Botrytis u winorosli

Bacterial rhamnolipids are novel MAMPs conferring

resistance to Botrytis cinerea in grapevine

ANNE-LISE VARNIER

*, LISA SANCHEZ

*, PARUL VATSA

, LESLIE BOUDESOCQUE

ANGELA GARCIA-BRUGGER

, FANJA RABENOELINA

, ALEXANDER SOROKIN

, JEAN-HUGUES RENAULT

SERGE KAUFFMANN

, ALAIN PUGIN

, CHRISTOPHE CLEMENT

, FABIENNE BAILLIEUL

& STEPHAN DOREY

URVVC-EA 2069, Stress, Défense et Reproduction des Plantes,

UMR CNRS 6229, IFR 53, Institut de Chimie Moléculaire

de Reims, Université de Reims Champagne-Ardenne, BP 1039, F-51687 Reims cedex 2, France,

UMR Plante-Microbe-

Environnement, INRA 1088, CNRS 5184, Université de Bourgogne, 17 rue Sully, BP 86510, 21065 Dijon cedex, France,

Institut de Biologie Moléculaire des Plantes du C.N.R.S., Université Louis Pasteur, 12 rue du Général Zimmer, 67084

Strasbourg, France and

Algentech Ltd, Institute of Food Research IFR2, Norwich Research Park, Colney Lane, Norwich

NR4 7UA, UK

ABSTRACT

Rhamnolipids produced by the bacteria Pseudomonas
aeruginosa are known as very efficient biosurfactant mol-
ecules. They are used for a wide range of industrial applica-
tions, especially in food, cosmetics and pharmaceutical
formulations as well as in bioremediation of pollutants. In
this paper, the role of rhamnolipids as novel molecules trig-
gering defence responses and protection against the fungus
Botrytis cinerea in grapevine is presented. The effect of
rhamnolipids was assessed in grapevine using cell suspen-
sion cultures and vitro-plantlets. Ca

influx, mitogen-

activated protein kinase activation and reactive oxygen
species production form part of early signalling events
leading from perception of rhamnolipids to the induction of
plant defences that include expression of a wide range of
defence genes and a hypersensitive response (HR)-like
response. In addition, rhamnolipids potentiated defence
responses induced by the chitosan elicitor and by the culture
filtrate of B. cinerea. We also demonstrated that rhamno-
lipids have direct antifungal properties by inhibiting spore
germination and mycelium growth of B. cinerea. Ultimately,
rhamnolipids efficiently protected grapevine against the
fungus. We propose that rhamnolipids are acting as microbe-
associated molecular patterns (MAMPs) in grapevine and
that the combination of rhamnolipid effects could partici-
pate in grapevine protection against grey mould disease.

Key-words: Vitis vinifera L. (grapevine); MAMPs; plant
defence; potentiation.

INTRODUCTION

In the course of their life, plants have to face a broad range
of micro-organisms which are potentially pathogens. To

counter these attacks, they have evolved a large set of
defence responses. These defences include pre-existing
physical and chemical barriers, as well as inducible
responses that are activated after pathogen perception
(Hammond-Kosack & Jones 1996). This recognition step
can be achieved by the means of molecules common to
many classes of microbes known as microbe-associated
molecular patterns (MAMPs) (Mackey & McFall 2006).
MAMPs, also known as general elicitors in plants, are
involved in non-specific immunity and the associated resis-
tance is effective against a broad range of pathogens (Jones
& Dangl 2006; Bent & Mackey 2007). MAMPs belong to
different families including proteins, glycans and lipids.
Some data have recently enlightened our knowledge
about MAMPs from perception to downstream defence
responses. Among proteins, elicitins secreted by the
oomycete Phytophthora sp. have been extensively studied
and remain one of the best example of MAMPs for which
early events cascade has been fully characterized (Baillieul,
de Ruffray & Kauffmann 2003; Garcia-Brugger et al. 2006).
Calcium influx, production of reactive oxygen species
(ROS) and mitogen-activated protein kinase (MAPK) acti-
vation are among the first responses occurring after elicitins
perception and play a key role in plant defence signal trans-
duction in tobacco (Garcia-Brugger et al. 2006). Some
MAMPs belong to oligosaccharides like chitin, which is
known to induce various defence responses in a wide range
of plant cells including both monocots and dicots (Shibuya
& Minami 2001; Kaku et al. 2006). Lipids or lipid derivatives
have also been described as potent MAMP elicitors. Cholic
acid, a bile acid, has recently been characterized as a general
elicitor inducing cell death, pathogenesis-related (PR)
protein synthesis and phytoalexin accumulation in rice
(Koga et al. 2006; Shimizu et al. 2008). Ergosterol elicits
extracellular alkalinization and activates several MAPKs in
alfalfa and tomato cells (Granado, Felix & Boller 1995;
Cardinale et al. 2000). Cerebrosides, compounds catego-
rized as glycosphingolipids, also lead to MAPK activation
prior to accumulation of phytoalexins and PR proteins in

Correspondence:

Dorey.

Fax:

+33 3 26 91 34 27;

e-mail:

stephan.dorey@univ-reims.fr

*These authors contributed equally to this work.

Plant, Cell and Environment (2009) 32, 178–193

doi: 10.1111/j.1365-3040.2008.01911.x

178

cell suspension culture of rice (Umemura et al. 2002). The
most described lipid MAMPs to date are lipopolysaccha-
rides (LPS). LPS are potent inducers of PR proteins in
Arabidopsis (Newman et al. 2002). This LPS-mediated acti-
vation of defence responses is coupled to nitric oxide syn-
thesis (Zeidler et al. 2004). In rice, LPS induce defence
responses associated with programmed cell death (PCD)
(Desaki et al. 2006). The lipid portion of LPS is necessary
and sufficient for its biological activity (Newman, Daniels &
Dow 1997; Zeidler et al. 2004; Silipo et al. 2008).

Among glycolipids, rhamnolipids have been widely

studied regarding their biological properties. Rhamnolipids
produced by Pseudomonas aeruginosa are amphiphilic
molecules typically composed of 3-hydroxyfatty acids linked
through a beta-glycosidic bond to mono- or di-rhamnoses
(Soberon-Chavez, Lépine & Déziel 2005). Rhamnolipids
are potent biosurfactants with applications related to envi-
ronmental concerns, such as bioremediation of hydrocar-
bon, organic pollutants and heavy-metal-contaminated sites
(Nitschke, Costa & Contiero 2005a; Nitschke et al. 2005b).
In addition, rhamnolipids have several potential industrial
applications including the production of fine chemicals,
characterization of surfaces and surface coatings, as well as
additives for food and cosmetics (Maier & Soberon-Chavez
2000). Rhamnolipids also have the ability to control some
zoosporic plant pathogens like oomycetes by inhibiting
spore germination (Stanghellini & Miller 1997; Perneel
et al. 2008). It has been previously suggested that rhamno-
lipids could inhibit the fungal growth of Botrytis cinerea
(Kim, Lee & Hwang 2000; Haba et al. 2003), but no consis-
tent data have been published so far.

To date, phytochemicals are largely used to reduce the

impact of fungi and oomycetes in vineyards. However, these
phytochemicals have a deleterious ecological impact and
some strains are becoming resistant (Coutos-Thevenot et al.
2001). An alternative strategy to replace pesticides could
consist of stimulation of plant defence by the mean of elici-
tors (Aziz et al. 2003, 2007; Aziz, Heyraud & Lambert 2004;
Vandelle et al. 2006). Unfortunately, most of the elicitors
described to date do not penetrate efficiently and directly
into the plant and they need to be formulated to be active in
the field. Because rhamnolipids are used as biosurfactants,
wetter, emulsifier and adjuvant for herbicidal and pesticidal
systems in agriculture, they have the potential to be directly
effective in the field. In this paper, we investigated the
role of rhamnolipids in plant defence and resistance to
the necrotrophic fungus B. cinerea in grapevine. Here, we
described for the first time the elicitor properties of rham-
nolipids and their ability to potentiate defence responses
induced by a second stimulus. We also demonstrated that
rhamnolipids protect grapevine plants against B. cinerea.

MATERIALS AND METHODS

Biological materials

Cell suspensions of the 41B rootstock (Vitis vinifera L. cv.
Chasselas x V. berlandieri) were cultured in a Murashige–

Skoog medium (Murashige & Skoog, 1962) containing vita-
mins (¥1.5), sucrose (30 g L

-1

), 2,4-D (0.2 mg L

-1

) and

cytokinin 6-benzylaminopurine (BAP) (0.5 mg L

-1

), equili-

brated to pH 5.8 and propagated in the dark at 25 °C under
shaking at 120 rpm in 500 mL flasks. They were subcultured
every 7 days to be maintained in exponential phase. For
inoculation experiments, 10 mL of cells subcultured for 6
days were transferred into 50 mL flasks. Before any treat-
ment, cells were allowed to adjust to the new condition
overnight. For some of the early events experiments, we also
used V. vinifera L. cv. Gamay cell suspensions as described by
Poinssot et al. (2003). Transformed V. vinifera L. cv. Gamay
cells expressing aequorin were used to generate cell suspen-
sions (Vandelle et al. 2006). Transformed cell suspensions
were subcultured every 8 days by transferring 30 mL of
aequorin-transformed cells to 70 mL of fresh liquid Nitsch–
Nitsch medium (Nitsch & Nitsch 1969) and were maintained
in suspension by continuous shaking at 150 rpm, at 25 °C and
under continuous light. Grapevine vitro-plantlets (V. vin-
ifera L. cv. Chardonnay 7535) were grown in a modified
Murashige–Skoog medium at 26 °C with a photoperiod of
16 h of light according to Bézier, Lambert & Baillieul (2002).
B. cinerea strain T4 (kind gift of C. Levis, INRA, Versailles,
France) was grown on solid tomato/agar medium [tomato
juice 25% (v/v), agar 2.5% (p/v)].

Plant treatments

Rhamnolipids

from

aeruginosa

were

purchased

from Jeneil Biosurfactant Company (Jeneil Biosurfac-
tant Co., LLC, Saukville, WI, USA, product data
sheet JBR 599, purity

>99%) and consist of a mix

two

rhamnolipid

species:

a-L-rhamnopyranosyl-

b-hydroxydecanoyl-b-hydroxydecanoate (RL-1,2

: 40%)

and 2 - O - a - L - rhamnopyranosyl - a - L - rhamnopyranosyl -

b -hydroxydecanoyl-b-hydroxydecanoate (RL-2,2

: 60%).

2-O-a-L-rhamnopyranosyl-a-L-rhamnopyranosyl-a(R)-3-
hydroxytetradecanoyl-(R)-3-hydroxytetradecanoate (RL-
2,2

) from Burkholderia plantarii (Andra et al. 2006) was

kindly supplied by Prof Klaus Brandenburg (Leibniz-
Zentrum für Medizin und Biowissenschaften, Borstel,
Germany). L-rhamnose was purchase from Sigma-Aldrich
(Saint Quentin Fallavier, France).

Water-diluted solutions of rhamnolipids were added

directly to the grapevine cell suspension media at the
indicated concentrations. Grapevine vitro-plantlets were
immersed for 1 min in the rhamnolipid solutions (0.1 or
1 mg mL

-1

) or water and then placed in growth chambers.

Leaves of vitro-plantlets were harvested at the times men-
tioned. At least five vitro-plantlets per condition were used
and results presented are a mean of three independent
experiments

⫾ SD. For potentiation experiments, a stock

solution of chitosan was prepared according to Ait Barka
et al. (2004). Culture filtrate (CF) of B. cinerea was prepared
according to Manteau et al. (2003). Diphenylene iodonium
(DPI) (Sigma) was dissolved in dimethyl sulphoxide
(DMSO) as a 10 mm stock solution. A total of 10 mm DPI
was applied to the cells.

Rhamnolipids as new MAMPs in plants

179

Isolation of mono- and di-rhamnolipids

Mono- and di-rhamnolipids were isolated from the com-
mercial mixture (Jeneil) by centrifugal partition chroma-
tography (CPC).

CPC apparatus and experimental conditions

The separations were performed on fast centrifugal parti-
tion chromatography (FCPC) Kromaton Technologies
apparatus (Angers, France) using a rotor made of 20 circu-
lar partition disks (1320 partition cells: 0.130 mL per cell;
total column capacity: 205 mL, dead volume: 32.3 mL).
Rotation speed could be adjusted from 200 to 2000 rpm,
producing a centrifugal force field in the partition cell of
ª120 g at 1000 rpm and 480 g at 2000 rpm. The solvents
were pumped by a Dionex P580HPG 4-way binary high-
pressure gradient pump (Sunnyvale, CA, USA). The
column was first filled with the aqueous stationary phase.
The sample (484 mg) was introduced into the column
through a low-pressure injection valve (Upchurch, CIL
Cluzeau, Sainte-Foy-La-Grande, France) equipped with a
20 mL sample loop. The organic mobile phase was then
pumped in the ascending mode. The flow rate was
8 mL min

-1

and the rotation speed was 1300 rpm, resulting

in 34 bar back pressure and 63% stationary-phase reten-
tion. Effluent detection was controlled by a Dionex UVD
170S detector at l = 200 nm equipped with a preparative
flow cell (6 mL internal volume, path length of 2 mm). Frac-
tions (4 mL) were collected by a Pharmacia Superfrac col-
lector (Uppsala, Sweden). The experiments were conducted
at room temperature (22

⫾ 1 °C).

Preparation of the biphasic solvent system for
CPC runs

Biphasic system (2 L) was prepared by mixing heptane,
ethylacetate, methanol (purchased as chromatographic
grade solvents from Carlo Erba, Rodano, Italy) and water
(purified by deionization and reverse osmosis) in the desi-
gnated proportions in a separatory funnel (1:1:1:1, v/v).
They were vigorously shaken and then allowed to settle
until the phases became limpid.

Fraction analyses

All CPC fractions were checked by thin-layer chromatog-
raphy (TLC) on Merck 60 F254 silica gel plates (Rahway,
NJ, USA) developed with chloroform/methanol/water
(70:30:5 v/v). The structure of the isolated compounds was
confirmed by nuclear magnetic resonance (NMR) and mass
spectroscopy. 1H, 13C, correlation spectroscopy (COSY),
heteronuclear single quantum correlation (HSQC) and
heteronuclear multiple bond correlation (HMBC) NMR
experiments were performed in CD

OD. They were

recorded on a Bruker (Wissembourg, France) Avance DRX
500 spectrometer (1H at 500 MHz and 13 °C at 125 MHz).
Electrospray ionization (ESI) mass spectra were obtained

on a Micromass (Guyancourt, France) Q-TOF (quadrupole
time-of-flight) micro-spectrometer. The chemical structures
of RL-1,2

and RL-2,2

are well established according to

NMR and ESI mass spectra data (Sharma et al. 2007). High
signal-to-noise ratio of our spectra unambiguously indi-
cated that the isolated rhamnolipids were in a pure state. At
the end of the procedure, the 484 mg of the commercial
extract yielded to 101 mg of mono-rhamnolipid and 88 mg
of di-rhamnolipid.

Free cytosolic calcium variation analysis, H

production measurement and MAPK assay

Bioluminescence measurements of calcium influx were
made using a digital luminometer (Lumat LB9507; Ber-
thold, Bad Wildbad, Germany). Cell culture aliquots
(250 mL) were transferred to a luminometer glass tube,
and the luminescence counts were recorded continuously at
1 s intervals [recorded as relative light units (RLU) per
second] and exported using Win term software (Berthold).
At the end of the experiment, residual functional aequorin
was quantified by adding 300 mL of lysis buffer [10 mm
CaCl

; 2% Nonidet P40 (v/v); 10% ethanol (v/v)] and the

resulting increase in luminescence was monitored. The
luminescence data were transformed into Ca

concentra-

tions as described by Lecourieux et al. (2002). H

produc-

tion was analysed according to Dorey et al. (1999). Briefly,
H

accumulation in the culture medium was measured

as the chemiluminescence of luminol. Luminescence,
expressed in RLU, is proportional to H

(linearity range

1 mm to 1 mm). In our plant system, 10 000 RLU correspond
to 25 mm of H

. In-gel kinase assay to monitor MAPK

activation was performed as described by Poinssot et al.
(2003).

Cell death assay

Cell death assay using Evans blue was monitored as
described by Dorey et al. (1999). For each sample, a 500 mL
aliquot of cells was incubated with 0.05% Evans blue for
20 min and then washed extensively. The dye bound to dead
cells was solubilized in 50% methanol with 1% sodium
dodecyl sulphate (SDS) for 30 min at 50 °C and quantified
by A600. Fluoresceine diacetate (FDA) staining was per-
formed according to Burbridge et al. (2007).

Protection assays

Conidia of B. cinerea were collected from 10-day-old
culture plates with 2 mL of growth culture medium
[KH

1.75 g L

-1

, MgSO

0.75 g L

-1

, glucose 4 g L

-1

peptone 4 g L

-1

, Tween 20 0.02% (v/v)], filtered to remove

mycelia and counted. Vitro-plantlets were immersed in
solutions of rhamnolipids (0.1 mg mL

-1

) or water for

control for 1 min and were placed in growth chambers.
Two days later, leaves were excised from grapevine
vitro-plantlets and placed on wet Whatman 3 MM paper in
plastic Petri dishes. A drop of 5 mL of a 1 ¥ 10

conidia mL

-1

180

A.-L. Varnier et al.

solution was inoculated on the lower face of the leaves.
Symptoms were observed from 24 to 96 h after inoculation.

B. cinerea growth and conidia inhibition assays

Mycelia from B. cinerea were excised from a plate with solid
tomato/agar medium and transferred to the centre of new
Petri dishes (diameter 9 cm) containing rhamnolipids at
concentrations: 0 (control); 0.1; 1 mg mL

-1

. Radial growth of

the fungus was measured 24, 48 and 72 h after fungus depo-
sition. Values corresponded to the average of measure-
ments from three independent plates for each condition.
For germination assays, conidia were cultured in 96-well
microplates (5000 conidia per well) in growth culture
medium without shaking at 28 °C in the dark. Rhamnolipids
were added to the conidia solution 16 h later. Germ tube
growth was observed using inverted light microscopy
(Leica, Wetzlar, Germany) 5, 8 and 24 h after rhamnolipid
addition.

RNA extraction and real-time
quantitative RT-PCR

For each sample, 100 mg of leaves were ground in liquid
nitrogen. Total RNA was isolated using Plant RNA purifi-
cation reagent (Invitrogen, Carlsbad, CA, USA) and 150 ng
were used for reverse-transcription using the ABsolute™
MAX 2-Step QRT-PCR SYBR® Green Kit (Thermo Elec-
tron, courteboeuf, France) according to the manufacturer.
RT-PCR was performed using an ABI Prism 5700 sequence
detection system (Applied Biosystems, Foster City, CA,
USA) according to (Aziz et al. 2007) with one modification:
the internal control corresponded to the gene encoding the
elongation factor 1 alpha (EF1-a: forward and reverse
primer, 5

′-GAACTGGGTGCTTGATAGGC-3′ and 5′-AA

CCAAAATATCCGGAGTAAAAGA-3

′, respectively).

RESULTS

Rhamnolipids induce free cytosolic calcium
variation, an oxidative burst, MAPKs activation
and cell death in grapevine cell suspensions

Given the role of free Ca

as a key second messenger, we

have first investigated variations of free cytosolic Ca

con-

centration {[Ca

]

cyt

}, using grapevine cells transformed with

the gene encoding aequorin (Vandelle et al. 2006). Dose–
response experiment was conducted on grapevine cells
using concentrations of rhamnolipids ranging from 0.005
to 0.05 mg mL

-1

. No calcium influx was detected at

0.005 mg mL

-1

(Fig. 1a). When cells were treated with

rhamnolipids at 0.01 mg mL

-1

, a biphasic elevation of

[Ca

]

cyt

was observed with a first peak at 2 min followed by

a second peak at 5 min (Fig. 1a). A similar profile was
observed after treatment with 0.025 mg mL

-1

of rhamno-

lipids except that the first peak was more pronounced,
maximum concentration of [Ca

]

cyt

culminating at 4 mm. A

rapid and transient burst of [Ca

]

cyt

was monitored 2 min

after treatment with 0.05 mg mL

-1

. At this concentration,

[Ca

]

cyt

reached 6 mm.

Elicitors like laminarin, oligogalacturonides or endopo-

lygalacturonase 1 (BcPG1) from B. cinerea have been
shown to activate at least two MAPKs in grapevine cell

(a)

(b)

kDa

0.025

–1

0.05

–1

Control

0.01

–1

(c)

Time after treament (min)

[Ca

]

cyt

(

)

0.01

–1

0.025

–1

0.05

–1

0.005

–1

(min)

20000

40000

60000

80000

Time after treatment (min)

RLU

Figure 1.

Early events induced by rhamnolipids in grapevine

cell suspensions. (a) [Ca

]

cyt

elevation was monitored in the

grapevine cells expressing aequorin in the cytosol and treated
with different concentrations of rhamnolipids. For each value,
data obtained in control cells were subtracted. Values are from
one representative experiment out of three. (b) Time-course
activation of two mitogen-activated protein kinases indicated by
arrows with the corresponding molecular weight in untreated
(control) or treated grapevine cells with different concentrations
of rhamnolipids. In-gel kinase assay was performed using myelin
basic protein as substrate. Results are from one representative
experiment out of three. (c) Kinetics of production of active
oxygen species by grapevine cells treated with water control
(closed square), 0.005 mg mL

-1

(open circles), 0.025 mg mL

-1

(closed circles) and 0.05 mg mL

-1

(closed triangles) of

rhamnolipids. H

production was determined using

chemiluminescence of luminol. Chemiluminescence, measured
within a 10 s period with a luminometer, was integrated and
expressed in relative light units (RLU). Results presented are
means of triplicate experiments

⫾ SD.

Rhamnolipids as new MAMPs in plants

181

suspensions (Aziz et al. 2003, 2004; Poinssot et al. 2003).
In-gel kinase assays using myelin basic protein as a sub-
strate revealed that rhamnolipids also activated the same
protein kinases with apparent molecular mass of 46 and
50 kDa (Fig. 1b). The activation of both MAPKs was
detected as soon as 5 min post-treatment. The activation of
the MAPKs was particularly strong at 30 min after treat-
ment with 0.05 mg mL

-1

of rhamnolipids.

It has been previously demonstrated that Ca

also par-

ticipates in the activation of enzymes like NADPH oxidase
which are involved in ROS production (Garcia-Brugger
et al. 2006; Vandelle et al. 2006). Accumulation of H

, the

most stable ROS in grapevine cells, was monitored using a
luminol-peroxidase based assay (Dorey et al. 1999). No pro-
duction of H

was detected after treatment with rhamno-

lipids at 0.005 mg mL

-1

(Fig. 1c). A slight increase in H

production (three times over the control) was reproducibly
detected after treatment with rhamnolipids at 0.01 mg mL

-1

(data not shown). A strong accumulation of H

was

measured 1 h after treatment with the concentration of
0.025 mg mL

-1

(Fig. 1c). Interestingly, H

accumulation

was also measured at 0.05 mg mL

-1

, but the levels were

noticeably lower compared with the treatment with
0.025 mg mL

-1

. Using DPI, a specific inhibitor of NADPH

oxidases, we completely abolished the oxidative burst
induced by rhamnolipids at 0.025 mg mL

-1

(data not

shown).

Using the Evans blue test, we did not detect any cell

death increase in the cell suspensions treated with concen-
trations of rhamnolipids at or below 0.01 mg mL

-1

com-

pared with the control (Fig. 2a). However, treatment of
grapevine cells with 0.025 mg mL

-1

of rhamnolipids induced

a level of cell death that was around 12% of the total cells.
In these experiments, 100% of cell death was observed
using the concentration 0.05 mg mL

-1

. In order to specify

the nature of the cell death induced by rhamnolipids at the
highest concentrations, we observed grapevine cells using
bright field microscopy and epifluorescence microscopy
after FDA staining of the cells. In agreement with the Evans
blue test, we did not observe any change in cell morphology
with up to 0.01 mg mL

-1

of rhamnolipids (Fig. 2b). Epifluo-

rescence images of the same cells showing FDA staining
indicated that the cells were viable. FDA staining re-
vealed an increase in cell death at the concentration of
0.025 mg mL

-1

and all the observed cells were affected

when treated with 0.05 mg mL

-1

of rhamnolipids. Interest-

ingly, cells that did not show any fluorescence after rham-
nolipids exposure also presented some morphological
changes like retraction of the protoplast from the cell wall,
a feature that is characteristic of cells undergoing PCD.

Rhamnolipids elicit plant defence gene
activation in grapevine

In order to specify the ability of rhamnolipids to induce
defence responses, we monitored the expression of defence
genes after treatment of grapevine cell suspensions or vitro-
plantlets with different concentrations of rhamnolipids.

Chitinase genes are classical markers used to monitor
defence induction in grapevine (Busam, Kassemeyer &
Matern 1997; Bézier et al. 2002; Robert et al. 2002). In the
present experiments, we used the chit4c gene, which is
strongly induced in our plant systems upon elicitation
with laminarin and oligogalacturonides (Aziz et al. 2003,
2004). Using real-time RT-PCR analysis, we did not detect
any change in chit4c gene expression after challenge of
grapevine cell suspensions with the lowest concentration
of rhamnolipids (0.005 mg mL

-1

) compared with the

control (Fig. 3). However, grapevine cells responded to
0.01 mg mL

-1

with a 30-fold increase in chit4c gene expres-

sion (Fig. 3). Rhamnolipids at 0.025 mg mL

-1

strongly

induced chit4c expression corresponding to a 320-fold
increase over the control.

We also monitored the chit4c gene expression level in

grapevine vitro-plantlets treated with rhamnolipids. In
this plant system, no significant expression of the gene was
detected below 0.05 mg mL

-1

over a time course (data not

shown). Moreover, we also observed that concentrations of
1 mg mL

-1

or higher were inducing some cell death that

could be visualized by small necrotic spots (data not
shown). The chit4c gene expression was strongly induced as
soon as 6 h after treatment with 0.1 mg mL

-1

of rhamnolip-

ids (Fig. 4a). A constant decrease in mRNA level was found
after 24 h and persisted until 4 days after treatment. In
order to have a better understanding of the defence
responses induced by rhamnolipids in grapevine plantlets,
we also monitored the expression of six genes covering a
large panel of defence markers (Aziz et al. 2007; Bézier
et al. 2007). In addition to the chit4c gene, two other PR
protein genes, a basic glucanase (gluc) and a protease
inhibitor (pin), were chosen for investigation (Aziz et al.
2007). Expression of stilbene synthase (sts), 9-lipoxygenase
(lox) and phenylalanine ammonia lyase (pal) gene was also
monitored (Bézier et al. 2002; Aziz et al. 2003). STS is a key
enzyme involved in the main grapevine phytoalexin res-
veratrol synthesis (Borie et al. 2004). LOX are implicated in
synthesis of oxylipins, ROS regulation and play an impor-
tant role in response to pathogen attack (Howe & Schilm-
iller 2002). PAL is a key enzyme from the phenylpropanoid
pathway involved in the biosynthesis of lignins and of the
salicylic acid signal molecule (Dixon & Palva 1995). Real-
time RT-PCR analysis was carried out to detect changes
in defence gene expression 24 h after treatment with
non-necrosing (0.1 mg mL

-1

) and cell death inducing

(1 mg mL

-1

) concentrations of rhamnolipids (Fig. 4b).

Expression of all genes was up-regulated following rham-
nolipid treatments. On the whole, the highest concentration
of glycolipids induced the strongest gene expression. PR
genes were more induced compared to other defence genes.
Overall, these results demonstrated that rhamnolipids are
potent elicitors that activate multiple defence responses in
grapevine.

Rhamnolipids that we used in the previous experiments

consist of a mix of mono- and di-rhamnolipids. In order to
investigate the MAMPs nature of each of these compounds,
we isolated the mono- and di-rhamnolipids from this

182

A.-L. Varnier et al.

100

0.005

0.01

0.025

0.05

Rhamnolipid concentration (mg

–1

)

% cell death

(a)

(b)

0.005

0.01

0.025

0.05

Rhamnolipid concentration (mg

–1

)

200

Figure 2.

Cell death assays. Cells were incubated with rhamnolipids at the concentrations indicated. (a) Quantitative measurement of

cell death using Evans blue. Control corresponds to water-treated cells. Cells were harvested 24 h post-treatment and stained with Evans
blue. Results presented are means of triplicate experiments. (b) Identification of live and dead cells using FDA and cell morphology 24 h
after treatment. Cells that are alive have the ability to cleave FDA and fluoresce under light at a wavelength of 490 nm. In cells that
undergo programmed cell death, the protoplast retracts from the cell wall. Left panels: observation made using white light. Right panels:
observation made after FDA staining at 490 nm light. Middle panel: observation made after superposition of white and 490 nm lights.

Rhamnolipids as new MAMPs in plants

183

mixture by CPC and tested their ability to induce plant
defence on grapevine cell suspensions. In addition, we also
tested the potential elicitor ability of L-rhamnose, the sugar
moiety of rhamnolipids, and of di-rhamnolipids from B.
plantarii, which are known as MAMPs perceived by human
mononuclear cells (Andra et al. 2006). Using chit4c, pin and
gluc genes as plant defence markers, we found that both
mono- and di-rhamnolipids from P. aeruginosa used sepa-
rately were active on grapevine cells (Fig. 5). The mixture of
mono- and di-rhamnolipids which was made to recreate the
original solution, with the same ratio, is as expected induc-
ing a 230-fold increase in chit4c expression (compare Figs 3
& 5) and strongly stimulates pin and gluc expression in cell
suspensions (Fig. 5). Interestingly, di-rhamnolipids from B.
plantarii are also stimulating plant defence genes but to a
less extent if compared with di-rhamnolipids from P. aerugi-
nosa. Dose–response experiments showed that significant
induction of the three defence markers was only detected
with 0.05 mg mL

-1

of B. plantarii rhamnolipids, a concentra-

tion which was lethal in the case of P. aeruginosa rhamno-
lipids (Fig. 2a). Finally, L-rhamnose itself is not active as
elicitor on grapevine cells. Because the best inductions of
plant defence genes were observed with the mix of mono-
and di-rhamnolipids from P. aeruginosa, we still used the
commercial solution for the following experiments.

Rhamnolipids potentiate defence responses in
grapevine cells in response to chitosan and
B. cinerea CF

Potentiation is characterized by the fact that following per-
ception of specific molecules plants are able to enhance or

more rapidly mount defence responses after a second chal-
lenge (Conrath, Pieterse & Mauch-Mani 2002; Conrath et al.
2006).We further tested the ability of rhamnolipids to poten-
tiate defence responses induced by a second elicitor in
grapevine. For this, we used chitosan, which has been char-
acterized as an efficient molecule stimulating defence reac-
tions in this plant (Trotel-Aziz, Couderchet & Vernet 2006),
and B. cinerea CF, which contains the BcPG1 elicitor (Poin-
ssot et al. 2003). Potentiation of chitosan and CF responses
by rhamnolipids was assayed in grapevine cell cultures using
the oxidative burst as plant defence marker. Plant cells were
simultaneously treated with rhamnolipids and the elicitor
solutions or pre-incubated for 6 h with rhamnolipids prior to
be challenged with the elicitor solutions (Fig. 6). In order to
highlight a putative effect of priming, we used a concentra-
tion of 0.005 mg mL

-1

of rhamnolipids that did not induce

early signalling responses including Ca

influx, the oxidative

burst nor defence-related gene expression. Figure 6a shows
that chitosan induced an oxidative burst in grapevine cell
suspensions. Production of H

by chitosan was signifi-

cantly potentiated by simultaneous addition of rhamnolip-
ids. The potentiation effect was even amplified when plant
cells were pre-treated 6 h prior to chitosan challenge. Rham-
nolipid potentiation of CF activity was also monitored in the
same conditions. Figure 6b shows that CF induced an oxida-
tive burst in grapevine cell suspensions. Co-treatment of cells
with rhamnolipids and CF solution also displayed a syner-
gistic effect with a threefold increase in H

production

compared with CF treatment alone. Potentiation was also
observed after pre-incubation of rhamnolipids prior to the
addition of CF, but unlike experiments carried out with
chitosan, longer pre-treatment did not result in stronger
potentiation effect over H

production. Similar poten-

tiation profiles were observed with a concentration of
0.01 mg mL

-1

of rhamnolipids that is slightly elicitor. We

also conducted dose–response experiments in order to find
the minimum concentration needed for the potentiation
response. We found that a concentration of 0.0025 mg mL

-1

was necessary and sufficient to enhance H

production

following elicitor treatments (data not shown).

Rhamnolipids inhibit spore germination and
mycelium growth of B. cinerea

Rhamnolipids have been shown to control zoosporic plant
pathogens like oomycetes by inhibiting spore germination
(Stanghellini & Miller 1997). In order to test the antifungal
activity of rhamnolipids on B. cinerea, we observed the for-
mation and the development of the germ tube at 5, 8 and
24 h after incubation of spores with rhamnolipids at the
concentration 0.1 and 1 mg mL

-1

(Fig. 7a). In the control,

most of the spores had germinated at 5 h and hyphae were
fully developed at 24 h. In the presence of rhamnolipids at
0.1 mg mL

-1

, some spores did not germinate at 5 h and the

spores that germinated exhibited small germ tubes com-
pared with control. These differences in hyphae develop-
ment were even more pronounced at 8 h. At 24 h, however,
most of the spores had germinated and the development of

100

150

200

250

300

350

400

0.005

0.01

0.025

Rhamnolipid concentration (mg

–1

)

Relative transcript accumulation of

chit4c

Figure 3.

Chitinase gene expression of cultured grapevine cells

treated with rhamnolipids. Chit4c expression was monitored 9 h
after treatment with rhamnolipids at the concentrations
indicated. Analyses were performed by real-time quantitative
polymerase chain reaction. Level of transcripts was calculated
using the standard curve method from duplicate data, with
grapevine EF-1a gene as internal control and non-treated cells as
reference sample. Results presented are means of triplicate
experiments

⫾ SD.

184

A.-L. Varnier et al.

hyphae was similar to the control. In the presence of rham-
nolipids at 1 mg mL

-1

, most of the spores did not germinate

at 5 h. After 24 h, only few spores presented a germ tube
and exhibited a strong inhibition of hyphae growth com-
pared with the control.

In a second set of experiments, we tested the effect of

rhamnolipids on mycelium growth. Petri dishes with tomato
agar medium supplemented with different concentrations
of rhamnolipids from 0 to 1 mg mL

-1

were inoculated with

B. cinerea. Time course of mycelium growth was measured
over 3 days. No mycelium growth inhibition was observed
below 0.1 mg mL

-1

(data not shown), but as shown in

Fig. 7b, a concentration of 0.1 mg mL

-1

was enough to

inhibit 40% of mycelium growth after 3 days. Inhibition
started as soon as 24 h for all the concentrations tested. One
mg mL

-1

of rhamnolipids inhibited more than 50% of radial

growth over the time course. These results showed that
rhamnolipids have a direct antifungal activity on B. cinerea.

Rhamnolipids protect grapevine plants against
B. cinerea

As rhamnolipids have a direct antifungal activity against B.
cinerea and they induce defence responses in grapevine, we
tested their efficiency to protect grapevine plants against
the fungal infection. Vitro-plantlets were immersed into the
solution of rhamnolipids at 0.1 mg mL

-1

, 48 h prior to infec-

tion with B. cinerea. Symptoms resulting from fungus colo-
nization were monitored for 4 days after inoculation. In
control plants inoculated with B. cinerea without pre-
treatment, the lesions characterized by the browning of the
tissues appeared at 24 hpi (data not shown). All lesions
expanded, resulting in full leaf maceration at 4 days post-
inoculation (Fig. 8b). At this time, most of the leaves pre-
sented the typical infection symptoms. From a total of 34
control leaves from three independent experiments, only
six did not show any symptom (data not shown). On the
contrary, most of the leaves pre-treated with rhamnolipids

exhibited no symptom and the few symptoms were
restricted to a small area of the leaves (Fig. 8a). From a total
of 36 pre-treated leaves from three independent experi-
ments, only seven exhibited some maceration area (data not
shown). These results clearly demonstrate that rhamnolip-
ids are able to protect grapevine plants against B. cinerea
infection.

DISCUSSION

Rhamnolipids produced by bacteria like Pseudomonas
species are glycolipids that have several properties of indus-
trial interest like biosurfactants, antimicrobials and mol-
ecules of bioremediation (Nitschke et al. 2005a). In this
paper, we present for the first time that rhamnolipids are
very effective molecules that elicit and potentiate defence
responses in grapevine and protect the plant against B.
cinerea. We first demonstrated that rhamnolipids at low
concentrations act like typical elicitor of plant defence.
Early events including Ca

influx, ROS production and

MAPK activation were detected in the first minutes after
rhamnolipid perception. Several elicitors have been
described to trigger a Ca

influx in grapevine, including the

endopolygalacturonase BcPG1 (Vandelle et al. 2006), lami-
narin (Aziz et al. 2003) and a-1,4 cellodextrins and oligoga-
lacturonides (Aziz et al. 2004, 2007). Ca

signal can be

characterized by its magnitude and duration (Trewavas &
Malho 1998). Interestingly, we observed a very strong influx
of calcium with rhamnolipids compared with oligosaccha-
rides. Cytosolic concentration peaked close to 6 mm after
addition of 0.05 mg mL

-1

of rhamnolipids when concentra-

tions of Ca

culminated from 0.4 to 0.6 mm with cell-

odextrins and oligogalacturonides at 0.5 mg mL

-1

(Aziz

et al. 2007). Concentrations of free cytosolic calcium was
even higher than concentrations detected after treatment of
tobacco cell suspensions with cryptogein, which is one of
the most potent elicitor characterized to date (Lamotte
et al. 2004). Moreover, cytosolic calcium influx induced by

Figure 4.

Defence gene expression in

grapevine vitro-plantlets treated with
rhamnolipids. (a) Transcript accumulation
of chitinase (chit4c) gene was monitored
over a time course after treatment
with 0.1 mg mL

-1

rhamnolipids (closed

circles) or water control (open circles).
(b) Transcript accumulation of genes
encoding lipoxygenase (lox),
phenylalanine ammonia lyase (pal),
protease inhibitor (pin), glucanase (gluc),
chitinase (chit4c) and stilbene synthase
(sts) was monitored 24 h after treatment
with rhamnolipids at 0.1 mg mL

-1

(grey

bars) or 1 mg mL

-1

(black bars). Analyses

were performed by real-time quantitative
polymerase chain reaction as described in
Fig. 3.

Time after treatment (h)

Relative transcript accumulation of

chit4c

(a)

(b)

lox

pal

pin

gluc

chit4c

sts

Relative transcript accumulation of defence genes

Rhamnolipids as new MAMPs in plants

185

rhamnolipids was extremely early and detected in the first
minutes after the molecule addition. This Ca

signature

seems to be very distinctive when compared with the signa-
tures described for other elicitors (Garcia-Brugger et al.
2006). Calcium influx caused by elicitors is usually followed
by activation of MAPKs and the production of ROS
(Garcia-Brugger et al. 2006). In grapevine cells, rhamnolip-
ids activated two MAPKs with apparent molecular mass of
46 and 50 kDa. MAPKs of similar weights were activated by
oligosaccharide elicitors like laminarin (Aziz et al. 2003) or

by protein elicitors like BcPG1 (Poinssot et al. 2003;
Vandelle et al. 2006) in grapevine suspension cells. Only few
studies pointed out the involvement of MAPKs in transduc-
tion pathways following lipid elicitor perception. Piater,
Nurnberger & Dubery (2004) showed that LPS from
Burkholderia cepacia activate a 43 kDa ERK-like MAPK
in tobacco. We also found that rhamnolipids induced an
oxidative burst in grapevine cell suspensions. Production of
ROS has often been involved as a key factor in plant cell
signalling following pathogen perception (Baker & Orlandi

chit4c

228

252

100

150

200

250

300

Relative transcript accumulation

of defence gene

pin

7446

10143

8078

2000

4000

6000

8000

10000

12000

14000

Relative transcript accumulation

of defence gene

gluc

131

123

100

120

140

160

180

control

Lrhamnose 0,05 RL

burkho 0,01

burkho

0,025

burkho

0,05

MonoRL

0,025

DiRL

0,025

MonoRL+DiRL

0,025

Relative transcript accumulation

of defence gene

Figure 5.

Defence gene expression of cultured grapevine cells treated with separated mono- and di-rhamnolipids, L-rhamnose or

di-rhamnolipids from Burkholderia plantarii. chit4c, pin and gluc expression was monitored 9 h after treatments. L-rhamnose,
rhamnolipids from B. plantarii (RL burkho), isolated mono-rhamnolipids (MonoRL) and di-rhamnolipids (DiRL) from Pseudomonas
aeruginosa, mixture of mono- and di-rhamnolipids (MonoRL + DiRL) were applied at the concentrations indicated (mg mL

-1

Control corresponds to water treatment. Analyses were performed by real-time quantitative polymerase chain reaction as described
in Fig. 3.

186

A.-L. Varnier et al.

1995; Neill et al. 2002). It is admitted that the origin of
the oxidative burst in tobacco and Arabidopsis involved
NADPH oxidases and/or peroxidases (Bindschedler et al.
2006). In grapevine, the origin of the oxidative burst is still
unclear. Our experiment using DPI, a specific inhibitor of
NADPH oxidases, suggests that these enzymes are involved
in the generation of the oxidative burst induced by rham-
nolipids. Using loss of function experiments, Aziz et al.
(2004) also found that treatment of grapevine cells with
DPI completely abolished the oligogalacturonide-induced
release of H

. Moreover, DPI addition lowered expres-

sion level of some defence genes and increased the suscep-
tibility of the plant to B. cinerea (Aziz et al. 2004).

Early events following elicitor perception often govern

later responses including defence gene expression and
sometimes cell death (Garcia-Brugger et al. 2006). Accord-
ingly, we found that in grapevine cell suspensions, rhamno-
lipids at 0.01 mg mL

-1

induced both early signalling events

and defence gene expression like chitinase gene and we did
not detect any changes in gene expression at 0.005 mg mL

-1

a concentration that did not induce early signalling events.
Both mono- and di-rhamnolipids were very efficient to
induce plant defence responses. Interestingly, L-rhamnose
alone did not stimulate defence genes, suggesting that the
lipid moiety of the molecules is necessary for the elicitor
activity. Rhamnolipids also stimulated a large set of defence
genes in vitro-plantlets. Pal, lox and sts encoding enzymes
that are involved in lignin, oxylipin and phytoalexin synthe-
sis respectively, and PR protein genes were up-regulated
following rhamnolipid perception. The differences in rham-
nolipid concentration necessary for elicitation in the two
plant systems used in this study can be explained at least in
part by a better accessibility of the molecule in cell suspen-
sions compared with vitro-plantlets.

In the cell suspensions and vitro-plantlets, we observed

some cell death at the highest concentration of rhamnolip-
ids. This cell death is characterized by a retraction or shrink-
age of the protoplast and the inability of the cell to cleave
the FDA and thus the cell does not fluoresce under light at
the wavelength of 490 nm. These features are characteristic
of a specific type of PCD (Reape, Molony & McCabe 2008).
This apoptotic-like PCD can be distinguished from necrosis
for which swilling rather than shrinkage is the defining
feature of the morphological change (Reape et al. 2008).
The cytoplasmic shrinkage is also a hallmark of the plant
hypersensitive response (HR) (Mur et al. 2008). It can thus
be postulated that the PCD observed after rhamnolipid
exposure is reminiscent to an HR-like response that has
been observed with some elicitors (Dorey et al. 1997). The
fact that defence gene activation is followed or not by
HR-like cell death depending on the elicitor concentrations
has already been described. For instance, a non-necrosing
dose of a fungal glycoprotein elicitor was shown to strongly

(a)

(b)

1000

2000

3000

4000

5000

6000

7000

8000

9000

Time (min)

RLU

500

1000

1500

2000

2500

3000

3500

4000

Time (min)

RLU

Figure 6.

Potentiation of elicitor-induced oxidative burst with

rhamnolipids. (a) Cell suspensions were treated with
rhamnolipids (0.005 mg mL

-1

) and chitosan 1 mg mL

-1

at the

same time (closed circles) or pre-incubated for 6 h (closed
triangles) with rhamnolipids prior to elicitation with chitosan. As
controls, grapevine cells were treated with chitosan alone at time
0 (open circles) or 6 h later (open triangles), with rhamnolipids
alone (open diamonds) or water (closed squares). (b) Cell
suspensions were treated with rhamnolipids (0.005 mg mL

-1

) and

Botrytis cinerea culture filtrate (CF) at the same time (closed
circles) or pre-incubated for 6 h (closed triangles) with
rhamnolipids prior to elicitation with B. cinerea CF. As controls,
grapevine cells were treated with B. cinerea CF alone at time 0
(open circles) or 6 h later (open triangles), with rhamnolipids
alone (open diamonds), or water (closed squares). Data shown
are from a duplicate out of three independent experiments with
similar results. H

production was determined as described in

Fig. 1.

Rhamnolipids as new MAMPs in plants

187

activate defence genes in tobacco (Costet et al. 1999). It
has been suggested that depending on the elicitor nature or
concentration, the initial signalling pathway can fork into
two branches, one leading to defence response expression
while the other ends in cell death and that a threshold
seems to be necessary for irrevocable commitment to death
(Morel & Dangl 1997). This threshold hypothesis could
account for the differences observed after rhamnolipid
perception.

To date, our report is the first to show that rhamnolipids

are potent elicitors of plant defence reactions. In addition,
rhamnolipids are the first glycolipids characterized as elici-
tor in grapevine. Recently, the elicitor properties of lipopep-
tide molecules like surfactine, fengycine and massetolide A
have been described in bean and tomato plants (Ongena

et al. 2007; Tran et al. 2007). Ergosterol has been found to
trigger WRKY, VvLTP1 and sts gene expression in grape
plantlets (Laquitaine et al. 2006). The mode of action of this
lipid elicitor in grapevine is still unclear. It has been sug-
gested that ergosterol is perceived by plant cells as a non-
specific elicitor that could bind to a plasma membrane
receptor and trigger a classical signal transduction pathways
in grapevine (Laquitaine et al. 2006). Elicitor properties of
ergosterol have also been demonstrated in tobacco and
tomato cell suspensions (Granado et al. 1995; Kasparovsky,
Blein & Mikes 2004). Like ergosterol, perception of rham-
nolipids by the plant cells is still unclear. We can hypoth-
esize that rhamnolipids are recognized by specific receptors
like it was demonstrated for other MAMPs including flagel-
lin, the elongation factor EF-Tu or chitin (Zipfel et al. 2004;

Figure 7.

Rhamnolipids inhibit spore

germination and mycelium growth of
Botrytis cinerea. (a) Conidia were placed
in growth medium supplemented with
solutions of rhamnolipids at the indicated
concentrations. Germ tubes were
observed by inverted light microscopy 5,
8 and 24 h later. (b) B. cinerea mycelium
excised from a solid culture in Petri
dishes was transferred to tomato agar
Petri dishes supplemented with
rhamnolipids at the concentrations
indicated. Radial growth of the fungus
was measured 24, 48 and 72 h after
inoculation.

(a)

0.1

Rhamnolipid concentration (mg

–1

)

Radial growth (cm)

(b)

0.1

Rhamnolipid concentration (mg

–1

)

188

A.-L. Varnier et al.

Kaku et al. 2006; Yamaguchi, Pearce & Ryan 2006; Zipfel
et al. 2006) and the endogenous peptide elicitor AtPEP1
(Huffaker & Ryan 2007; Yamaguchi et al. 2006). There are
other indications that lipid or glycolipid elicitors are per-
ceived by plant receptors. In mammalian systems, LPS
recognition is mediated by a receptor complex comprising
Toll-like receptor 4 (TLR4), MD2 and CD14 (Desaki et al.
2006), and in plants, there are cytological indications of the
presence of LPS-binding sites on the plant cell surface
(Gross et al. 2005). Specific binding of the glycolipid syrin-
golide elicitors to a soluble protein has also been docu-
mented (Ji et al. 1998). Interestingly, syringolides like
rhamnolipids are able to induce plant defence genes and
cell death following perception (Hagihara et al. 2004). Nev-
ertheless, the existence of similarities in the perception and
the mode of action of the two glycolipids remain to be
elucidated. Alternatively, rhamnolipids could directly inter-
act with the lipid bilayer of the plasma membrane and lead
to plasma membrane destabilization, which in turn could
activate the plant defence signalling pathways. Interaction
of rhamnolipids with the plasma membrane of oomycete
zoospores has previously been described (Stanghellini
& Miller 1997). However, major lipid components of
zoospores plasma membranes are glycolipids and neutral
lipids, rather than the phospholipids normally found in
most eukaryotic plasma membranes (Stanghellini & Miller
1997). MAMPs are usually conserved patterns in microbes
from different origins. Rhamnolipids from B. plantarii and
P. aeruginosa are both recognized by the grapevine plant
cells, suggesting that rhamnolipids can be considered as a
new category of MAMPs. Interestingly, the rhamnolipids
from B. plantarii have endotoxin-like properties and stimu-
late the immune system in human mononuclear cells
(Andra et al. 2006). These findings and our data reinforce
the fact that MAMPs (including the typical LPS endotox-
ins) can be recognized by both the plant and the animal
innate immunity systems.

In addition to their elicitor properties, rhamnolipids

potentiated the oxidative burst following the challenge with

a second elicitor. Oxidative burst is widely used as a plant
defence marker to monitor potentiation responses (Shirasu
et al. 1997) including priming (Ortmann, Conrath & Moer-
schbacher 2006). We found that while rhamnolipids acted
synergistically with both chitosan and CF, they also exhib-
ited a potentiation effect when applied as pre-treatment.
Moreover, the potentiation effect was displayed whatever
the concentration of rhamnolipids induced or not defence
responses when applied alone. Typically, a synergistic effect
has been described in the case of simultaneous addition of
two elicitors that display a combined effect, which is greater
than the sum of the individual effects (Boller 1995). For
instance, it has been shown that treatment of cell suspension
cultures of rice with cerebroside and chitin oligomers, each
inducing phytoalexin accumulation, resulted in a synergistic
induction of this defence response (Umemura et al. 2002).
Recently, a synergistic effect between a non-elicitor dose
of laminarin and its sulphated derivative PS3 has been
described (Menard et al. 2005). Unfortunately, the duration
of the synergistic effect has not been tested in the case of
laminarin/PS3 and cerebroside/chitin models. The physi-
ological differences between synergy and priming are still
unclear. A prime state is defined as a physiological condi-
tion in which plants are able to better or more rapidly
mount defence responses, or both, to biotic or abiotic
stresses (Conrath et al. 2006; Beckers & Conrath 2007).
Typically, priming is induced by molecules without elicitor
properties like b-aminobutyric acid (Ton et al. 2005) or used
at concentrations that do not stimulate defence responses
as in the case of salicylic acid or benzothiadiazole (Thulke
& Conrath 1998; Kohler, Schwindling & Conrath 2002). In
addition, priming response is usually detected following a
pre-treatment of the priming-inducing molecule for at least
several hours before the second challenge (Conrath et al.
2006; Ortmann et al. 2006). Interestingly, our results high-
lighted differences in potentiation profiles induced by
rhamnolipids, depending on the nature of the second stimu-
lus. While the synergistic effect was predominant in the case
of CF, we observed a better potentiation of chitosan

Figure 8.

Rhamnolipids induce

protection of grapevine leaves against
Botrytis cinerea. Vitro-plantlets were
immersed in a solution of rhamnolipids at
0.1 mg mL

-1

(a) or in water (b) for 1 min.

Two days later, leaves were excised,
placed on Petri dishes and inoculated
with B. cinerea conidia. Symptoms were
observed 96 h after inoculation. Brown
tissues correspond to maceration
symptoms caused by the fungus.

(a)

(b)

Rhamnolipids as new MAMPs in plants

189

response with a 6 h pre-treatment of grapevine cell suspen-
sions with rhamnolipids compared with a co-treatment
with the same molecules. In this later case, the phenom-
enon is reminiscent to a typical priming response
as described by Ortmann et al. (2006) for Pantoea
exopolysaccharides in rice and wheat cells. Our results
suggest that the mechanisms of potentiation induced by
the same molecule could significantly differ depending on
the nature of the second stimulus. Moreover, the fact that
low concentrations of rhamnolipids did not induce any
detectable changes in signalling events or gene induction
while promoting potentiation of defences induced by a
second stimulus could also suggest that two distinct mecha-
nisms are involved for elicitation and potentiation of
defence responses by these molecules. Our data could rein-
force the hypothesis of a post-transcriptional modification
of some components of the defence machinery or the
activation of some transcription factors that would be
necessary for the prime state or potentiation via synergy
(Conrath et al. 2006).

In this paper, we also demonstrated that rhamnolipids

protect grapevine plantlets against the necrotrophic
fungus B. cinerea. Enhanced protection can be correlated
with induced responses like it was previously shown in
grapevine after treatment with cellodextrins (Aziz et al.
2007), oligogalacturonides (Aziz et al. 2004), laminarin
(Aziz et al. 2003) and ergosterol (Laquitaine et al. 2006).
In addition, potentiation of the defence responses induced
by B. cinerea perception could also participate to the
increased resistance to the fungus. Given the fact that
rhamnolipids have also a direct effect on the fungus
growth and spore germination, we can also assume that
both plant defence induction and antimicrobial properties
are involved in B. cinerea restriction. Similar results have
been published with chitosan which also protects grape-
vine against the fungus. Chitosan acts both by inhibiting
mycelium growth (Ait Barka et al. 2004) and stimulat-
ing defence reactions (Aziz et al. 2006; Trotel-Aziz et al.
2006). Direct antimicrobial activity of rhamnolipids is
not restricted to B. cinerea. Therefore, Phytophthora
sp., Pythium sp., Plasmopara sp. and Colletotrichum sp.
zoospores and mycelium growth are strongly affected by
rhamnolipids (Stanghellini & Miller 1997; Kim et al. 2000;
Haba et al. 2003; De Jonghe et al. 2005; Yoo, Lee & Kim
2005). The mode of action of rhamnolipids on zoospore is
well documented (Stanghellini & Miller 1997) but the
mechanisms involved in mycelium growth inhibition
remain to be elucidated. Overall, rhamnolipids seem to be
effective against a large set of pathogen including some
bacteria (Haba et al. 2003).

As rhamnolipids are efficient in stimulating plant defence

and in bioremediation processes (Nitschke et al. 2005a) and
given their ecological acceptance owing to their low toxicity
for human and biodegradable nature, they have the poten-
tial to be part of alternative strategies in order to reduce or
replace pesticides. Further experiments will need to be con-
ducted to access the potential of rhamnolipids under field
conditions.

ACKNOWLEDGMENTS

This work was supported by funds from Europôl’ Agro and
Chambre d’Agriculture de la Marne (France). The authors
are particularly grateful to the Professor Klaus Branden-
burg from Forschungszentrum Borstel, Leibniz-Zentrum
für Medizin und Biowissenschaften, Borstel, Germany who
kindly provided us rhamnolipids from B. plantarii. We also
thank Dr Sandrine Dhondt-Cordelier from the SDRP labo-
ratory, University of Reims for her technical expertise and
support on cell death experiments.

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193

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