N-Acyl-Homoserine Lactone Confers Resistance toward Biotrophic and Hemibiotrophic Pathogens via Altered Activation of AtMPK6

Abstract

Pathogenic and symbiotic bacteria rely on quorum sensing to coordinate the collective behavior during the interactions with their eukaryotic hosts. Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as signals in such communication. Here we show that plants have evolved means to perceive AHLs and that the length of acyl moiety and the functional group at the γ position specify the plant's response. Root treatment with the N-3-oxo-tetradecanoyl-L-homoserine lactone (oxo-C14-HSL) reinforced the systemic resistance to the obligate biotrophic fungi Golovinomyces orontii in Arabidopsis (Arabidopsis thaliana) and Blumeria graminis f. sp. hordei in barley (Hordeum vulgare) plants. In addition, oxo-C14-HSL-treated Arabidopsis plants were more resistant toward the hemibiotrophic bacterial pathogen Pseudomonas syringae pv tomato DC3000. Oxo-C14-HSL promoted a stronger activation of mitogen-activated protein kinases AtMPK3 and AtMPK6 when challenged with flg22, followed by a higher expression of the defense-related transcription factors WRKY22 and WRKY29, as well as the PATHOGENESIS-RELATED1 gene. In contrast to wild-type Arabidopsis and mpk3 mutant, the mpk6 mutant is compromised in the AHL effect, suggesting that AtMPK6 is required for AHL-induced resistance. Results of this study show that AHLs commonly produced in the rhizosphere are crucial factors in plant pathology and could be an agronomic issue whose full impact has to be elucidated in future analyses.

Full text

N-Acyl-Homoserine Lactone Confers Resistance toward
Biotrophic and Hemibiotrophic Pathogens via
Altered Activation of AtMPK61[C][W]
Adam Schikora2*, Sebastian T. Schenk2, Elke Stein2, Alexandra Molitor3,AlgaZuccaro
4,
and Karl-Heinz Kogel
Institute of Phytopathology and Applied Zoology, Research Center for BioSystems, Land Use and Nutrition
(IFZ), Justus Liebig University Giessen, 35392 Giessen, Germany
Pathogenic and symbiotic bacteria rely on quorum sensing to coordinate the collective behavior during the interactions with
their eukaryotic hosts. Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as signals in such communi-
cation. Here we show that plants have evolved means to perceive AHLs and that the length of acyl moiety and the functional
group at the gposition specify the plant’s response. Root treatment with the N-3-oxo-tetradecanoyl-L-homoserine lactone (oxo-
C14-HSL) reinforced the systemic resistance to the obligate biotrophic fungi Golovinomyces orontii in Arabidopsis (Arabidopsis
thaliana) and Blumeria graminis f. sp. hordei in barley (Hordeum vulgare) plants. In addition, oxo-C14-HSL-treated Arabidopsis
plants were more resistant toward the hemibiotrophic bacterial pathogen Pseudomonas syringae pv tomato DC3000. Oxo-C14-
HSL promoted a stronger activation of mitogen-activated protein kinases AtMPK3 and AtMPK6 when challenged with flg22,
followed by a higher expression of the defense-related transcription factors WRKY22 and WRKY29, as well as the
PATHOGENESIS-RELATED1 gene. In contrast to wild-type Arabidopsis and mpk3 mutant, the mpk6 mutant is compromised
in the AHL effect, suggesting that AtMPK6 is required for AHL-induced resistance. Results of this study show that AHLs
commonly produced in the rhizosphere are crucial factors in plant pathology and could be an agronomic issue whose full
impact has to be elucidated in future analyses.
Rhizosphere communication is based on a com-
plex exchange and perception of molecules originating
from interacting organisms. Many bacteria use N-acyl-
homo-Ser lactones (AHLs) to coordinate the behavior of
individual cells in a population. This communication
phenomenon is called quorum sensing (QS), and was
first described in Vibrio fisheri (Engebrecht and Silverman,
1984). V. fisheri secretes N-3-oxo-hexanoyl-L-homo-Ser
lactone (oxo-C6-HSL), which is synthesized by the
enzyme LuxI from S-adenosyl Met and an acyl chain
carrier protein (Schaefer et al., 1996) and sensed by a
LuxR-type receptor (Hanzelka and Greenberg, 1995).
In many Gram-negative bacteria, the control of the
population density relies on production of diverse
AHLs that influence gene transcription once detected
by a companion. Numerous AHL derivatives varying
in the length of the acyl chain (from 4–18 carbons) and
in the substitution at the gposition of the chain with
hydroxyl (OH) or oxo (O) groups have been so far
identified (for review, see Williams, 2007).
AHLs also have an impact on eukaryotic cells. N-3-
oxo-dodecanoyl-L-homo-Ser lactone (oxo-C12-HSL),
produced by the opportunistic plant and human path-
ogen Pseudomonas aeruginosa, induces distension of
mitochondria and endoplasmic reticulum (Kravchenko
et al., 2006). In addition, it increases phosphorylation of
the mitogen-activated protein kinase (MAPK) p38 and
the translation initiation factor elF2a. In human NCI-
H292 cells, treatment with oxo-C12-HSL enhances the
phosphorylation of the negative regulator of NF-kB, the
I-kB, and the ERK1/2 MAP kinases (Imamura et al.,
2004). Moreover, oxo-C12-HSL modulates the NFkB
signaling and abolishes the TOLL-LIKE RECEPTOR4-
dependent immune response in mice (Kravchenko et al.,
2008). It was suggested that mammalian cells perceive
oxo-C12-HSL in a TOLL-LIKE RECEPTOR-independent
manner (Kravchenko et al., 2006). Jahoor et al. (2008)
proposed the peroxisome proliferator-activated recep-
tors PPARgand PPARb, members of the nuclear hor-
mone receptor family as potential candidates for AHLs
receptorsinanimals.Ontheotherhand,AHLswere
shown to interact directly with different cellular and
artificial membranes, which suggest another possible
1
This work was supported by the Bundesministerium fu
¨r Bil-
dung und Forschung, Germany, in the frame of the GABI-forte
program.
2
These authors contributed equally to the article.
3
Present address: KWS SAAT AG, Grimselstrasse 31, 37555
Einbeck, Germany.
4
Present address: Max Planck Institute for Terrestrial Microbiol-
ogy, Karl-von-Frisch-Strasse 10, 35043 Marburg, Germany.
* Corresponding author; e-mail adam.schikora@agrar.uni-giessen.de.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Adam Schikora (adam.schikora@agrar.uni-giessen.de).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.111.180604
Plant PhysiologyÒ,November 2011, Vol. 157, pp. 1407–1418, www.plantphysiol.org Ó2011 American Society of Plant Biologists. All Rights Reserved. 1407

membrane-located AHL-reception system (Davis et al.,
2010).
In the plant-interacting Agrobacterium tumefaciens,N-3-
oxo-octoanoyl-L-homo-Ser lactone is produced by the
TraI enzyme and perceived by the LuxR-homologous
receptor TraR (Fuqua and Winans, 1994). Both traI and
traR genes are located on the Ti plasmid and are induced
by the tumor-released octopine; TraR transcriptionally
induces all genes required for conjugation and intra-
bacterial transfer of the Ti plasmid. N-3-oxo-octoanoyl-
L-homo-Ser lactone also induces the repABC operon,
obligatory for stable vegetative replication of the octo-
pine-type Ti plasmid (Fuqua and Winans, 1994; Pappas
and Winans, 2003). On the other hand, plants may
interact with the Agrobacterium QS. Different plant sig-
nals induce the transcription of bacterial attM and aiiB
genes coding for two lactonases, AttM and AiiB. They
cleave the g-butyrolactone ring of AHLs and might in
this way modulate the QS-dependent virulence of Agro-
bacterium (Haudecoeur et al., 2009a, 2009b).
In recent years, several reports have shown that plants
evolved means to perceive AHLs and respond to them
withchangesingeneexpressionormodificationsin
development (Mathesius et al., 2003; Schuhegger et al.,
2006; Go
¨tz et al., 2007; Ortı
´z-Castro et al., 2008; von Rad
et al., 2008; Pang et al., 2009). Assessment of AHLs’
impact on root development revealed that C6-HSL
promotes root growth (von Rad et al., 2008), C10-HSL
alters root architecture similarly to auxin, but in an
auxin-independent way, and C12-HSL strongly induces
root hair formation (Ortı
´z-Castro et al., 2008). Several
reports provided indirect evidences that AHLs play a
role in plant immunity. Serratia liquefaciens MG1 in-
creases systemic resistance of tomato (Solanum lycoper-
sicum) plants against the fungal leaf pathogen Alternaria
alternata. However, the AHL negative mutant S. lique-
faciens MG44 is less effective (Schuhegger et al., 2006).
Similarly, colonization with the Serratia plymuthica protects
cucumber (Cucumis sativus) plants from the damping-
off disease caused by Pythium aphanidermatum, as well
as tomato and bean (Phaseolus vulgaris) plants from
infection with the gray mold fungus Botrytis cinerea
(Pang et al., 2009). The splI2mutant of S. plymuthica,
impaired in the production of AHLs, could not pro-
vide this protection (Pang et al., 2009). However, the
molecular basis of AHLs’ influence on the plant im-
mune system is still unknown.
In this report we addressed the question how AHLs
affect the plant immune system. We tested AHL-induced
resistance toward different pathogens, using a monocot-
yledonous (barley [Hordeum vulgare]) and a dicotyledon-
ous (Arabidopsis) host. Our results show that even
though the long-chained AHLs cannot be transported
within the plant, their perception in roots enhances the
systemic resistance toward biotrophic and hemibiotro-
phic pathogens. Furthermore, in Arabidopsis plants
pretreated with N-3-oxo-tetradecanoyl-L-homo-Ser lac-
tone (oxo-C14-HSL), we observed prolonged activities of
the MAPKs AtMPK3 and AtMPK6 after induction with
the bacterial pathogen-associated molecular pattern
(PAMP) flg22. The modified activities of MAPKs pre-
ceded enhanced transcription of the defense-related
WRKY22 and WRKR29 transcription factors, as well as
the PATHOGENESIS-RELATED1 (PR1) gene. Taken to-
gether, results presented here suggest that perception of
long-chained AHLs has a positive impact on plant
resistance.
RESULTS
Long-Chain AHLs Have No Impact on Plant Growth
Several AHLs have been reported to modify root
development and promote plant growth (Ortı
´z-Castro
et al., 2008; von Rad et al., 2008). Therefore, to study how
oxo-C14-HSL modulates plant physiology, we first mon-
itored the impact of oxo-C14-HSL on growth and devel-
opment of Arabidopsis Columbia-0 (Col-0). Ten-day-old
seedlings grown on standard one-half Murashige and
Skoog (MS) medium were transferred to medium sup-
plied with 6 mMoxo-C14-HSL, and cultivated for addi-
tional 4 or 8 d. No alteration in the shoot or root
development was observed (Fig. 1A). However, in ac-
cordance with previous reports (von Rad et al., 2008), the
short-chained C6-HSL, used here as a positive control,
promoted shoot and root biomass accumulation (Fig.
1B), as well as the root length (Fig. 1, A and C).
Application of Oxo-C14-HSL to Roots Does Not Result in
AHL Detection in Leaves
Next, we addressed the question whether AHLs can
be transported within the plant. To this end, we
recorded the presence of AHL exogenously applied to
the root medium, in different plant tissues. We used
reporter bacteria carrying either the GFP gene or the
lux operon from V. fi s h e ri under AHL-inducible promot-
ers (Supplemental Fig. S1, A and D). Cleared acetone
extracts from either roots or shoots were prepared and
applied to lawns of reporter bacteria (Supplemental Fig.
S1B). GFP or luminescence signals were recorded 2 h
after application. The most sensitive reporter bacterium
was Pseudomonas putida F117 pKR C12 GFP (Steidle et al.,
2001; carrying the lasR+lasB::gfp construct) with a detec-
tion limit of approximately 60 nMfor oxo-C14-HSL
(Supplemental Fig. S1A). In accord with previous re-
ports (von Rad et al., 2008), if Arabidopsis roots were
treated with C6-HSL, the AHL was detectable in
extracts from roots and shoots of the treated plants
(Supplemental Fig. S1B). In contrast, the long-chained
oxo-C14-HSL was not detected in leaf extracts even
though it was detectable in extracts from the roots of
oxo-C14-HSL-treated plants. Neither the P. putida F117
pKR C12 GFP strain, nor the Escherichia coli strain
pSB403 (Winson et al., 1998) carrying the lux operon
from V. fisheri, were able to detect oxo-C14-HSL in the
leaf extracts (Fig. 1D; Supplemental Fig. S1B). These
results support the notion that long-chain AHLs are
not transported systemically throughout the plant
organism.
Schikora et al.
1408 Plant Physiol. Vol. 157, 2011

AHLs Confer Resistance against Hemibiotrophic and
Biotrophic Pathogens
Based on the strong effect of AHLs on the mamma-
lian immune system (Kravchenko et al., 2008), we
followed the working hypothesis that AHLs may
induce plant resistance to microbial pathogens. Arabi-
dopsis plants were grown for 5 weeks in sterile hy-
droponics culture, and then the growing medium was
supplied with 6 mMoxo-C14-HSL for 3 d. Subsequently
plants were spray inoculated with Pseudomonas syrin-
gae pv tomato DC3000 (Pst). Bacterial population on
leaves was monitored during 96 h post inoculation
(hpi). Plants pretreated with oxo-C14-HSL developed
significantly smaller populations of Pst bacteria then
the control plants (Fig. 2A).
To exclude the possibility that oxo-C14-HSL has a
direct effect on bacterial fitness, we cultivated bacteria
in the presence of 6 mMoxo-C14-HSL and monitored
population density over 48 h. No change in bacterial
duplication time was observed (Supplemental Fig.
S2A). In addition, we examined the virulence of bac-
teria grown in the presence of oxo-C14-HSL and
subsequently sprayed onto Arabidopsis leaves (Sup-
plemental Fig. S2B), or grown in standard Kings B
medium and sprayed onto leaves together with the
AHL (Supplemental Fig. S2C). None of these tests
showed that oxo-C14-HSL lowers bacterial fitness or
the virulence. Hence, results presented above suggest
that oxo-C14-HSL enhances resistance of Arabidopsis
toward Pst bacteria. Interestingly, not only oxo-C14-
HSL, but also OH-C14-HSL and oxo-C12-HSL reduce
Pst proliferation, though to lesser extent than oxo-C14-
HSL (Fig. 2B; Supplemental Fig. S7A).
To expand our observations to another pathogen, we
analyzed the impact of oxo-C14-HSL on the develop-
ment of the fungus Golovinomyces orontii, the causal
agent of powdery mildew in Arabidopsis. Hydropon-
ically grown Arabidopsis plants were pretreated with
6mMoxo-C14-HSL and leaves were subsequently
inoculated with G. orontii (560 conidia/cm2leaf). The
number of mycelia per leaf was calculated 5 d after
inoculation. We found that the number of mycelia
developing on leaves was significantly reduced in
plants pretreated with oxo-C14-HSL (Fig. 3A). On the
other hand, the morphology of G. orontii mycelium
was not affected by the AHL treatment (Fig. 3, B–D).
These results support the concept that long-chained
AHL exhibits a positive effect on the resistance toward
hemibiotrophic and biotrophic pathogens.
Oxo-C14-HSL Induces Resistance against the Biotrophic
Powdery Mildew Fungus in Barley Plants
In addition to experiments with Arabidopsis, we
assessed oxo-C14-HSL-induced resistance in the mono-
cotyledonous crop plant barley. Barley seedlings were
grown for 5 d and then pretreated with 6 mMoxo-C14-
HSL for 3 d in hydroponics culture. Subsequently first
leaves were inoculated with the casual agent of barley
powdery mildew Blumeria graminis f. sp. hordei (450
conidia/cm2of leaf). As shown by the increased rate of
fungal penetration failure (Fig. 3E) the effect of oxo-
C14-HSL on barley is similar to that on Arabidopsis.
The frequency of elongating secondary hyphae (ESH)
shown in Figure 3F, indicative of successful infection,
decreased from 43% of all interactions in control plants
to 14% in oxo-C14-HSL-treated plants, while the fre-
quency of papillae (Fig. 3G), indicative of reduced host
cell accessibility, increased from about 54% in control to
over 77% of all interactions in AHL-treated plants (Fig.
Figure 1. Long-chained oxo-C14-HSL has no im-
pact on plant growth and is not transported within
the plant. A to C, Col-0 plants were grown for 10
d on one-half MS medium and afterward trans-
ferred to one-half MS medium supplied with 6 mM
oxo-C14-HSL, or 6 mMC6-HSL. A, Col-0 plants
grown for 4 d on C6-HSL or oxo-C14-HSL, bar =
1 cm. B, Fresh weights (mg/plant) of plants treated
with different AHLs, n$80. C, Root length of
Arabidopsis plants on media supplied with differ-
ent AHLs for 4 d, n$40, *P#0.05 (Student’s t
test). D, Oxo-C14-HSL is not transported system-
ically throughout the plant. Six micromolar oxo-
C14-HSL was added to the root medium for 3 d.
Subsequently, fresh plant material was extracted
in acetone and detection assays were performed
by using the reporter bacterium E. coli Top10
pSB403 lxR+luxI::luxCDABE. [See online article
for color version of this figure.]
Quorum Sensing Molecule Induces Plant Immunity
Plant Physiol. Vol. 157, 2011 1409

3E). The third possibility to respond is the hypersensi-
tivity response (HR; Fig. 3H), also this resistance mech-
anism was enhanced in AHL-treated plants (Fig. 3E). In
conclusion, oxo-C14-HSL affects the basal immune
system of Arabidopsis and barley.
Oxo-C14-HSL Does Not Induce Resistance against
Necrotrophic Pathogens
To examine the hypothesis thatoxo-C14-HSL induces
resistance particularly against hemibiotrophic and bio-
trophic pathogens, we performed a series of tests with
necrotrophic fungal pathogens. Arabidopsis plants
were pretreated as above for 3 d with 6 mMoxo-C14-
HSL, and detached leaves were subsequently inocu-
lated with B. cinerea or Plectosphaerella cucumerina BMM.
Disease symptoms were analyzed after 5 d. Both path-
ogens caused maceration of leaf tissue, which can be
classified into different symptom categories (B. cinerea;
Fig. 4, A–D) or measured by lesion diameter (P. cucu-
merina BMM; Fig. 4, E–H). No significant differences in
symptoms caused by B. cinerea or P. c u c u m e r i n a BMM,
as compared with water and acetone controls were
observed (Fig. 4). To evaluate the possibility that
resistance against necrotrophic fungi may be induced
by different AHL concentration, we conducted a
dose-response experiment with B. cinerea,usingvar-
ious concentrations of oxo-C14-HSL (0.6–12 mM). Yet,
none of the tested concentration significantly en-
hanced resistance toward this fungus (Supplemental
Fig. S3).
AHL Modulates the Response to the Bacterial
Pathogen-Associated Molecular Pattern flg22
To study the mechanism by which AHL induces
resistance in more detail, we examined the response
of AHL-treated Arabidopsis plants to flg22. First, we
monitored the accumulation of reactive oxygen spe-
cies (ROS) after solely treatment with oxo-C14-HSL.
For this, leaves of soil-grown plants were treated for 3
d with 6 mMoxo-C14-HSL in floating conditions and
stained with diaminobenzidine (DAB). DAB staining
revealed no changes in the steady-state level of hydro-
gen peroxide (H2O2; Supplemental Fig. S4). Next, we
analyzed the production of ROS (oxidative burst) in
those leaves after elicitation with 100 nMflg22. Like-
wise, ROS production induced by flg22 in Arabidopsis
leaves was neither enhanced nor inhibited by pretreat-
ment with AHL (Fig. 5A).
In the following step, we analyzed the activation of
the MAPKs AtMPK3 and AtMPK6. Both kinases have a
high degree of functional redundancy and are involved
in plant defense mechanisms. Total protein extract was
prepared from 2-week-old seedlings grown on agar
plates and pretreated with oxo-C14-HSL in liquid one-
half MS medium for additional 3 d. MAPKs were
activated by treatment with 100 nMflg22. Phosphory-
lated forms of AtMPK3 and AtMPK6 were visualized
using the apERK1/2 antibody (Hamel et al., 2005). Con-
sistent with earlier reports, flg22 triggered a short tran-
sient activation of AtMPK3 and AtMPK6 at 15 min after
application (Fig. 5B). Intriguingly, AHL-pretreated seed-
lings showed stronger activation of both kinases, which
lasted until 60 min after treatment (Fig. 5B). Immuno-
blots with respective aAtMPK3 and aAtMPK6 anti-
bodies showed no changes in AtMPK3 and AtMPK6
quantities during 1 h after treatment (Fig. 5B). Similarly,
quantitative reverse transcription (qRT-PCR) analysis
detected no changes in AtMPK3 and AtMPK6 transcript
levels after pretreatment with AHLs (Supplemental Fig.
S5A), and only slightly changes after the subsequent
treatment with flg22 (Supplemental Fig. S5, B and C).
Although we cannot exclude a short transcriptional
induction of AtMPK3/6between 1 and 2 h after flg22
treatment (those intermediate time points were not
examined), the results obtained in our tests indicate
rather that oxo-C14-HSL modulates the flg22-induced
phosphorylation status of AtMPK3 and AtMPK6 and
not the amount of the kinases.
Owing to the fact that numerous WRKY transcription
factors are induced upon pathogen attack and some
WRKY are direct targets of activated MAPKs, we
assessed whether oxo-C14-HSL-mediated enhanced ac-
tivation of MAPKs influences the expression levels of
defense-related WRKYs. We examined the relative ex-
pression levels of WRKY18,WKRY22,andWRKY29
Figure 2. C14-HSL derivatives induce resistance against P. syringae.A,
Proliferation of Pst on 5-week-old Arabidopsis was analyzed at hpi as
indicated. Plants were untreated (control) or pretreated with acetone or
6mMoxo-C14-HSL, 3 d prior to infection with Pst bacteria. **P#
0.005; ***P#0.0005. B, Colony formingunits (cfu) ofPst harvested from
5-week-old Arabidopsis plants pretreated for 3 d with acetone, 6 mMoxo-
C14-HSL, or 6 mMOH-C14-HSL, and subsequently inoculated with Pst
bacteria for 96 h. a = P,5.2E-6, b = P,9.7E-11 (Student’s ttest).
Schikora et al.
1410 Plant Physiol. Vol. 157, 2011

whose activation is pathogenesis associated or induced
by flg22 (Asai et al., 2002; Xu et al., 2006; Zheng et al.,
2006). Two-week-old Arabidopsis seedlings were pre-
treated with 6 mMoxo-C14-HSL as described above and
subsequently elicited with 100 nMflg22. Total RNA was
extracted and transcript levels of WRKYs normalized to
the expression of UBQ (At5g25760). Pretreatment with
the AHL alone did not induce the expression of either
WRKY (Supplemental Fig. S6, A–C). However, a sub-
sequent treatment with 100 nMflg22 resulted in differ-
ent expression patterns. Both WRKY22 and WRKY29
show induction already 30 min after treatment with
elicitor (Libault et al., 2007). Here we show a prolonged
and stronger transcriptional activation of WRKY22 and
WRKY29 in plants pretreated with oxo-C14-HSL even
at 2, 6, and 12 h after flg22 treatment (Fig. 5, C and D).
WRKY18 expression did not change after the subse-
quent flg22 treatment (Fig. 5E).
PR genes such as PR1, are prominent example of
defense-associated gene in Arabidopsis. Consequently,
we examined the expression level of PR1 after treat-
ment with oxo-C14-HSL. Similar to the expression of
WRKY22 and WRKY29, AHL treatment alone does not
induce the expression of PR1 (Supplemental Fig. S6D).
However, expression of PR1 is induced after treatment
with 100 nMflg22, and pretreatment with oxo-C14-HSL
enhances it even more (Fig. 5F).
Active Kinases Are Necessary for
AHL-Induced Resistance
To test the assumption that AtMPK3 and/or
AtMPK6 activations are required for AHL-induced
resistance we analyzed the mpk3 and mpk6 mutants.
First we monitored the phosphorylation status of the
kinasesinbothmutants.Two-week-oldCol-0wild-
type and mutant seedlings were pretreated for 3 d
with 6 mMoxo-C14-HSL in liquid medium and MAPK’s
activation was triggered by addition of 100 nMflg22.
In the mpk3 mutant the phosphorylation pattern of
AtMPK6 is similar to that in Col-0 plants: The 45-kD
band representing the double-phosphorylated form
of AtMPK6 is stronger in AHL-treated mpk3 plants
(Fig. 6A). Interestingly, in the mpk6 mutant, the
pAtMPK3 signal is much weaker if compared to
pAtMPK3 in the Col-0 plants (Fig. 6A). To investigate
this in further detail, we tested AHL-induced resis-
tance toward Pst in the mpk3 and the mpk6 mutants.
Mutant plants were grown for 5 weeks in hydropon-
ics culture and pretreated for 3 d with 6 mMoxo-C14-
HSL prior to a subsequent inoculation with Pst.In
clear contrast to Col-0 wild-type and mpk3 mutant
plants, the mpk6 mutant was compromised in the in-
duced resistance toward Pst, suggesting that AtMPK6
plays a critical role in the AHL-induced resistance
(Fig. 6B).
Figure 3. Oxo-C14-HSL induces resistance
against G. orontii and B. graminis. Five-week-
old Arabidopsis or 5-d-old barley plants were
pretreated for 3 d with 6 mMoxo-C14-HSL in
hydroponics cultures and then fungal conidia
suspension was sprayed on their leaves. A to D,
Proliferation of G. orontii on Arabidopsis leaves.
A, Number of mycelia per leaf at 5 d post
inoculation (dpi). Student’s ttest performed on
the raw data resulted in P= 0.33 (control versus
acetone); P= 0.04 (control versus oxo); and P=
0.01 (acetone versus oxo). B to D, Mycelia for-
mation on untreated Arabidopsis leaves (control;
B), pretreated with acetone (C), or oxo-C14-HSL
(D), shows no alternation in fungal development.
E to H, Proliferation of B. graminis on barley. E,
Percent of interaction sites with ESH, papillae,
and HR, on first leaves at 5 dpi. F, ESH formation,
haustorium (arrow). G, Papillae formation (arrow-
head). H, HR. *P#0.05; **P#0.005; ***P#
0.0005 (Student’s ttest).
Quorum Sensing Molecule Induces Plant Immunity
Plant Physiol. Vol. 157, 2011 1411

Next, we analyzed the expression pattern of WRKY22
and WRKY29 in the mpk3 and mpk6 mutants. In wild-
type Col-0 plants, expression of both WRKYsisup-
regulated upon treatment with flg22, and could be
even more induced by pretreatment with AHL (Fig. 5,
C and D). mpk3 and mpk6 mutants were grown for 2
weeks on standard medium and transferred to liquid
one-half MS medium supplied with AHL 3 d before
the treatment with flg22. In both the mpk3 and the mpk6
mutant, expression of WRKYs is induced after treat-
ment with flg22, yet it is not further modified by the
pretreatment with oxo-C14-HSL (Fig. 6, C–F).
AHL Pretreatment Enhances Effector-Triggered
Immunity-Based Defense Mechanism in Arabidopsis
In plants, recognition of a pathogen occurs at two
levels: The pattern recognition receptors detect PAMPs
and induce the PAMP-triggered immunity; the host-
encoded R proteins, on the other hand, recognize (directly
or indirectly) microbial effectors and initiate the effector-
triggered immunity (ETI). We wondered whether AHL
might influence also the second level of defense: the
ETI. An increased frequency of HR is often associated
with ETI as the consequence of the recognition of bac-
terial effectors via the R proteins. Therefore, we used
the Pst strain expressing the AvrRpt2 gene, whose
product is recognized in Arabidopsis by the RPS2
receptor (Kunkel et al., 1993; Yu et al., 1993). Arabi-
dopsis Col-0 plants were grown in soil for 4 weeks and
detached leaves were pretreated for 3 d with 6 mMoxo-
C14-HSL in floating conditions. Leaves were then
spray inoculated with the Pst AvrRpt2 strain and the
accumulation of H2O2, as well as the cell death rate
were analyzed during 48 h. Pretreatment with oxo-
C14-HSL resulted in a strong accumulation of H2O2
after Pst AvrRpt2 inoculation, as visualized by the DAB
staining (Fig. 7, A–C), and evidenced by the increased
number of DAB-positive clusters per leaf area (Fig.
7D). Cell death was visualized with trypan blue (TB)
stain. Similar to the DAB stain, the positively stained
clusters were counted in the 48-h time range following
infection (Fig. 7H). Like H2O2production, the number
of dead cell clusters was enhanced in leaves pretreated
with AHL as indicated by the increased numbers of
blue-stained clusters in those leaves (Fig. 7, I–N). In
conclusion, this result supports the notion that the
AHL affects defense mechanisms on different levels.
Moreover, the systemic action (Figs. 2 and 3) might be
accompanied with local effects (Fig. 7).
DISCUSSION
In this report we show that AHL modulates plant-
pathogen interactions. Until now AHLs were iden-
Figure 4. Oxo-C14-HSL does not induce resis-
tance against B. cinerea or P. cucumerina BMM.
Five-week-old Arabidopsis plants were untreated
(A and E), pretreated with acetone (B and F), or
6mMoxo-C14-HSL (C and G) for 3 d. Conidial
suspensions were dropped onto detached leaves.
Necrotic symptoms or lesion diameters were
analyzed at 5 dpi. Classification of symptoms: I,
healthy leaf; II, leaf necrotic at 25%; III, 50% of
leaf surface show necrosis; IV, 75% necrotic
surface; V, 100% of the leaf surface is necrotic.
D, Necrotic symptoms caused by B. cinerea.H,
Lesions caused by P. cucumerina BMM.
Schikora et al.
1412 Plant Physiol. Vol. 157, 2011

tified in over 70 species of Gram-negative bacteria.
Among the numerous QS systems, the AHL-based
mechanism is best understood (Whitehead et al., 2001;
Zhang and Dong, 2004; Waters and Bassler, 2005; Dong
et al., 2007). Several studies show that also eukaryotes
(plants as well as animals) respond to the presence of
AHLs (Mathesius et al., 2003; Jahoor et al., 2008;
Kravchenko et al., 2008; Ortı
´z-Castro et al., 2008; von
Rad et al., 2008). Nevertheless, a direct evidence for
their involvement in plant defense reactions has been
missing. In this report we show that perception of oxo-
C14-HSL significantly increases the resistance toward
hemibiotrophic bacteria and biotrophic fungi, while it
appears rather ineffective to microbes with a necrotro-
phic life style. Furthermore, our data suggest that
activation of MAPKs, especially AtMPK6, is crucial for
AHL-induced resistance.
Plants React to Bacterial QS Molecules
AHLs trigger diverse plant reactions: C6-HSL pro-
motes growth (von Rad et al., 2008), while C10-HSL
and C12-HSL modulate root development and root
hair formation, respectively (Ortı
´z-Castro et al., 2008).
Here we show that the long-chained AHL (oxo-C14-
HSL) increased Arabidopsis resistance toward Pst and
powdery mildew fungi in Arabidopsis and barley
(Figs. 2 and 3). Diverse bacteria such as Acidithiobacil-
lus ferrooxidans,Burkholderia pseudomallei,Rhodobacter
spheroides,Sinorhizobium meliloti, and Yersinia enteroco-
litica produce C14-HSL derivatives (Williams, 2007). In
biofilms of the opportunistic plant and human path-
ogen P. aeruginosa, oxo-C14-HSL can reach concentra-
tion of 40 mM(Charlton et al., 2000). Interestingly, the
length of the fatty acid chain is not the only determi-
nant for induced resistance. Modifications at the g
positions in the fatty acid chain are also critical.
Substitution by a OH or O group at this position has
impact on the plant response; in our tests the oxo
group appeared to be more efficient in resistance
induction (Fig. 2B). The strongest effect on resistance
was observed in plants pretreated with C14 or C12
derivatives of AHLs (Figs. 2 and 3; Supplemental Fig.
S7A). In contrast, similarly to the previous report by
Figure 5. Response to flg22 in plants pretreated
with AHL. A, Oxidative burst in response to 100
nMflg22. Production of H2O2was measured
using the luminol-based assay in detached leaf
discs from soil-grown plants. Leaves were un-
treated (control), or pretreated with acetone or
oxo-C14-HSL for 3 d before treatment with flg22.
Arrow shows the time of flg22 addition. B, Time
course of phosphorylation status of AtMPK6 and
AtMPK3. Whole 2-week-old Arabidopsis Col-0
seedlings were pretreated with 6 mMoxo-C14-
HSL for 3 d. Responses were analyzed in seed-
lings during minutes after treatment with 100 nM
flg22. Top section shows an immunoblot with
apERK1/2 antibody, the bottom section shows
Coomassie stain of the membrane. Sections be-
low show immunoblots with the specific aMAPKs
antibodies. C to F, Transcriptional regulation of
WRKY and PR1 genes after pretreatment with
oxo-C14-HSL. Total mRNA was extracted from
2-week-old Arabidopsis Col-0 seedlings, pretrea-
ted for 3 d with 6 mMAHL plants, and subsequent
treatment with 100 nMflg22 at hour as indicated.
qRT-PCR analysis was performed using WRKY-
and PR1-specific primers (Supplemental Table
S1).
Quorum Sensing Molecule Induces Plant Immunity
Plant Physiol. Vol. 157, 2011 1413

von Rad et al. (2008), the growth-promoting effect was
present only in plants pretreated with the short-
chained C6-HSL (Fig. 1; Supplemental Fig. S7B).
Homo-Ser Lactones Induce Systemic Reaction
The transport of AHLs within plants has been
addressed in several previous reports. Go
¨tz et al.
(2007) demonstrated move of both C6-HSL and C8-
HSL from roots into barley shoots, whereas C10-HSL
was not transported. Similarly, C6-HSL but not the
C10-HSL was translocated from roots to shoots in
Arabidopsis plants (von Rad et al., 2008). Using dif-
ferent marker bacteria with sensitivity as low as 60 nM
AHL, we confirmed that C6-HSL is systemically trans-
ported in Arabidopsis plants from root to shoot. How-
ever, we were unable to detect oxo-C14-HSL in shoots
after root treatment (Fig. 1; Supplemental Fig. S1B),
showing that increased chain length reduces the mo-
tility of AHLs within the plant. Accordingly, when
roots were treated with long-chained AHL, the result-
ing change of the resistance status in leaves toward
leaf pathogens is consistent with an inducible, sys-
temic disease resistance.
Active AtMPK6 Is Required for AHL-Induced Resistance
Induction of different MAPK cascades is one of the
first steps in pathogen perception. The involvement of
AtMPK6 and upstream compounds of the MAPK
cascade in prompt response to pathogen attack is
well documented (Asai et al., 2002). AtMPK6 together
with its closest homolog AtMPK3, plays an important
role in signaling cascades downstream of several pat-
tern recognition receptors being crucial in induction of
defense mechanisms, reviewed by Pitzschke et al.
Figure 6. AHL fails to modulate the response to
flg22 in MAPK mutants. A, Time course of phos-
phorylation status of AtMPK3 and AtMPK6 in Col-
0, mpk3, and mpk6. Whole 2-week-old mpk3,
mpk6, or Col-0 seedlings were pretreated with 6
mMoxo-C14-HSL for 3 d. Responses were ana-
lyzed during minutes after treatment with 100 nM
flg22. Top sections show an immunoblot with
apERK1/2 antibody, bottom sections show Coo-
massie stain of the membrane. B, Pst proliferation
on mpk3 and mpk6 plants. Plants were pretreated
as indicated for 3 d and spray inoculated with
bacteria. Cfu numbers were analyzed after 48 h.
Col-0 plants were used as a control. C to F,
Transcriptional regulation of WRKY transcription
factors after pretreatment with oxo-C14-HSL. To-
tal mRNA was extracted from 2-week-old Arabi-
dopsis mpk3 or mpk6 seedlings, pretreated for 3 d
with 6 mMAHL plants, and subsequent treatment
with 100 nMflg22 at hour as indicated. qRT-PCR
analysis was performed using WRKY-specific
primers (Supplemental Table S1). The fold induc-
tion was normalized to 0 h after flg22 treatment,
note that mpk3 mutant has approximately 4 and
144 times lower basal level of WRKY22 and
WRKY29 than Col-0 plants, respectively. The
basal level of WRKY22 expression in mpk6 plants
is slightly higher; the WRKY29 is 3 times lower
than in Col-0 plants.
Schikora et al.
1414 Plant Physiol. Vol. 157, 2011

(2009). Nonetheless, little is known about the role of
MAPK cascades in systemic resistance mechanisms
such as systemic acquired resistance (SAR) or induced
systemic resistance (Vlot et al., 2008; Shah, 2009).
Activation of the MAP kinase kinase 7 (MKK7) is
required for SAR against P. syringae pv maculicola
ES4326 and Hyaloperonospora parasitica Noco2 (syn.
Hyaloperonospora arabidopsidis) in Arabidopsis (Zhang
et al., 2007). The authors demonstrated that ectopic
expression of MKK7 is sufficient to generate a SAR-
inducing signal. By showing that the edr1 mutant
constitutively expresses SAR, the EDR1, a CRT1-like
MAPK kinase kinase, was suggested to be a negative
regulator of SAR (Frye and Innes, 1998; Frye et al.,
2001). Here we show that already local pretreatment of
Arabidopsis with AHL modulates the inductions of
AtMPK6 and AtMPK3. Moreover, the AHL-induced
systemic resistance to Pst is compromised in mpk6 but
not in the mpk3, supporting the assumption that the
AtMPK6 is predominantly required for resistance re-
inforcement caused by bacterial QS molecules. This is
further supported by the transcriptional induction of
PR1 gene in oxo-C14-HSL-pretreated Col-0 and mpk3,
but not in mpk6 mutant, after subsequent flg22 induc-
tion (Supplemental Fig. S8).
Interestingly, in direct response to oxo-C14-HSL,
Arabidopsis plants did not accumulate ROS, nor was
SAR-associated PR1 gene induced (Supplemental Figs.
S4 and S6). Nevertheless, if oxo-C14-HSL-pretreated
plants are challenged with biotrophic or hemibiotrophic
pathogens they show higher resistance if compared to
control plants. This phenomenon is comparable to the
priming response induced in many plants by chemical
resistance inducers such as benzo-(1,2,3)-thiadiazole-7-
carbothioic acid S-methyl ester (BTH; Conrath et al.,
2006). Recently it was suggested that AtMPK3 is the
molecular base of priming responses (Beckers et al.,
2009). The authors showed that AtMPK3 initially accu-
mulates in an inactive form in the response to BTH, and
that, upon inoculation with Pst DC3000, the kinase is
activated and shows a prolonged activity. Results
presented in this report are very similar to those
Figure 7. Oxo-C14-HSL promotes ac-
cumulation of ROS and induces HR
rate after challenge with Pst in Arabi-
dopsis plant. Arabidopsis plants were
grown on soil and detached leaves
were pretreated for 3 d with water
(control; A, E, I, L), acetone (B, F, J,
M), or 6 mMoxo-C14-HSL (C, G, K, N).
Pretreated leaves were inoculated with
Pst strain AvrRpt2 (avirulent) by spray-
ing. Leaves were stained with DAB to
visualize the accumulation of H2O2
(A–G), or with TB to visualize the
dead cells (H–N). A to C, DAB stain
of Arabidopsis leaves inoculated with
Pst AvrRpt2 for 48 h. E to G show
magnifications of respective A to C.
Bar = 0.1 cm. D, Representation of
DAB-positive stained clusters in Pst
AvrRpt2-inoculated leaves. Pretreated
leaves were infected with bacteria and
stained with DAB at hpi as indicated.
Number of stained clusters was cal-
culated per cm2, 60 clusters were
analyzed in three independent experi-
ments. H, Numbers of TB-positive
stained clusters in Pst AvrRpt2-inocu-
lated leaves. Pretreated leaves were
inoculated with bacteria and stained
with TB at hpi as indicated. Clusters
presence was calculated per cm2,60
clusters were analyzed in three inde-
pendent experiments. I to K, TB stain of
Arabidopsis leaves inoculated with Pst
AvrRpt2 for 48 h. L to N show respec-
tive magnifications of I to K. [See on-
line article for color version of this
figure.]
Quorum Sensing Molecule Induces Plant Immunity
Plant Physiol. Vol. 157, 2011 1415

presented by Beckers et al. (2009), suggesting that also
AHL may induce priming-like state in Arabidopsis and
barley. However, we did observe some differences. The
lack of enhanced accumulation of transcripts or inactive
forms of either AtMPK3 or AtMPK6 upon AHL treat-
ments or the fact that AtMPK6 seems to play the major
role in AHL-induced resistance, whereas AtMPK3 ap-
pears to be the major player in BTH priming (Beckers
et al., 2009) suggest that AHL-induced resistance may
differ from the BTH-induced priming mechanism.
An interesting possibility to explain the mechanism
of AHL-enforced activation of MAPKs may be the inhi-
bition of phosphatases. In Arabidopsis, several dual-
specificity phosphatases and at least one of protein
phosphatases 2C interact with AtMPK6 or AtMPK3
(Schweighofer et al., 2007; Bartels et al., 2009; Lumbreras
et al., 2010). The Arabidopsis mkp2 mutant exhibits
delayed wilting symptoms after infection with the
biotrophic bacterium Ralstonia solanacearum, albeit stron-
ger disease symptoms were observed after infection
with the necrotrophic B. cinerea (Lumbreras et al., 2010).
CONCLUSION
Together with other reports, the results presented
here provide strong evidence that plants evolved
potent ways to take advantage of the bacterial QS.
The data show that the specificity of the plant’s re-
sponse to AHLs depends on the length of the acyl
moiety and on the functional group at the gposition.
We suggest that long-chained AHLs have the ability to
induce resistance against microbial pathogens. Finally
we demonstrate that the mechanism of systemic resis-
tance induced by oxo-C14-HSL relies on the presence
of AtMPK6, and is most probably different from other
chemical-induced priming effects.
MATERIALS AND METHODS
Plant Growth
For pathogenicity assays Arabidopsis (Arabidopsis thaliana) Col-0, mpk3,
and mpk6 plants were grown in a sterile hydroponics system. Surface-
sterilized seeds (3 min with 50% ethanol/0.5%Tritron X-100 mix and briefly
rinsed with 95% ethanol) were germinated and grown for 5 weeks at 22°C
with 150 mmol m22s21light in 8/16 h day/night photoperiod. Seeds were
directly planted into 96-well plates with one-half MS medium (Murashige and
Skoog, 1962) supplemented with vitamins, 0.7% agar, and 1% Suc. Plates were
placed on 200 mL liquid one-half MS in a sterile box. Medium was exchanged
every second week. AHLs were added directly into the medium.
For transcriptional and biochemical analyses, Arabidopsis seeds were
surface sterilized and germinated on sterile one-half MS with 0.4% gelrite and
1% Suc for 2 weeks. Seedlings were then transferred into six-well plates with
5 mL liquid one-half MS medium, prior to the pretreatment with AHLs.
For oxidative burst, ROS accumulation, and HR rate assays Arabidopsis
plants were grown on soil in short-day condition (8/16 h day/night photo-
period) at 21°C for 4 weeks. Detached leaves were floated on sterile medium
(10 mMphosphate buffer pH 8.0), supplied with AHLs for 3 d, then treated
with flg22 or spray inoculated with Pst bacteria.
Barley (Hordeum vulgare ‘Golden Promise’) seeds were surface sterilized
with 70% ethanol and 6% NaClO and germinated for 2 d in the dark. Seedlings
were transferred for 5 d on plant nutrient medium (Scha
¨fer et al., 2009)
supplied with 0.4% gelrite, and then for 3 additional days to liquid plant
nutrient medium for AHL treatment.
AHL Treatment
AHLs (Sigma-Aldrich) were solved in acetone as 60 mMstock. AHL
pretreatment occurred either directly in the hydroponics system, or AHLs
were added into liquid or solid (0.4% gelrite) one-half MS medium. Plants
were pretreated for 3 d. All experiments were performed with two controls:
untreated plants (control) and solvent control (acetone).
AHL Detection
The detection of AHLs was done using transgenic bacterial strains:
Pseudomonas putida (F117 pKR C12 GFP; Steidle et al., 2001), Serratia liquefaciens
(MG44 pBAH9 GFP; Li, 2010), Escherichia coli (MT102 GFP pJBA89; Andersen
et al., 2001), and the E. coli strain (Top10 pSB403; Winson et al., 1998)
expressing luxR+luxI::luxCDABE from Vibrio fisheri, detecting a range of
AHL from C6-HSL to oxo-C14-HSL (Supplemental Fig. S2). Reporter bacteria
were grown on Luria-Bertani medium with specific ant ibiotics. Leaves (70 mg)
or roots (30 mg) were homogenized in acetone. Ten microliters of cleared
acetone extract was dropped on the bacteria lawn. Fluorescence was observed
2 h after incubation using an ex: 480/40 nm, em: 510 nm filter.
Pathogenicity Tests
Pst and Pst expressing AvrRpt2 were cultured overnightin Kings B medium,
washed in 10 mMMgSO4. Inoculation solution was adjusted to OD600 =0.1.
Bacterial solution with 0.02% Silwet was sprayed uniformly over the plants.
After: 1, 24, 48, and 96 h 100-mg leaves were homogenized in 10 mMMgSO4.
Samples were diluted and plated for colony forming units counting.
Golovinomyces orontii spore suspension (40,000 spores/mL) was sprayed
uniformly onto leaves from plants grown in hydroponics culture and incu-
bated for 5 d. Infected leaves were stained with calcofluor, and mycelia
formation was counted in fluorescence Axioplan2 (Zeiss) microscope using an
ex: 360/40 nm, em: 460/50 nm filter.
Five microliters of spore suspension of Plectosphaerella cucumerina BMM
(for isolate Brigitte Mauch-Mani; 20,000 spores/mL) or Botrytis cinerea (20,000
spores/mL) were dropped directly onto the leaf surface. Conidia of Blumeria
graminis f. sp. hordei race A6 (450 conidia/cm2) were directly sprayed onto
barley leaves.
Oxidative Burst Assay
Oxidative burst was monitored after treatment with 100 nMflg22 using
luminol-based assay. Leaves from soil-grown, 4-week-old plants were floated
for 3 d on AHL-supplied medium and used to cut out segments, which were
incubated overnight in sterile water in 96-well plates. Water was then replaced
with 180 mL of luminol working solution (1 mL luminol stock, 50 mL
peroxidase, 50 mL distillated water), and 5 mLof200m
Mphosphate buffer
pH 8.0. Luminol stock: 1.77 mg luminol (Sigma-Aldich) dissolved in 1 mL 10
mMNaOH and 9 mL water. The background was measured for 10 min, and
then 20 mL of 0.25 mMflg22 were added into each well and measured for
additional 60 min.
Immunodetection of Phosphorylated MAPKs
Arabidopsis seedlings pretreated with AHLs (Col-0, mpk3,andmpk6) were
treated as indicated with 100 nMflg22. Total proteins were extracted and
separated on 12% SDS-polyacrylamide gel. Western-blot analyses were done
using aMPK3, aMPK6, aMPK4 (Sigma-Aldrich), and the apERK1/2 (Cell
Signaling) antibodies.
Transcriptional Analysis
Seedlings were treated with 100 nMflg22 and harvested as indicated. Fifty
to one hundred milligrams of plant material was homogenized and RNA
extracted using the Trizol system. Two micrograms of total RNA was used for
DNAse digestion. cDNA synthesis was done according to the qScript cDNA
synthesis kit from Quanta BioScience Inc. RT efficiency was verified with
semiquantitative amplification of the actin 2 transcript. qRT-PCR was done
Schikora et al.
1416 Plant Physiol. Vol. 157, 2011

using primers listed in Supplemental Table S1. All expression values were
normalized to expression of UBQ4 (At5g25760). The fold induction was
normalized to 0 h after flg22 treatment, note that mpk3 mutant has approx-
imately 4 and 144 times lower basal level of WRKY22 and WRKY29 then Col-0
plants, respectively. The basal level of WRKY22 expression in mpk6 plants is
slightly higher; the WRKY29 is 3 times lower then in Col-0 plants.
TB and DAB Staining
Leaves of 4-week-old plants were floated for 3 d on AHL-supplied
medium and afterward spray inoculated with Pst AvrRpt2. Samples were
harvested after 0, 24, and 48 hpi. Leaves were either: (1) incubated in TB
solution for 1 min at 60°C followed by 30 min at 22°C and destained in 25 mM
chloral hydrate; or (2) incubated in DAB solution (1 mg/mL 3,3#-DAB in
water) overnight at 22°C and destained in ethanol/chloroform/trichloroacetic
acid (4:1:0.15) for 24 h.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Detection of AHLs using reporter bacteria.
Supplemental Figure S2. AHL has no impact on bacterial virulence.
Supplemental Figure S3. Different concentrations of oxo-C14-HSL have
no effect on the resistance against B. cinerea.
Supplemental Figure S4. Oxo-C14-HSL has no effect on the accumulation
of ROS or the spontaneous cell death.
Supplemental Figure S5. Transcriptional regulation of the MAP kinases
AtMPK6 and AtMPK3 after pretreatment with oxo-C14-HSL.
Supplemental Figure S6. Pretreatment with oxo-C14-HSL is not sufficient
to induce the transcription of WRKY or PR1 genes.
Supplemental Figure S7. Long-chained AHLs impact on plants resistance
and development.
Supplemental Figure S8. Transcriptional analysis of PR1 gene in mpk3/6
mutants.
Supplemental Table S1. List of primers used in quantitative RT-PCR.
ACKNOWLEDGMENTS
We thank Prof. Anton Hartmann (Hemholtz Zentrum Mu
¨nchen, Germany)
for the kind gift of reporter bacteria used in the study, Prof. Brigitte Mauch-
Mani (University of Neuchatel, Switzerland) for the kind gift of P. cucumrina
BMM, and Christina Neumann (Justus Liebig University Giessen, Germany) for
help with expression analysis.
Received May 27, 2011; accepted September 20, 2011; published September 22,
2011.
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