways

would explain the greater levels of resistance to Pst

displayed by ssi2-1 NPR1 plants as compared with ssi2-1

npr1-5 plants (Figure 3). This model can also explain the

observation that SA enhances but is not required for

resistance. Expression of the nahG transgene would not

prevent ssi2-1-mediated activation of the NPR1-independ-

ent pathway; however, it would inactivate the NPR1-

dependent pathway and thereby preclude full resistance

to either pathogen.

An alternative explanation for why the ssi2-1-conferred

enhanced resistance to Pst and P. parasitica is not com-

pletely abolished in ssi2-1 NPR1 nahG and ssi2-1 npr1-5

nahG plants is that SSI2 or an SSI2-generated signal

inhibits signaling of an SA-independent defense pathway

(Figure 7b). In this scenario, the combined action of the

SA-dependent and -independent pathways contributes to

full resistance against these pathogens. ssi2-1-conferred

constitutive activation of this SA-independent pathway

could account for the partial resistance observed in ssi2-1

nahG plants. Recently, an SA-independent pathway(s) that

is regulated by ethylene and jasmonic acid was shown to

activate induced systemic resistance and expression of

the defensin gene PDF1.2 in Arabidopsis (Penninckx

et al., 1998; Pieterse et al., 1996, Pieterse et al., 1998).

Resistance against P. parasitica in the cpr5 mutant also

occurs independently of NPR1 and is correlated with

enhanced PDF1.2 expression (Bowling et al., 1997).

However, ssi2-1-conferred enhanced resistance to P.

parasitica is not associated with elevated levels of

PDF1.2; PDF1.2 is not constitutively expressed in ssi2-1

(Figure 1c). On the contrary, unlike wt, ssi2-1 plants are

unable to express PDF1.2 after infection with Alternaria

brassicicola or in response to jasmonic acid application (P.

Kachroo, J. Shah and D. F. Klessig, unpublished results).

Figure 7. Possible interactions among SA, SSI2 and NPR1 that regulate PR gene expression and resistance.Expression of PR genes and resistance is induced via NPR1-dependent and NPR1-independent pathways. Based on our results, resistance to Pst isprimarily conferred by the NPR1-dependent pathway, while that to P. parasitica is governed by the NPR1-independent pathway.(a) SA is shown to regulate the NPR1-dependent and -independent pathways. Loss of both pathways (as in the SA-de®cient NahG plants) results inhypersusceptibility. Because exogenously applied SA cannot induce PR gene expression and resistance in npr1 plants, a second pathogen derived/activated signal `X' appears to be required, in addition to SA, for the activation of the NPR1-independent pathway. SSI2 or an SSI2-derived factor is shownto inhibit the NPR1-independent pathway since PR genes are expressed in ssi2-1 plants in the absence of pathogen infection. The infection-inducedsignals SA and `X' overcome SSI2's repressive effect on this pathway, either directly by inhibiting SSI2, or indirectly by overriding SSI2's negative effecton this pathway.(b) Expression of PR genes and resistance are shown to be regulated by SA-dependent and SA-independent pathways. NPR1 is required for functioning ofthe SA-dependent pathway, while SSI2 or an SSI2-derived factor is shown to modulate the SA- and NPR1-independent defense pathway. An infectioninduced signal(s) override SSI2's repressive activity on this pathway, either directly by inhibiting SSI2, or indirectly by overriding SSI2's negative effect onthis pathway.In both models, SSI2 also is shown negatively to regulate cell death, as loss of SSI2 activity in the ssi2-1 mutant causes the spontaneous appearance ofHR-like lesions. Infection is shown to inhibit or override SSI2's negative effect on cell death. Cell death is shown upstream of SA as the ssi2-1-conferredlesions develop independently of SA. However, it is equally plausible that cell death and SA accumulation are not interdependent.



Constitutive defense responses are associated with cell

death in various lesion mimic mutants of Arabidopsis. It is

plausible that constitutive PR expression and enhanced

resistance in these mutants and in ssi2-1 is due to stress

caused by cell death. However, we have genetic evidence

that unlinks ssi2-1-conferred constitutive expression of PR

genes from the ssi2-1-conferred cell death and stunted

plant phenotypes. We have identi®ed a mutant (sup21ssi2)

in which ssi2-1-conferred lesion mimic and stunted growth

phenotypes are suppressed (Figure 8a; data not shown).

However, ssi2-1-conferred constitutive expression of PR

genes is retained in the sup21ssi2 mutant (Figure 8b), thus

strongly arguing against ssi2-1-conferred constitutive

defense responses being an indirect effect of growth

defects of ssi2-1.

The ability of ssi2-1 plants to develop spontaneous

lesions was also observed to be an SA- and NPR1-

independent phenomenon. Similarly, spontaneous lesion

development in the lsd2, lsd4 (Hunt et al., 1997) and cpr5

mutants (Bowling et al., 1997) is regulated in an SA-

independent manner. By contrast, elevated SA levels are

required for spontaneous lesion formation in the mutants

ssi1 (Shah et al., 1999); acd6 (Rate et al., 1999); cep (Silva

et al., 1999); lsd1 (Dangl et al., 1996); lsd6 and lsd7

(Weymann et al., 1995). The mechanism through which

ssi2-1 induces lesion formation is unclear. Cell death and

SA accumulation have been shown in the past to be

involved in a feedback ampli®cation loop (Draper, 1997;

Shirasu et al., 1997; Van Camp et al., 1998). Consistent with

this possibility, ssi2-1 plants, as well as the other spon-

taneous lesion-forming mutants, contain constitutively

elevated levels of SA. As SA is not required for lesion

formation, cell death might induce SA accumulation in

ssi2-1 plants, rather than the other way round.

Alternatively, both SA accumulation and cell death may

occur independently of each other in ssi2-1 plants.

In summary, we have shown that suppressor screens are

useful for identifying not only additional components of a

given pathway, but also components of parallel pathways.

Through isolation of the ssi2-1 mutant we have identi®ed a

novel gene whose product appears to directly or indirectly

modulate signaling through a NPR1-independent defense

pathway. Mutations in SSI2 also appear to activate the

NPR1-dependent defense pathway, presumably by caus-

ing constitutive accumulation of SA. Based on our results,

both the NPR1-dependent and -independent defense

pathways are required for full ssi2-1-conferred resistance.

However, the amount either pathway contributes to the

overall level of resistance depends on the identity of the

attacking pathogen. Unlike all other mutations identi®ed

thus far, only ssi2-1 activates resistance to Pst and P.

parasitica in NahG plants. Future studies should help

elucidate whether SSI2 functions in a previously uncharted

portion of the SA-dependent, NPR1-independent signaling

pathway, or whether it targets an uncharacterized SA- and

NPR1-independent defense pathway.

Experimental procedures

Growth conditions for plants and bacteria

Arabidopsis plants were grown in soil at 22°C in growth chambersprogrammed for a 16 h light (8000±10 000 lux) and 8 h dark cycle,unless otherwise stated. Pst DC3000 carrying a plasmid-borneavrRpt2 gene was propagated as previously described (Shahet al., 1997). Peronospora parasitica isolate Emco5 was cultivatedon the susceptible ecotype NoÈ as described by Dangl et al. (1992).

Pathogen infection of plants

Plants for infection with Pst DC3000 were grown in soil at 22°C ina growth chamber programmed for a 12 h light/12 h dark cycle.Three days before infection, the plants were transferred to a 16 h

Figure 8. PR gene expression and loss of cell death in sup21ssi2.(a) Trypan blue staining of an ssi2-1 npr1-5 leaf reveals an intenselystained, dark cluster of cells (white arrow) that are indicative of celldeath. In comparison, trypan blue staining of a sup21ssi2 ssi2-1 npr1-5leaf does not reveal the presence of dead cells.(b) Constitutive expression of the PR-1 and BGL2 genes in ssi2-1 npr1-5and sup21ssi2 ssi2-1 npr1-5 plants. RNA was extracted from leaves of soil-grown plants. Gel loading was monitored by photographing the ethidiumbromide-stained gel (EtBr) before transferring the RNA to a Nytran Plusmembrane. The blot was sequentially probed for the indicated genes.All plants were sampled when 4 weeks old.



light/8 h dark cycle. Infection with Pst DC3000 carrying a plasmid-borne avrRpt2 gene was performed as described earlier (Shahet al., 1997). Four leaves per plant were in®ltrated with a suspen-sion (OD600 = 0.001) in 10 mM MgCl2. Twelve leaf discs, 0.5 cm indiameter (0.20 cm2) were harvested at the indicated time andplaced in pre-weighed tubes. After the weight of each sample wasdetermined, the samples were processed for bacterial counts andRNA extraction as described earlier (Shah et al., 1997). Bacterialcounts were expressed as colony-forming units per mg leaftissue.

Inoculation with the virulent P. parasitica isolate Emco5 wasdone on 7-day-old soil-grown plants. Spore suspensions of P.parasitica were prepared as described (Dangl et al., 1992). Plantswere sprayed with a freshly prepared suspension of conidios-pores in water (106 spores ml±1). Inoculated plants were keptcovered with a clear plastic dome to maintain high humiditythroughout the course of the experiment, and fungal growth wasevaluated under a dissecting microscope 8 days post-inoculationby counting the number of sporangiophores per leaf. Plants withcotyledons containing ®ve or more sporangiophores were scoredas infected.

Chemical treatment of plants

Four-week-old plants were sprayed and sub-irrigated with asolution of SA (500 mM) in water, as previously described (Shahet al., 1997). As controls, plants were similarly treated with water.Leaves were harvested at the indicated times after treatment andquick-frozen in liquid nitrogen. Leaf samples were stored at ±80°C.

RNA extraction, Northern and dot blot analyses

Large-scale preparation of RNA from Arabidopsis was carried outaccording to Das et al. (1990). Small-scale extraction of RNA fromone or two leaves was performed in the TRIzol reagent (GIBCO-BRL, Gaithersburg, MD, USA) following the manufacturer'sinstructions. Northern blot analysis and synthesis of randomprimed probes for PR-1, BGL2, PR-5 and nahG were synthesizedas described earlier (Shah et al., 1997; Shah et al., 1999).

Histochemistry and microscopy

Leaf samples for trypan blue staining and epi¯uorescencemicroscopy were obtained from 4-week-old soil-grown plants.Trypan blue staining on P. parasitica-infected leaves was carriedout on samples harvested 8 days post-inoculation. Samples wereprocessed and analyzed as described by Bowling et al. (1997).

SA and SAG estimations

SA and SAG were extracted and estimated from 0.25±0.5 g FWleaf tissue as described by Bowling et al. (1994).

Mutagenesis and selection of ssi2-1 mutant

M2 seeds derived from ethyl methylsulfonate mutagenized npr1-5seeds (ecotype NoÈ ) were screened for constitutive PR geneexpression as previously described (Shah et al., 1999).

Genetic analysis

Backcrosses were performed by pollinating ¯owers of an npr1-5(SSI2 npr1-5) plant with pollen from an ssi2-1 npr1-5 doublemutant plant. For all other genetic analyses, progeny from abackcrossed line homozygous for the ssi2-1 and npr1-5 alleleswere used. To generate ssi2-1 plants homozygous for the NPR1wt allele, pollen from an ssi2-1 npr1-5 double mutant was used topollinate ¯owers from an Arabidopsis ecotype NoÈ line 1/8E/5(Shah et al., 1997) which is wild type at both the SSI2 and NPR1loci. Likewise, to generate ssi2-1 plants homozygous for the nim1-1 mutant allele, pollen from an ssi2-1 npr1-5 double mutant wasused to pollinate ¯owers from an SSI2 nim1-1 plant (ecotype Ws).Success of the cross was con®rmed by CAPS analysis on F1 plantsfor heterozygosity at the NPR1 locus. Segregation of the ssi2-1mutant allele was monitored in the F2 progeny by the presence ofthe small lesion+ plant phenotype and by Northern blot or dot-blot analysis for constitutive PR-1 gene expression. CAPS analysiswas performed as previously described (Shah et al., 1999) on DNAfrom these phenotypically ssi2-1 plants, in order to identify plantshomozygous for the wt NPR1 or the nim1-1 mutant allele. Formapping analysis, pollen from an ssi2-1 npr1-5 double mutant(ecotype NoÈ ) was used to pollinate ¯owers from a wild-type plantof ecotype Columbia. F2 progeny plants from the above crosswere monitored for spontaneous lesion and constitutive PR-1expression phenotype by dot-blot analysis. ssi2-1 mutant linescontaining the nahG transgene were generated by fertilizing¯owers from an ssi2-1 npr1-5 plant with pollen from a transgenicNahG plant (ecotype NoÈ ). Success of the cross was con®rmed byanalyzing expression of the nahG gene in the F1 plants. A quarterof the F2 plants had the ssi2-1-conferred lesion+ phenotype,suggesting that the nahG gene did not suppress the lesion+

phenotype of ssi2-1 plants. Northern blot analysis showedconstitutive expression of PR genes in these plants, reaf®rmingthat these were truly ssi2-1 plants. Northern blot analysis alsoidenti®ed expression of the nahG gene in roughly three-quartersof these plants. Analysis of the F3 progeny of some of these F2

lines identi®ed F2 plants that were homozygous for ssi2-1, NPR1or npr1-5 and nahG.

Acknowledgements

We thank Robert Dietrich, Eric Ward and John Ryals for providingthe nahG clone and nim1-1 seeds, Barbara Kunkel for providingPst DC3000, and Jeff Dangl for the P. parasitica isolate Emco5. Weare grateful to Frank Tsui for carrying out HPLC analyses for SAand SAG quanti®cation, and to Matt Burkhart and RachelBuf®ngton for cultivation of plants for bacterial infection. Wegratefully acknowledge D'Maris Dempsey and Jian-Min Zhou forcritical reading of this manuscript. This work was supported bygrant # MCB 9723952 from the National Science Foundation toD.F.K. and funding from the Agricultural Experiment Station atKansas State University to J.S.

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A recessive mutation in the Arabidopsis SSI2 gene confers SA- and NPR1-independent expression of PR...

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