Plastid x3-fatty acid desaturase-dependent accumulation of asystemic acquired resistance inducing activity in petioleexudates of Arabidopsis thaliana is independent ofjasmonic acid

Ratnesh Chaturvedi1,2,‡, Kartikeya Krothapalli2,‡, Ragiba Makandar1,2, Ashis Nandi2,†, Alexis A. Sparks2,

Mary R. Roth2, Ruth Welti2 and Jyoti Shah1,2,*

1Department of Biological Sciences, University of North Texas, Denton, TX 76203, USA, and2Division of Biology and the Molecular Cellular and Developmental Biology Program, Kansas State University,

Manhattan, KS 66506, USA

Received 22 October 2007; accepted 6 December 2007.*For correspondence (fax +1 940 565 4136; e-mail shah@unt.edu).†Present address: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.‡These authors contributed equally to this work.

Summary

Systemic acquired resistance (SAR) is an inducible defense mechanism that is activated throughout the plant,

subsequent to localized inoculation with a pathogen. The establishment of SAR requires translocation of an

unknown signal from the pathogen-inoculated leaf to the distal organs, where salicylic acid-dependent

defenses are activated. We demonstrate here that petiole exudates (PeXs) collected from Arabidopsis leaves

inoculated with an avirulent (Avr) Pseudomonas syringae strain promote resistance when applied to

Arabidopsis, tomato (Lycopersicum esculentum) and wheat (Triticum aestivum). Arabidopsis FATTY ACID

DESATURASE7 (FAD7), SUPPRESSOR OF FATTY ACID DESATURASE DEFICIENCY1 (SFD1) and SFD2 genes are

required for accumulation of the SAR-inducing activity. In contrast to Avr PeX from wild-type plants, Avr PeXs

from fad7, sfd1 and sfd2 mutants were unable to activate SAR when applied to wild-type plants. However, the

SAR-inducing activity was reconstituted by mixing Avr PeXs collected from fad7 and sfd1 with Avr PeX from

the SAR-deficient dir1 mutant. Since FAD7, SFD1 and SFD2 are involved in plastid glycerolipid biosynthesis and

SAR is also compromised in the Arabidopsis monogalactosyldiacylglycerol synthase1 mutant we suggest that

a plastid glycerolipid-dependent factor is required in Avr PeX along with the DIR1-encoded lipid transfer

protein for long-distance signaling in SAR. FAD7-synthesized lipids provide fatty acids for synthesis of

jasmonic acid (JA). However, co-infiltration of JA and methylJA with Avr PeX from fad7 and sfd1 did not

reconstitute the SAR-inducing activity. In addition, JA did not co-purify with the SAR-inducing activity

confirming that JA is not the mobile signal in SAR.

Keywords: inducible defense, long-distance signaling, salicylic acid, systemic acquired resistance, jasmonic

acid.

Introduction

Plants have pre-formed and inducible mechanisms that

protect them from pathogens. Systemic acquired resistance

(SAR) is an inducible defense mechanism that confers

enhanced resistance against a broad-spectrum of pathogens

(Chaturvedi and Shah, 2006; Durrant and Dong, 2004;

Metraux et al., 2002). SAR is activated in the healthy leaves

of a plant subsequent to the exposure of another leaf to a

pathogen. The pathogen-inoculated leaf is the source of a

long-distance SAR signal that is translocated to the other

organs (Dean and Kuc, 1986). Allograft experiments between

cucumber (Cucumis sativus), muskmelon (Cucumis melo)

and watermelon (Citrullus lanatus) demonstrated that the

SAR signal can function across Cucurbit species (Kuc, 1982).

Furthermore, girdling experiments in cotton (Gossypium

106 ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd

The Plant Journal (2008) 54, 106–117 doi: 10.1111/j.1365-313X.2007.03400.x

hirsutum) suggested that the SAR-signal is translocated

through the phloem (Guedes et al., 1980). However, experi-

ments with Arabidopsis thaliana have indicated that phloem

is not the exclusive conduit for translocation of the SAR

signal; some signal can be translocated via other routes

(Kiefer and Slusarenko, 2003).

The activation of SAR is accompanied by an increase in

the level of salicylic acid (SA) and elevated expression of the

pathogenesis-related PR1 gene in the pathogen inoculated

and the distal organs in which SAR is manifested (Chaturv-

edi and Shah, 2006; Durrant and Dong, 2004; Metraux et al.,

2002). Studies in tobacco (Nicotiana tabaccum) and Arabid-

opsis have confirmed an important role for SA in the

manifestation of SAR (Chaturvedi and Shah, 2006; Durrant

and Dong, 2004; Metraux et al., 2002). For example, appli-

cation of SA induces a SAR-like mechanism. Furthermore,

preventing accumulation of SA by either blocking its

synthesis, as in the Arabidopsis salicylic acid-induction

deficient2 (sid2) and enhanced disease susceptibility5

(eds5) mutants, or promoting its breakdown by expression

of the bacterial nahG-encoded salicylate hydroxylase in

transgenic plants compromised SAR (Nawrath and Metraux,

1999; Vernooij et al., 1994). SAR was also attenuated in the

Arabidopsis nonexpresser of PR genes1 (npr1) mutant,

which is defective in SA signaling (Maldonado et al., 2002;

Nandi et al., 2004). Although SA moves from the pathogen-

inoculated organ to the distal organs (Molders et al., 1996;

Shulaev et al., 1995), leaf excision experiments in cucumber

and grafting experiments involving tobacco NahG plants

suggested that SA is not the primary mobile signal that is

translocated from the pathogen-inoculated leaf to the distal

organs for establishment of SAR (Rasmussen et al., 1991;

Vernooij et al., 1994). However, Darby et al. (2000) demon-

strated that nahG expression when targeted to the phloem

tissue compromised SAR. Furthermore, a recent study with

SA-binding protein2 (SABP2), which possesses methylSA

(MeSA) esterase activity, indicated that MeSA is required for

long-distance signaling associated with Tobacco mosaic

virus-induced SAR in N. tabaccum (Park et al., 2007).

Jasmonic acid (JA) is another important signal molecule

in plant stress responses (Devoto and Turner, 2003; Howe

and Schilmiller, 2002; Pieterse et al., 2002). In tomato, JA

functions as a graft transmissible signal in the wound

response (Li et al., 2002) and in Arabidopsis, JA signaling

is required for systemic disease resistance induced by root-

colonizing bacteria (Devoto and Turner, 2003; Pieterse et al.,

2002). A recent study showed that JA signaling is also

involved in SAR that was induced by localized inoculation

with an avirulent (Avr) strain of Pseudomonas syringae

(Truman et al., 2007); systemic resistance was attenuated in

the JA-response jasmonate insensitive1 (jin1) mutant and

the oxophytodienoic acid reductase3 (opr3) mutant, which is

JA deficient. However, two other studies showed that SAR

was not compromised in the JA-insensitive Arabidopsis

coronatine-insensitive1 (coi1) and jasmonate resistant1

(jar1) mutants (Cui et al., 2005; Mishina and Zeier, 2007).

Genetic studies in Arabidopsis have suggested the

involvement of lipids in long-distance signaling in the

establishment of SAR (Chaturvedi and Shah, 2006; Durrant

and Dong, 2004; Shah, 2005). The DEFECTIVE IN INDUCED

RESISTANCE1 (DIR1) gene, which encodes a putative apo-

plastic protein with homology to non-specific lipid transfer

proteins (nsLTPs), is required for the activation of SAR

(Maldonado et al., 2002). Unlike petiole exudates (PeXs)

collected from Avr pathogen inoculated wild-type (WT)

leaves, Avr PeX collected from the dir1 mutant were unable

to induce PR1 expression when applied to WT leaves.

However, the dir1 mutant leaves were responsive to the

PR1 expression-inducing activity present in Avr PeXs from

WT leaves. These results suggest that DIR1 is required for

the synthesis and/or translocation of the SAR signal. In

contrast to dir1, SAR was constitutively manifested in the

Arabidopsis suppressor of SA-insensitivity2 (ssi2) mutant

that constitutively expressed PR1 and exhibited heightened

resistance to bacterial, viral and oomycete pathogens

(Kachroo et al., 2003; Nandi et al., 2003; Sekine et al., 2004;

Shah et al., 2001). SSI2 encodes a stearoyl-acyl carrier

protein desaturase involved in lipid metabolism (Kachroo

et al., 2001). ssi2-conditioned constitutive PR1 expression

and resistance were attenuated by the suppressor of fatty

acid desaturase deficiency1 (sfd1) mutant allele in the ssi2

sfd1 double-mutant plants (Nandi et al., 2003). Studies with

the sfd1 single mutant indicated that SFD1 is essential for

SAR, but not basal resistance to pathogen (Nandi et al.,

2004). Although SA accumulation was not adversely affected

in pathogen-infected sfd1 leaves, SAR-associated accumu-

lation of SA in the distal leaves was blocked in sfd1,

suggesting that SFD1 is required prior to accumulation of

SA in the organs exhibiting SAR (Nandi et al., 2004). SFD1

encodes a dihydroxyacetonephosphate (DHAP) reductase

that catalyzes the interconversion between DHAP and glyc-

erol-3-phosphate (G3P). Glycerol-3-phosphate provides car-

bon for a variety of macromolecules including the glycerol

backbone in complex lipids. Leaf galactolipid composition

was altered in the sfd1 mutant. The level of the 34:6 (18:3

plus 16:3 acyl composition)-monogalactosyldiacylglycerol

(MGD) was 45% lower, while the level of 36:6 (18:3 plus 18:3

acyl composition)-MGD was two-fold higher in the leaves of

the sfd1 mutant than in the WT plant (Nandi et al., 2004).

Since, SFD1 function is required in the plastids it was

suggested that a plastid-synthesized lipid may be critical for

SAR (Nandi et al., 2004).

In this study, we demonstrate that in addition to SFD1, the

Arabidopsis FATTY ACID DESATURASE7 (FAD7), SFD2

and MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE1

(MGD1) genes, which are also involved in the synthesis of

plastidic glycerolipids, are required for the activation of SAR.

These genes are vital for the accumulation of a factor that,

Plant defense signaling 107

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 106–117

along with the DIR1-encoded nsLTP, is required in PeXs for

the activation of SAR. Although FAD7-synthesized lipids

provide fatty acids for JA synthesis, we provide evidence

that JA is not a component of this SAR inducing activity in

Arabidopsis PeXs.

Results

SAR is compromised in the Arabidopsis fad7, sfd2 and

mgd1 mutants

To determine whether a plastid-synthesized lipid is involved

in SAR, we studied the activation of SAR-conferred resis-

tance in the Arabidopsis fad7 and sfd2 mutants in which the

composition of plastid-synthesized phosphatidylglycerol

(PG), MGD and digalactosyldiacylglycerol (DGD) are altered

relative to the WT plant (Tables S1 and S2) (Nandi et al.,

2003; Wallis and Browse, 2002). In addition, the fad7 muta-

tion also affects plastid synthesized sulfoglycerolipid (SL)

composition (Wallis and Browse, 2002). Figure 1 summa-

rizes the involvement of the FAD7 and SFD2 genes in Ara-

bidopsis lipid biosynthesis. To monitor SAR-conferred

resistance, 3 days after the primary inoculation with the Avr

pathogen, P. syringae pv. tomato DC3000 expressing

avrRpt2, four upper leaves of the plant were challenged

(secondary inoculation) with the virulent pathogen P. sy-

ringae pv. maculicola ES4326 (Psm) and bacterial numbers

monitored 3 days post the secondary inoculation. Psm-

challenged plants that received a primary inoculation with

10 mM MgCl2 (Mock) served as the control. As expected of

SAR, Psm numbers were lower in leaves of WT accessions

Nossen (No) and Columbia (Col), and Col containing the

glabra1 allele (gl) that received a primary inoculation of the

Avr pathogen, than in plants that received a Mock primary

inoculation (Figure 2a). In contrast, comparable numbers

of Psm were present in the upper leaves of the Mock and

Avr pathogen-treated fad7 and sfd2 mutants (Figure 2a).

SAR-conferred induction of PR1 expression in the upper

leaves was also attenuated in the fad7 and sfd2 mutants

(Figure 2b). Growth of Psm in fad7 and sfd2 that received a

Mock primary inoculation was comparable to that in the

Col-gl and No, which contain the WT FAD7 and SFD2 alleles,

suggesting that these mutations do not have any effect on

basal resistance against Psm. Furthermore, the onset of

the hypersensitive response (HR) induced in response to

primary inoculation with the Avr pathogen was comparable

between the WT and the mutant plants (data not shown).

The fad7 mutant allele also attenuated the ssi2-conditioned

constitutive PR1 expression and resistance to Psm in two

independent ssi2 fad7 double-mutant lines (Figure 2c),

confirming that FAD7 is required for SAR. Our previous

studies had showed that the ssi2-conferred constitutive

SAR phenotype was similarly attenuated in the ssi2 sfd1

and ssi2 sfd2 double-mutant plants (Nandi et al., 2003).

The level of 34:4 PG, the major PG species in Arabidopsis

leaves is 30% lower in the fad7 mutant than in the WT plant

(Table S1) (Wallis and Browse, 2002). Similarly, in compar-

ison to the WT, levels of PG species containing polyunsat-

urated fatty acids were lower in the sfd2 mutant. To

determine whether PG contributes to SAR, SAR was

evaluated in the fad4 mutant in which 34:4 PG level is

reduced by 99% and total PG content is 40% lower than in

the WT plant; however, the fad4 allele does not affect

galactolipid and SL content (Table S1) (Wallis and Browse,

2002). As shown in Figure 2(a), SAR-conferred resistance to

Psm was comparable between the WT accession Col and

the fad4 mutant, indicating that PG is not important

for SAR.

The involvement of plastid glycerolipids in SAR was

further tested by studying SAR-conferred resistance against

Psm in the Arabidopsis mgd1 mutant (Jarvis et al., 2000).

MGD1 encodes a MGD synthase activity that catalyzes the

transfer of a D-galactose moiety from UDP-galactose to

diacylglycerol to synthesize MGD in the plastids (Figure 1)

(Arai et al., 2001; Jarvis et al., 2000). Levels of MGD in the

leaves of the mgd1 mutant are reduced by 75% compared

with the WT (Jarvis et al., 2000). However, levels of PG and

SL are not reduced in the mgd1 mutant (Jarvis et al., 2000).

Similar to the fad7, sfd1 and sfd2 mutants, SAR, but not

basal resistance, was compromised in the mgd1 mutant

(Figure 2a). The above results establish that a plastid

synthesized glycerolipid, most likely a galactolipid, is

required for the establishment of SAR.

sfd1

DHAP

G3P LPA PA DAG

MGDPG

mgd1

fad4sfd2

fad7

MGDPG

fad7

DGD

DGD

fad7

sfd2

SL

fad7

sfd2

Figure 1. Schematic of steps in phosphatidylglycerol (PG), galactolipid and

sulfoglycerolipid (SL) synthesis in plastids affected by the sfd1, sfd2, fad4,

fad7 and mgd1 mutants.

Steps leading to synthesis of PG, the galactolipids monogalactosyldiacyl-

glycerol (MGD) and digalactosyldiacylglycerol (DGD) and SL in the plastids

are shown. The acyl chains on PG, MGD, DGD and SL are desaturated by

specific fatty acyl desaturases. SFD1 encodes a dihydroxyacetonephosphate

(DHAP) reductase that provides glycerol-3-phosphate (G3P) for glycerolipid

synthesis in the plastids. MGD1 encodes an enzyme that transfers a

D-galactose moiety onto diacylglycerol (DAG) to yield MGD. FAD4-encoded

desaturase primarily acts on 16:0 acyl chains at the sn2 position in PG to yield

16:1. The identity of the SFD2 gene is not known. However, lipid profiling

indicates that the sfd2 allele affects the desaturation of 16:0 acyl chains

present in MGD and DGD to 16:1. The FAD7-encoded desaturase catalyzes the

conversion of 18:2 and 16:2 acyl chains in PG, MGD, DGD and SL to 18:3 and

16:3, respectively.

LPA, lysophosphatidic acid; PA, phosphatidic acid.

108 Ratnesh Chaturvedi et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 106–117

Petiole exudates from avirulent pathogen-inoculated

wild- type leaves contain a SAR-inducing activity

Previously, Maldonado et al. (2002) showed that PeXs, which

are enriched in phloem sap, collected from Avr pathogen-

inoculated leaves of WT Arabidopsis contained an activity

that, when infiltrated into Arabidopsis leaves, induced accu-

mulation of PR1 transcript. However, whether these exudates

(Avr PeX) from Avr pathogen-inoculated leaves promote a

SAR-like mechanism was not known. We similarly observed

that in comparison to PeX collected from 10 mM MgCl2-

infiltrated (Mock PeX) WT leaves, Avr PeX contained an

activity that enhanced PR1 transcript accumulation in the

infiltrated leaf (Figure 3a). In addition, PR1 transcripts also

accumulated at elevated levels in the upper leaves of Avr

PeX-treated plants. More significantly, resistance to Psm was

enhanced in the local Avr PeX-infiltrated (Figure 3b) and

upper leaves of WT plants (Figure 4a–c and Figure S3). When

applied to healthy plants, Avr PeX did not induce necrosis.

Furthermore, Avr PeX did not exhibit bacteriostatic activity

against Psm (data not shown), suggesting that Avr PeX-

conferred suppression of P. syringae growth in planta was

due to the activation of plant defense responses. The ability of

Avr PeX to promote disease resistance required SA synthesis

and signaling in the recipient plant; Avr PeX did not promote

resistance in the SA-deficient Arabidopsis sid2 and eds5

mutants, and the SA-insensitive npr1 mutant plants (Fig-

ure 3b). The Avr PeX from WT leaves also restricted Fusarium

head blight disease of wheat caused by the fungal pathogen

Fusarium graminearum (Figure 3c) and Psm growth in

tomato leaves (Figure S1). These results indicate that Avr

PeX from WT Arabidopsis leaves contains a SAR-inducing

activity. No difference in the salicylate content between

the Avr PeX (0.26 � 0.10 ng per leaf) and Mock PeX

(0.27 � 0.10 ng per leaf) were found.

AvrPeX from fad7, sfd1 and sfd2 lack the

SAR-inducing activity

The FAD7, SFD1 and SFD2 genes may be involved in either

the accumulation of a SAR-inducing activity in the Avr

pathogen-inoculated leaf or perception of this activity in the

upper leaves which exhibit SAR. To distinguish between the

above possibilities we collected Avr PeX and Mock PeX from

the fad7, sfd1 and sfd2 mutants and the corresponding WT

plants. Petiole exudates from the WT plant were infiltrated

into leaves of the fad7, sfd1 and sfd2 plants, and as a control

into the WT plant, to determine whether they could promote

fad7WT(Col-gl)

WT(Nö) sfd2C

FU

leaf

dis

c–1WT(Col) fad4

105

106

107

105

106

107

105

104

106

107mgd1WT(Col)

106

107

108(a)

(b) (c)

106

107

108

CF

U le

af d

isc–1

CF

U le

af d

isc–1

WT(Nö)

fad7 ssi2 fad7ssi2

PR1

ACT8

Mock Avr Mock Avr

WT(Col-gl) fad7

PR1

ACT8

Mock Avr Mock Avr

WT(Nö) sfd2

PR1

ACT8

Figure 2. Systemic acquired resistance (SAR) is

compromised in the fad7, mgd1 and sfd2

mutants.

(a) Growth of the virulent pathogen P. syringae

pv. maculicola (Psm) in wild type (WT), fad7,

mgd1, sfd2 and fad4 mutant plants. Pseudomo-

nas syringae pv. tomato DC3000 carrying the

avrRpt2 avirulence gene was infiltrated into three

lower leaves of fad7, sfd2, mgd1 and fad4 mutant

plants and their corresponding WT plants. Plants

similarly treated with 10 mM MgCl2 provided

controls. Three days later, four upper leaves in

each plant were infiltrated with Psm and bacterial

numbers monitored 3 days post-inoculation

(dpi). Each bar represents the average Psm count

in 15 leaf discs � SD. White bars, primary inoc-

ulation with 10 mM MgCl2; black bars, primary

inoculation with Avr pathogen. The mgd1 and

fad4 mutants are in the accession Columbia

(Col), the fad7 mutant is in accession Col con-

taining the glabra allele (gl), and the sfd2 mutant

is in the accession Nossen (No) background.

(b) Reverse transcription-PCR analysis for PR1

expression in the upper leaves of WT, fad7 and

sfd2 mutants, 2 days post-infiltration of three

lower leaves with 10 mM MgCl2 (Mock) or the

avirulent pathogen (Avr).

(c) Upper panel: bacterial counts in Psm-inocu-

lated leaves of WT accession No, the fad7 and

ssi2 single mutant and two ssi2 fad7 double-

mutant lines, 3 dpi. Each bar represents the

average Psm count in 15 leaf discs � SD. Lower

panel: RT-PCR analysis for PR1 expression in

leaves of the WT, fad7, ssi2 and ssi2 fad7 mutant

plants.

In (b) and (c) ACT8 provided a control for RT-PCR.

Plant defense signaling 109

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 106–117

resistance against Psm in the upper leaves. In parallel, PeXs

from the mutant leaves were infiltrated into WT leaves, and

as a control into the corresponding mutant plant, to deter-

mine whether they contained a factor that could activate

SAR-associated resistance in WT leaves. Resistance against

Psm was enhanced in the fad7, sfd1 and sfd2 mutant plants

that were treated with Avr PeX collected from the corre-

sponding WT plant (Figure 4a–c), demonstrating that these

mutants are capable of responding to the SAR signal. In

contrast, Avr PeX collected from the fad7, sfd1 and sfd2

CF

U le

af d

isc–1

104

105

106

107

WT(Col)

sid2 eds5 npr1

PR1ACT8

Inf Up

Mock Pe(a)

(b)

(c)

X Avr PeX

Inf UpIn

fect

ed s

pike

lets

(%

)

WT PeX sfd1 PeX

Mock Avr 0

20

40

60

80

3 DPI9 DPI

15 DPI21 DPI

Mock Avr

Mock PeX Avr PeX

Figure 3. Systemic resistance conferred by petiole exudates (PeXs) from

avirulent (Avr) pathogen-inoculated Arabidopsis leaves requires salicylic acid

(SA) signaling.

(a) Petiole exudates (Mock PeX and Avr PeX) were collected from MgCl2- and

Avr pathogen-inoculated leaves of WT accession No plants. The PeXs were

infiltrated into three leaves of each WT plant of accession No. Twenty-four

hours later RNA was extracted from the infiltrated (Inf) and upper (Up) leaves

and analyzed by RT-PCR for PR1 transcript accumulation. Reverse transcrip-

tase-PCR for ACT8 provided control for the quality of RNA.

(b) Petiole exudates from WT accession No leaves were infiltrated into leaves

of the WT accession Col and sid2, eds5 and npr1 mutant plants, which are in

the accession Col background. Three days later, these leaves were challenged

with Pseudomonas syringae pv. maculicola (Psm) and bacterial numbers

determined 3 days post-inoculation (dpi). Each bar represents the average

Psm count in 15 leaf discs � SD. White bars, fractions from Mock PeX; black

bars, fractions from Avr PeX.

(c) The SAR-competent Avr PeXs from Arabidopsis promote Fusarium head

blight resistance in wheat. Top panel: Fusarium graminearum growth on

spikes that were pre-treated with either 10 ll of Mock PeX or Avr PeX from WT

Arabidopsis plants of accession No. A single spikelet on each spike was

treated with PeX and 3 days later inoculated with 300 spores of Fusarium

graminearum. The inoculated spikes were photographed 7 dpi. Bottom panel:

quantification of severity of Fusarium head blight disease in F. graminearum-

inoculated spikes that were pre-treated with Mock PeX and Avr PeX from WT

plants of accession No and sfd1 mutant. Head blight severity was measured

as the percentage of spikelets on each spike showing blight symptoms at 3, 9,

15 and 21 dpi with fungal spores. All values are mean of seven spikes per

treatment.

WT(Col-gl) fad7

WT(Col-gl)

WT(Col-gl)

fad7

fad7104

105

10

(a)

(b)

(c)

6

CF

U le

af d

isc–1

CF

U le

af d

isc–1

CF

U le

af d

isc–1

104

WT(Nö) sfd1

WT(Nö) sfd1 sfd1WT(Nö)

105

106

WT(Nö) sfd2

WT(Nö) sfd2 sfd2WT(Nö)104

105

106

Source of PeX

Source of PeX

Source of PeX

Figure 4. Avirulent (Avr) petiole exudates (PeXs) from fad7, sfd1 and sfd2 lack

a systemic acquired resistance-inducing activity.

Mock PeX and Avr PeX were collected from the fad7, sfd1 and sfd2 mutants

and the corresponding wild-type (WT) plants. These PeXs were infiltrated into

three lower leaves of WT (left panel) and the mutant (right panel) plants. Three

days later, four upper leaves in each plant were challenged with Pseudomo-

nas syringae pv. maculicola (Psm) and bacterial counts determined 3 days

post-inoculation (dpi). Each bar represents the average Psm count in 15 leaf

discs � SD. White bars, fractions from Mock PeX; black bars, fractions from

Avr PeX.

(a) PeXs collected from Col-gl, which contains the WT FAD7 allele, and the

fad7 mutant.

(b) PeXs collected from WT plants of accession No and the sfd1 mutant.

(c) PeXs collected from WT plants of accession No and the sfd2 mutant.

110 Ratnesh Chaturvedi et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 106–117

mutants, when applied to the corresponding WT plant did

not promote resistance against Psm (Figure 4a–c). The Avr

PeX from sfd1 did not promote resistance in wheat and

tomato either (Figure 3c and Figure S1). Furthermore,

co-infiltration of fad7 plus sfd1 Avr PeXs and fad7 plus sfd2

Avr PeXs also did not activate SAR in Arabidopsis (Figure 5a

and Figure S2). We conclude that the SAR deficiency of the

fad7, sfd1 and sfd2 mutants is due to their inability to

accumulate a SAR-inducing activity in Avr PeX.

The SAR-activating activity is reconstituted by mixing

Avr petiole exudates from dir1 with Avr petiole

exudates from fad7 and sfd1

Like the fad7, sfd1 and sfd2 mutant alleles, the SAR-incom-

petent dir1 is also epistatic to ssi2. The ssi2-conditioned

constitutive PR1 expression and heightened resistance to

Psm were suppressed in the ssi2 dir1 double mutant

(Figure 6a,b), suggesting that DIR1 and the plastid glycer-

olipid-dependent factor are both required for a common

defense mechanism. Like the sfd1, sfd2 and fad7 mutants,

the dir1 mutant is unable to accumulate a SAR-promoting

activity in Avr PeX (Figure S3) (Maldonado et al., 2002).

However, the dir1 mutant was responsive to the SAR-pro-

moting activity present in Avr PeX collected from WT leaves.

To characterize the relationship between DIR1 and the plastid

glycerolipid-dependent factor in SAR long-distance signal-

ing we mixed Avr PeX from dir1 with Avr PeX collected from

the sfd1 mutant. These mixed PeXs were then infiltrated into

leaves of WT No plants and after 3 days the upper leaves

were challenged with Psm. As negative controls, Mock PeX

from dir1 was mixed with Mock PeX from sfd1 and similarly

infiltrated into WT No plants. The Mock and Avr PeXs from

the sfd1 and dir1 mutants when injected individually, and

Mock PeXs from the mutants when mixed together, did

not enhance resistance to Psm (Figure 5b). In contrast,

co-application of Avr PeXs from sfd1 plus dir1 to WT No

plants resulted in heightened resistance to Psm (Figure 5b)

and also activated PR1 expression (Figure S4). Similarly,

co-application of Avr PeXs from fad7 plus dir1 also enhanced

resistance to Psm (Figure 5c). These results suggest that the

accumulation of the DIR1-derived factor and the plastid

glycerolipid-dependent factor in Avr PeX is independent of

the other. Furthermore, both factors are required together in

the Avr PeX for long-distance signaling in the establishment

of SAR.

JA is not a component of the SAR-inducing activity

present in Arabidopsis petiole exudates

Recently, it was shown that JA signaling is required for the

SAR-conferred restriction of pathogen growth in Arabidop-

sis (Truman et al., 2007). Moreover, as shown in Figure 7(a),

the JA level was 15-fold higher in Avr PeX than in Mock PeX

of WT leaves, raising the possibility that JA is a component

of the SAR-inducing activity present in Avr PeX. Since FAD7-

synthesized glycerolipids provide fatty acids for the

10 4

10 5

10 6

10 7

WT (Col-gl)

fad7 dir1 fad7 +

dir 1 Source of PeX

Source of PeX

104

10 5

10 6

WT (Nö)

sfd1 dir1 sf d1 +

dir1

WT (Nö)

sf d1 fad7 10 5

10 6

10

(a)

(b)

(c)

7

sf d1 +

fad7 Source of PeX

CF

U le

af d

isc–1

C

FU

leaf

dis

c–1

CF

U le

af d

isc–1

Figure 5. Co-application of avirulent (Avr) petiole exudate (PeX) from sfd1

and fad7 with Avr PeX from dir1 activates systemic acquired resistance in

wild-type (WT) plants.

Mock PeX and Avr PeX were collected from WT and mutant plants. These

PeXs were applied to three lower leaves of WT accession No plant. In addition,

equal volumes of PeXs from the mutant plants were mixed and infiltrated into

leaves of the WT accession No plant. Three days later four upper leaves of

each plant were challenged with Pseudomonas syringae pv. maculicola (Psm)

and bacterial numbers were determined 3 days post-inoculation (dpi). Each

bar represents the average Psm count in 15 leaf discs � SD. White bars,

fractions from Mock PeX; black bars, fractions from Avr PeX.

(a) The PeXs were collected from WT plants of accession No and the sfd1 and

fad7 mutant plants.

(b) The PeXs were collected from No and the sfd1 and dir1 mutant plants.

(c) The PeXs were collected from WT plants of accession Col-gl and the fad7

and dir1 mutant plants.

Plant defense signaling 111

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 106–117

synthesis of JA we tested whether the inability of the Avr

PeX from fad7 and sfd1 mutant to activate SAR could be

complemented by the addition of JA or methylJA (MeJA).

As shown in Figure 8(a,b), JA and MeJA did not comple-

ment the inability of the Avr PeXs from fad7 and sfd1 leaves

to activate SAR. Furthermore, JA and MeJA when infiltrated

individually into WT leaves did not increase resistance to

Psm either (Figure 8a,b). As expected, expression of the

PDF1.2 gene, which is a molecular marker for the activation

of JA signaling, was higher in the MeJA-treated leaves than

the control ethanol-treated leaves (Figure S5), confirming

the activation of JA signaling in the treated leaves.

The SAR-inducing activity in Avr PeX from WT leaves was

fractionated over a gel-filtration column and JA levels

determined in the fractions. The majority of JA was found

in a fraction (no. 24) that lacked the SAR-inducing activity

(Figure 7a,b). The JA content in fraction 12, which contained

the majority of the SAR-inducing activity, was comparable to

that in Mock PeX. Together, the above results confirm that

JA is not a component of the SAR-activating factor present

in Avr PeX.

Discussion

We have shown that the genes FAD7, SFD2 and MGD1 are

required for the activation of SAR. Previously, the SFD1 gene

(also known as GLY1) was shown to be similarly required for

SAR (Nandi et al., 2004). The ability of the fad7, sfd1 and sfd2

mutant alleles to attenuate the ssi2-conferred constitutive

PR1 expression and resistance to Psm supports the role of

these genes in SAR (Figure 2c) (Nandi et al., 2003). Similarly,

mutations in the SFD1 gene also attenuate ssi2-dependent

heightened resistance to Cucumber mosaic virus and the

oomycete Hyaloperonospora parasitica (Kachroo et al.,

2003; Sekine et al., 2004). fad7, sfd1 and sfd2 also diminish

ssi2-dependent accumulation of SA in Arabidopsis leaves

(Kachroo et al., 2005; Nandi et al., 2003). The fad7, sfd1, sfd2

and mgd1 mutants have altered plastid glycerolipid com-

position (Tables S1 and S2) (Jarvis et al., 2000; Nandi et al.,

2003, 2004), suggesting a role for plastid glycerolipids in the

activation of SAR. While SFD1 provides the G3P required for

glycerolipid synthesis in plastids, the x-3 desaturase

encoded by FAD7 catalyzes the desaturation of dienoic (16:2

and 18:2) to trienoic (16:3 and 18:3) fatty acids (Figure 1,

Table S1) (Wallis and Browse, 2002) and SFD2 is required for

the desaturation of 16:0 to 16:1 in plastid glycerolipids

(Figure 1, Table S2). The MGD synthase encoded by MGD1

catalyzes the synthesis of MGD from diacylglycerol in the

TE104

105

106

107

Fr #12 Fr #24

Jasm

onat

e co

nten

t(n

g le

af–1

)

0

10

20

30(a)

(b)

WT(Nö)

sfd1 Fr#12

Fr#24

CF

U le

af d

isc–1

Figure 7. Jasmonic acid (JA) does not co-purify with the systemic acquired

resistance (SAR)-inducing activity in avirulent (Avr) petiole exudate (PeX).

(a) The JA content in Mock PeX (white bars) and Avr PeX (black bars) collected

from wild-type (WT) accession No and sfd1 mutant leaves and in fractions 12

and 24 collected by passing Avr PeX of WT leaves over a gel-filtration column.

The JA content is given as ng JA per leaf from which PeX was collected.

(b) Fractions 12 and 24 collected after passing Mock PeX (white bars) and Avr

PeX (black bars) over the gel-filtration column were infiltrated into three lower

leaves per WT plant of accession No. Three days later, four upper leaves in

each plant were infiltrated with Pseudomonas syringae pv. maculicola (Psm).

As a control (TE), four leaves from 2-amino-2-(hydroxymethyl)-1,3-propane-

diol (TRIS)-EDTA-infiltrated WT plants (hatched bar) were inoculated with

Psm. Bacterial numbers in Psm-infiltrated leaves were determined 3 days

post-inoculation. Each bar represents the average Psm count in 15 leaf

discs � SD. White bars: fractions from Mock PeX; black bars: fractions from

Avr PeX.

104

105

106

ssi2 dir1

WT(Nö)

dir1 ssi2 #1 #2

WT(a)

(b)

(Nö) dir1 ssi2ssi2dir1

PR1

ACT8

CF

U le

af d

isc–1

Figure 6. dir1 suppresses ssi2-conferred PR1 expression and resistance.

(a) Reverse transcriptase-PCR analysis of PR1 transcript accumulation in

leaves of the wild-type (WT) accession No, dir1, ssi2 and ssi2 dir1 double-

mutant line 1. Reverse transcriptase-PCR for ACT8 provided a control for the

quality of the RNA.

(b) Bacterial numbers in Pseudomonas syringae pv. maculicola (Psm)-

inoculated leaves of WT accession No, dir1, ssi2 and two ssi2 dir1 mutant

lines, 3 days post-inoculation. Each bar represents the average Psm count in

15 leaf discs � SD.

112 Ratnesh Chaturvedi et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 106–117

inner membrane of plastids (Arai et al., 2001; Jarvis et al.,

2000). A mutation in the FAD4 gene, which is involved in the

synthesis of PG, the second largest class of glycerolipid in

Arabidopsis plastids, does not adversely affect the activation

of SAR (Figure 2a) and does not attenuate the ssi2-conferred

defense phenotype (Kachroo et al., 2005). In addition, the PG

and SL contents are not affected in the mgd1 mutant (Jarvis

et al., 2000). Together, these results suggest that a trienoic

fatty acid-containing galactolipid is the most likely source of

this SAR-promoting activity. However, considering that loss

of MGD1 activity affects chlorophyll content (Jarvis et al.,

2000) we cannot rule out the possibility that the impact of the

mgd1 allele on SAR is due to a general defect in plastid

function.

Salicylic acid is synthesized in the plastids. Hence, muta-

tions in genes affecting plastid lipid biosynthesis could

indirectly compromise pathogen infection induced SA syn-

thesis and thus affect the activation of SAR. However,

comparable levels of SA accumulated in the Avr pathogen-

inoculated Col-gl (6.9 � 0.2 lg g)1 fresh weight) and fad7

(7.7 � 2.2 lg g)1 fresh weight) leaves. Similarly, we had

previously demonstrated that the sfd1 allele does not

compromise the accumulation of SA in the Avr pathogen-

inoculated leaves (Nandi et al., 2004). Together, these

results suggest that the SAR deficiency of the sfd1 and

fad7 mutants is not due to impairment of SA synthesis in the

Avr pathogen-inoculated leaves. Furthermore, basal resis-

tance to Psm was not compromised in the fad7, sfd1, sfd2

and mgd1 mutants (Figure 2a), confirming that the involve-

ment of plastid glycerolipids is specific to the activation of

SAR. Indeed, experiments with PeXs indicate that SAR

deficiency of the fad7, sfd1 and sfd2 mutants is due to the

absence of SAR-inducing activity in the Avr PeXs of these

mutants (Figure 4a–c). The inability to reconstitute the

SAR-inducing activity by mixing Avr PeXs from sfd1 and

fad7, or sfd2 and fad7 mutant plants (Figure 5a and

Figure S2) indicates that these genes are required for a

common step in SAR that affects accumulation of the SAR-

inducing activity in Avr PeXs. However, the SAR-inducing

activity was reconstituted when Avr PeXs from either

the sfd1 or fad7 mutants were mixed with Avr PeX from

the dir1 mutant (Figure 5b,c). These results indicate that

the DIR1-encoded activity and the plastid glycerolipid-

dependent factor accumulate in the Avr PeX, independent

of each other. Once in the Avr PeX, the DIR1-dependent

activity and the glycerolipid-derived factor could function in

parallel to activate SAR. Alternatively, once in the Avr PeX,

the glycerolipid-derived factor may interact with DIR1, which

has sequence similarity to nsLTPs. The nsLTPs have

active sites large enough to accommodate fatty acids,

lysophospholipids, sterols and oxylipins (Bakan et al.,

2006; Blein et al., 2002; Osman et al., 2001). Indeed, sterol

binding was shown to be essential for the elicitor activity of

cryptogein, a nsLTP produced by Phytophthora spp., which

is transported systemically through the plant (Osman et al.,

2001).

We observed a higher content of JA in the Avr PeX than in

Mock PeX from WT leaves (Figure 7a). Furthermore, Truman

et al. (2007) showed that JA-inducible genes were expressed

at elevated level in the SAR-expressing leaves of WT

Arabidopsis plants and systemic disease resistance was

Avrfad7

Avrsfd1

-MockWT(Nö)

AvrWT(Nö)

- - MeJAEtOH MeJAEtOH MeJAEtOH chemical104

105

106

104

105

106

10

(a)

(b)

7

- -

AvrWT(Nö)

MockWT(Nö)

Avrsfd1

Avrfad7-

JAMeOH chemicalJAMeOH JAMeOH

PeX source

PeX source

CF

U le

af d

isc–1

CF

U le

af d

isc–1

Figure 8. Co-infiltration of jasmonic acid (JA) or methylJA (MeJA) with

avirulent (Avr) petiole exudate (PeX) from sfd1 and fad7 does not reconstitute

the systemic acquired resistance (SAR)-inducing activity.

(a) The Avr PeX from sfd1 (Avr sfd1) and fad7 (Avr fad7) were mixed with an

equal volume of 100 lM JA solution in 0.01% methanol (MeOH) to give a final

JA concentration of 50 lM. These were infiltrated into three lower leaves of

wild-type (WT) plants of accession No. As a negative control, Avr PeX from

sfd1 and fad7 were mixed with an equal volume of 0.01% MeOH and infiltrated

into leaves of WT plants. Wild-type accession No plants pre-treated on three

lower leaves with 0.01% MeOH, 50 lM JA, Mock PeX (Mock WT) and Avr PeX

(Avr WT) from WT accession No leaves provided additional controls for this

experiment.

(b) The Avr PeX from sfd1 (Avr sfd1) and fad7 (Avr fad7) were mixed with an

equal volume of 100 lM MeJA solution in 0.01% ethanol (EtOH) to give a final

MeJA concentration of 50 lM. These were infiltrated into three lower leaves of

WT accession No plants. As a negative control, Avr PeX from sfd1 and fad7

were mixed with an equal volume of 0.01% EtOH and infiltrated into leaves of

WT accession No plants. Wild-type accession No plants pre-treated on three

lower leaves with 0.01% EtOH, 50 lM MeJA, Mock PeX (Mock WT) and Avr

PeX (Avr WT) from WT accession No leaves provided additional controls for

this experiment.

In (a) and (b) 3 days subsequent to the treatment of the lower leaves as

described above, four upper leaves were challenged with Pseudomonas

syringae pv. maculicola (Psm) and bacterial counts monitored 3 days post-

inoculation. Each bar represents the average Psm count in 15 leaf discs � SD.

Plant defense signaling 113

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 106–117

attenuated in the JA-insensitive jin1 and JA-biosynthesis

opr3 mutants. These observations raise the hypothesis that

JA may be the plastid glycerolipid-derived factor that is

required in Avr PeX for the establishment of SAR. However,

several lines of evidence counter this possibility. First, the JA

content of Avr PeX from sfd1 leaves was comparable to that

in Avr PeX from WT leaves (Figure 7a). Second, co-infiltra-

tion of JA or MeJA with Avr PeXs collected from the fad7 and

sfd1 mutant leaves did not alter the inability of these PeXs to

activate SAR (Figure 8a,b), indicating that the SAR defi-

ciency of the fad7 and sfd1 mutants is not a result of JA

deficiency. Furthermore, JA and MeJA when individually

applied to WT plants did not activate PR1 expression and the

SAR-associated systemic resistance to Psm (Figure 8a and

Figure S5). This is in striking contrast to the ability of Avr

PeX from WT leaves to increase PR1 transcript accumula-

tion (Figure 3a) and enhance systemic resistance to Psm

(Figures 3b, 4a–c and 5a–c). Finally, JA did not co-purify

with the SAR-inducing activity that was obtained after

fractionation of Avr PeX over a gel-filtration column

(Figure 7a,b). Together, these results argue against a role

for JA as the primary mobile signal in SAR. However, they

are not in disagreement with the involvement of JA-depen-

dent defenses in restricting pathogen growth in the distal

leaves subsequent to the perception of the SAR signal by

these organs. But, since the coi1 and jar1 mutants are SAR-

competent (Cui et al., 2005; Mishina and Zeier, 2007), the

requirement for JA in SAR-conferred resistance seems to be

independent of COI1 and JAR1 but dependent on JIN1.

When Avr PeX collected from WT leaves was infiltrated

into leaves of the sid2, eds5 and npr1 mutant plants, which

are defective in SA synthesis/signaling, SAR was not

observed (Figure 3b). Rather, on the contrary, bacterial

growth was slightly higher in these Avr Pex-infiltrated sid2,

eds5 and npr1 plants than in the corresponding Mock PeX-

infiltrated plants. The increased growth of Psm in the Avr

Pex-infiltrated sid2, eds5 and npr1 mutants as compared to

the corresponding Mock PeX-infiltrated plants could be a

result of systemic induced susceptibility (SIS), a phenome-

non that previously has been reported in Arabidopsis–

P. syringae interaction (Cui et al., 2005). The prior inocula-

tion of Arabidopsis with coronatine-producing P. syringae

results in increased susceptibility of the distal organs to

subsequent challenge by pathogen. Infiltration of purified

coronatine into the apoplast was sufficient to induce SIS in

the untreated leaves of the same plant (Cui et al., 2005).

Systemic induced susceptibility and SAR are counteracting

processes. The Avr PeX used in our experiments may

contain a SIS-inducing factor, presumably coronatine, in

addition to the SAR-inducing factor. The effects of SIS may

become more prominent when the plant is unable to activate

SAR, as is the case with the sid2, eds5 and npr1 mutants.

In conclusion, we have shown that plastid glycerolipid

metabolism is required for the accumulation of a

SAR-inducing activity in the Avr PeX of Arabidopsis leaves.

This glycerolipid-dependent factor is required along with the

DIR1-encoded nsLTP in Avr PeX for the establishment of

SAR in Arabidopsis. The identification of this plastid glyc-

erolipid-dependent factor will aid in understanding systemic

signaling in plants and in promoting resistance to a broad

spectrum of pathogens.

Experimental procedures

Plant and pathogen cultivation

Arabidopsis was grown at 22�C in growth chambers programmedfor 14 h light (100 lE m)2 sec)1) and 10 h dark cycle. Approximately4-week-old plants were used for all experiments. Pseudomonassyringae pv. maculicola ES4326 and P. syringae pv. tomato DC3000containing the avrRpt2 avirulence gene were propagated at 28�C onKing’s B medium containing appropriate antibiotics (Nandi et al.,2003, 2004). Cultivation of tomato, wheat and F. graminearumwere as previously described (Makandar et al., 2006; Tang et al.,1999).

Bacterial and fungal inoculations

Systemic acquired resistance was activated by infiltrating threelower fully expanded leaves with a suspension (107 colony-forming units (CFU) ml)1) in 10 mM MgCl2 of the avirulentpathogen P. syringae pv. tomato DC3000 avrRpt2. Plants infil-trated with 10 mM MgCl2 provided the mock controls. Three dayslater, four upper leaves were challenged with a suspension(105 CFU ml)1) of the virulent pathogen P. syringae pv. maculi-cola ES4326 (Psm). The Psm-challenged leaves were harvested3 days post inoculation (dpi) and rinsed in 10 mM MgCl2. A corkborer was used to punch out leaf discs (area = 0.28 cm2), whichwere ground in 10 mM MgCl2, and serial 10-fold dilutions platedon King’s medium containing streptomycin (100 lg ml)1). Plateswith bacteria were incubated at 28�C for 2 days before countingthe bacterial colonies. A total of 15 leaf disks (three replicationsof five leaf disks in each sample) were analyzed per treatment.Fusarium graminearum inoculation of wheat and Fusarium headblight disease evaluation were done as previously described(Makandar et al., 2006).

Arabidopsis mutants

The sfd1-1, sfd2-2 and ssi2 mutants are in the accession Nossen(No) (Nandi et al., 2003, 2004; Shah et al., 2001), the dir1 mutant(contains a T-DNA insertion) is in the accession Wassilewskija(Ws) background (Maldonado et al., 2002), the fad7-1 mutant is inthe accession Columbia (Col) glabra1, and the mgd1 mutant is inthe accession Col background (Jarvis et al., 2000). To generatethe ssi2 fad7-1 double mutant, the ssi2 mutant was crossed withthe fad7-1 mutant plant and the F2 progeny were screened fordouble-mutant plants. The ssi2 mutant allele was followed byPCR (see below) and plants homozygous for fad7-1 allelewere identified based on their lipid profile (reduction in 34:6- and36:6-MGD levels). The ssi2 dir1 plants were identified from a F2

population derived from a cross between the ssi2 and dir1mutant plants. Segregants homozygous for ssi2 and dir1 wereidentified by PCR (see below).

114 Ratnesh Chaturvedi et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 106–117

Lipid and phytohormone analysis

Lipid extraction and electrospray ionization tandem mass spec-trometry analysis of leaf lipids were performed as previouslydescribed (Devaiah et al., 2006; Welti et al., 2002). The JA and SAlevels were determined by gas chromatography-mass spectrometry(Schmelz et al., 2003).

RNA extraction and RT-PCR analysis

Leaf tissue was ground under liquid nitrogen, and RNA wasextracted using acid guanidinium thiocyanate-phenol-chloroformas previously described (Chomczynski and Sacchi, 1987). The iso-lated RNA was purified and used in the reverse-transcription poly-merase chain reactions (RT-PCR) (Pegadaraju et al., 2007). The PCRprimers for the ACT8 gene (At1g49240) are as previously described(Pegadaraju et al., 2005). The At-PR1-F 5¢-GCTCTTGTAGGTGCTC-TTGTTC-3¢ and At-PR1-R 5¢-CAGCTCTTATTTGTATTATTTG-3¢primers were used for PCR amplification of PR1 (At2g14610). ThePCR was performed with the following conditions: 95�C for 5 minfollowed by 25 cycles of 95�C for 30 sec, 60�C for 45 sec, 72�C for1 min, with final extension at 72�C for 10 min.

DNA extraction and PCR analysis

Arabidopsis genomic DNA from leaf tissue was isolated as previ-ously described (Konieczny and Ausubel, 1993). ssi2 was distin-guished from SSI2 using a derived-cleaved amplified polymorphicsequence (dCAPS). The primers, ssi2dCAPS-F 5¢-TTGTTTTGGTGGGGGACATGATCACAGAAGGTGCA-3¢ and ssi2dCAPS-R 5¢-TCGATCTGCCTCATGTCAACACG-3¢ were used in the PCR reaction. ThePCR was performed with the following conditions: 95�C for 5 minfollowed by 35 cycles of 95�C for 45 sec, 65�C for 45 sec, 72�C for45 sec, with final extension at 72�C for 5 min. The 200-bp PCRproduct derived from WT DNA contains one ApaL1 (New EnglandBiolabs, http://www.neb.com/) site, which on restriction with Apal1yields two products of 175 and 25 bp. In contrast, the PCR productderived from the ssi2 allele lacks the ApaL1 site. To identify dir1homozygous plants the T-DNA left border primer T-DNA-LBb15¢-GCGTGGACCGCTTGCTGCAACT-3¢ was used along with aprimer designed to the 3¢ untranslated region (UTR) of DIR1(5¢-GGGGTTAACCCATAAACGCCATTTGGCAG-3¢).

Collection of petiole exudates

The PeXs were collected essentially as previously described(Maldonado et al., 2002). A suspension (107 CFU ml)1)ofP. syringae pv. tomato containing the avrRpt2 gene wasinfiltrated into the abaxial surface of leaves. For the controls(Mock), 10 mM MgCl2 was similarly infiltrated into leaves. Plantswere placed in the growth chamber, covered with a plastic dome.Eight hours after infiltration, leaves were harvested for collectionof PeX. Leaves were cut at the base of their petiole using sharpscissors and the petiole immediately dipped in 50% ethanol fol-lowed by 0.0005% bleach and finally in 1 mM EDTA (pH 8.0)solution. The base of each petiole was immersed in 2.5 mlcollection solution (1 mM EDTA, pH 8.0) contained in a well of a24-well tissue culture plate (ICN Biomedical Inc.; http://www.mpbio.com). Five harvested leaves were placed in eachwell. Wet paper towels were placed under the tissue culture traysand the entire set-up was covered with a transparent plasticdome and incubated in a growth chamber exposed to a 14/10 h

light/dark period. For each treatment 50–100 leaves were used. Atthe end of the exudation period, PeXs from multiple wells werepooled. The absence of any contaminating P. syringae pv.tomato DC3000 avrRpt2 in the PeXs was tested by plating analiquot of the exudates on King’s medium (King et al., 1954). Thebiological activity of the PeX was determined by infiltratingexudates diluted two-fold in sterile water into leaves of healthyplants. The treated plants were placed in the growth chamber for3 days to allow for SAR to be activated. At the end of theincubation, the upper uninfiltrated leaves of the plant werechallenged with Psm and bacterial counts determined 3 dayslater. For experiments that involved mixing PeXs from twodifferent genotypes, equal volumes of each PeX were mixedand diluted two-fold in sterile water before infiltration intoArabidopsis leaves.

Gel-filtration fractionation of SAR-inducing activity

The PeX collected from 10 mM MgCl2- and Avr pathogen-inocu-lated plants were lyophilized at )70�C. The lyophilized powderwas reconstituted in 1 ml 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS)-EDTA buffer (10 mM TRIS-HCl, 1 mM EDTA,pH 7.5) and centrifuged at 12 000g for 5 min to sediment par-ticulate matter. The supernatant was loaded on a 2 · 25 cm col-umn packed with Bio-Gel A-5m matrix (Bio-Rad, http://www.bio-rad.com/) that was pre-equilibrated with TRIS-EDTA buffer.The column was eluted with TRIS-EDTA buffer. One-milliliterfractions were collected at a flow rate of 1 ml min)1. Biologicalactivity of individual fractions was tested by infiltrating two-folddiluted fractions into leaves of 4-week-old WT plants. Threedays after infiltration, upper leaves of the infiltrated plantswere challenged with Psm and bacterial growth monitored asdescribed above.

Acknowledgements

We thank Fred Ausubel, Robin Cameron and Henrik Aronsson forproviding the sid2, dir1 and mgd1 mutant, respectively, and theABRC stock center at Ohio State University for providing the fad7-1mutants. We thank Eric Schmelz for training AAS in the measure-ment of phytohormones. This work was supported at varied timesby grants from the National Science Foundation (IOB0543862 andMCB0455318) and awards from the NIH Grant Number P20RR016475 from the INBRE Program of the National Center forResearch Resources. Additional support of the Kansas LipidomicsResearch Center was from NSF grants (EPS-0236913 andDBI0521587), Kansas Technology Enterprise Corporation andKansas State University. This is Kansas Agricultural ExperimentalStation contribution 07-151J.

Supplementary Material

The following supplementary material is available for this articleonline:Figure S1. Systemic acquired resistance competent petioleexudates from Arabidopsis promote resistance to Pseudomonassyringae in tomato.Figure S2. Co-application of avirulent petiole exudates collectedfrom the Arabidopsis sfd2 and fad7 mutants does not activate SARin wild-type plants.Figure S3. Avirulent petiole exudates from dir1 lack a SAR inducingactivity.

Plant defense signaling 115

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 106–117

Figure S4. PR1 expression in wild-type leaves infiltrated with petioleexudates from sfd1 and dir1 mutants.Figure S5. PR1 and PDF1.2 transcript accumulation in methyljasmonic acid-infiltrated leaves.Table S1. Comparison of digalactosyldiacylglycerol (DGD), monog-alactosyldiacylglycerol (MGD) and phosphatidylglycerol (PG) com-position (nmol mg)1 dry weight) in leaves of wild-type Arabidopsisaccession Columbia and the fad4 and fad7 mutants.Table S2. Comparison of digalactosyldiacylglycerol (DGD),monogalactosyldiacylglycerol (MGD) and phosphatidylglycerol(PG) composition (nmol mg)1 dry weight) in leaves of wild-typeArabidopsis accession Nossen and the sfd2-2 mutant.This material is available as part of the online article from http://www.blackwell-synergy.comPlease note: Blackwell Publishing are not responsible for the contentor functionality of any supplementary materials supplied by theauthors. Any queries (other than missing material) should bedirected to the corresponding author for the article.

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