Laminarin Elicits Defense Responses in Grapevine and Induces Protection Against Botrytis cinerea and Plasmopara viticola

Abstract

Grapevine (Vitis vinifera L.) is susceptible to many pathogens, such as Botrytis cinerea, Plasmopara viticola, Uncinula necator, and Eutypa lata. Phytochemicals are used intensively in vineyards to limit pathogen infections, but the appearance of pesticide-resistant pathogen strains and a desire to protect the environment require that alternative strategies be found. In the present study, the beta-1,3-glucan laminarin derived from the brown algae Laminaria digitata was shown both to be an efficient elicitor of defense responses in grapevine cells and plants and to effectively reduce B. cinerea and P. viticola development on infected grapevine plants. Defense reactions elicited by laminarin in grapevine cells include calcium influx, alkalinization of the extracellular medium, an oxidative burst, activation of two mitogen-activated protein kinases, expression of 10 defense-related genes with different kinetics and intensities, increases in chitinase and beta-1,3-glucanase activities, and the production of two phytoalexins (resveratrol and epsilon-viniferin). Several of these effects were checked and confirmed in whole plants. Laminarin did not induce cell death. When applied to grapevine plants, laminarin reduced infection by B. cinerea and P. viticola by approximately 55 and 75%, respectively. Our data describing a large set of defense reactions in grapevine indicate that the activation of defense responses using elicitors could be a valuable strategy to protect plants against pathogens.

Full text

1118 / Molecular Plant-Microbe Interactions
MPMI Vol. 16, No. 12, 2003, pp. 1118–1128. Publication no. M-2003-0915-01R. © 2003 The American Phytopathological Society
Laminarin Elicits Defense Responses
in Grapevine and Induces Protection
Against Botrytis cinerea and Plasmopara viticola
Aziz Aziz,
1
Benoît Poinssot,
2
Xavier Daire,
2
Marielle Adrian
2,3
, Annie Bézier,
1
B. Lambert,
1
Jean-Marie
Joubert,
4
and Alain Pugin
2
1
Unité de Recherche Vignes et Vins de Champagne, URVVC - UPRES EA 2069, UFR Sciences, Moulin de la Housse,
Université de Reims Champagne-Ardenne, BP 1039, F-51687 Reims cedex 2 France;
2
Unité Mixte de Recherche, Plante-
Microbe-Environnement, INRA 1088/CNRS 2625/ Université de Bourgogne, 17 rue Sully, BP 86510, 21065 Dijon cedex,
France;
3
Institut Jules Guyot, UMR 1088 INRA/Université de Bourgogne, 17 rue Sully, BP 86510, 21065 Dijon cedex,
France;
4
Société Goëmar, Avenue du Général Patton, BP 55, 35413 Saint Malo cedex, France
Submitted 21 April 2003. Accepted 30 June 2003.
Grapevine (Vitis vinifera L.) is susceptible to many patho-
gens, such as Botrytis cinerea, Plasmopara viticola, Uncinula
necator, and Eutypa lata. Phytochemicals are used intensively
in vineyards to limit pathogen infections, but the appearance
of pesticide-resistant pathogen strains and a desire to protect
the environment require that alternative strategies be found.
In the present study, the b-1,3-glucan laminarin derived
from the brown algae Laminaria digitata was shown both to
be an efficient elicitor of defense responses in grapevine cells
and plants and to effectively reduce B. cinerea and P. viticola
development on infected grapevine plants. Defense reactions
elicited by laminarin in grapevine cells include calcium in-
flux, alkalinization of the extracellular medium, an oxidative
burst, activation of two mitogen-activated protein kinases,
expression of 10 defense-related genes with different kinetics
and intensities, increases in chitinase and b-1,3-glucanase ac-
tivities, and the production of two phytoalexins (resveratrol
and e-viniferin). Several of these effects were checked and
confirmed in whole plants. Laminarin did not induce cell
death. When applied to grapevine plants, laminarin reduced
infection by B. cinerea and P. viticola by approximately 55
and 75%, respectively. Our data describing a large set of
defense reactions in grapevine indicate that the activation of
defense responses using elicitors could be a valuable strategy
to protect plants against pathogens.
The spread of a plant disease is governed by the abilities of the
plant and its potential pathogen to react quickly to new signals
generated during the molecular dialogue between both partners.
In plants, a complex array of defense responses is induced after
detection of a microorganism via the recognition of elicitor mole-
cules released during the plant–pathogen interaction (Ebel and
Cosio 1994). Following elicitor perception, the activation of sig-
nal transduction pathways generally leads to the production of
active oxygen species (AOS), phytoalexin biosynthesis, rein-
forcement of plant cell walls, and the accumulation of patho-
genesis-related (PR) proteins, some of which possess antimicro-
bial properties (Fritig et al. 1998; Hammerschmidt 1999;
Somssich and Hahlbrock 1998; Van Loon and Van Strien 1999).
All these defense reactions, which sometimes are associated with
a localized cell death known as the hypersensitive reaction (HR),
are considered to be important responses for delimiting the
pathogen’s growth. Moreover, plants have the ability to develop
systemic acquired resistance (SAR), which reduces subsequent
infection of healthy tissues by a broad range of pathogens. How-
ever, if these defense reactions occur too late, the infection proc-
ess will spread successfully.
Various types of elicitors have been characterized, including
carbohydrate polymers, lipids, (glyco)peptides, and
(glyco)proteins. These products are secreted by microorgan-
isms or derived from the cell walls of fungi, bacteria, or host
plants (Côté and Hahn 1994; Ebel and Cosio 1994) or from
seaweed (Bouarab et al. 1999; Klarzynski et al. 2000; Potin et
al. 1999). Among them, ramified b-(1,3)-(1,6)-glucans, xylo-
glucans, oligogalacturonides, and chitin or chitosan oligomers
exhibit elicitor activity across different plant species and evoke
pathogen defense responses (Côté and Hahn 1994; Côté et al.
1998; Darvill et al. 1992; John et al. 1997; Sharp et al. 1984).
The chemically well-characterized heptaglucoside from the
pathogenic oomycete Phytophthora sojae (Cheong and Hahn
1991; Sharp et al. 1984) has been shown to highly induce phy-
toalexin biosynthesis even when applied at low nanomolar con-
centrations (Brady et al. 1993; Côté and Hahn 1994). Similarly,
linear b-1,3-glucan oligomers are recognized as elicitors by a
variety of plants, such as alfalfa (Cardinale et al. 2000), bean
(Mithöfer et al. 1999), and rice (Inui et al. 1997). The linear b-
1,3-glucan laminarin derived from the brown algae Laminaria
digitata elicits a variety of defense reactions in tobacco plants,
such as the stimulation of phenylalanine ammonia lyase, caf-
feic acid O-methyl transferase, and lipoxygenase activities, as
well as the accumulation of salicylic acid and PR proteins
(Klarzynski et al. 2000). Furthermore, certain glucans have
been reported to enhance resistance against viruses (Rouhier et
al. 1995) and bacteria (Klarzynski et al. 2000).
Grapevine (Vitis vinifera L.) is susceptible to many diseases,
such as gray mould (Botrytis cinerea), downy mildew (Plasmo-
para viticola), powdery mildew (Uncinula necator), and dieback
(Eutypa lata). Phytochemicals commonly are used in vineyards
to prevent and limit pathogen infections. However, because of
the development of pesticide-resistant pathogen strains (Leroux
Corresponding author: A. Pugin, E-mail: pugin@dijon.inra.fr; Fax: 33-3-
80-69-32-26.
Current address of B. Poinssot: Department of Plant Biology, University
of Fribourg, 1700, Switzerland.
A. Aziz and B. Poinssot contributed equally to this article.

Vol. 16, No. 12, 2003 / 1119
et al. 1999), and in an effort to preserve wine quality and yet
reduce the impact of pesticides on the environment, considerable
interest has been focused on the replacement of chemicals by
efficient, alternative strategies. Grapevine resistance could be
increased by genetic improvement, but hybrids and transformed
grapevine (Coutos-Thévenot et al. 2001) are forbidden in French
vineyards. Thus, the alternative strategies most commonly
involve biological control of pathogens, or else the activation of
plant defense mechanisms using elicitors.
In V. vinifera, only a few defense markers have been investi-
gated in leaves and berries. Most of studies have concentrated on
gene expression and activities of chitinases and glucanases
(Bézier et al. 2002a; Busam et al. 1997; Derckel et al. 1996,
1998; Robert et al. 2002; Robinson et al. 1997; Salzman et al.
1998) and the production of stilbenic phytoalexins (Adrian et al.
1997; Bais et al. 2000; Jeandet et al. 1991; Langcake and Pryce
1977a and b; Pryce and Langcake 1977). Recently, we reported a
large set of defense responses triggered by the endopolygalactu-
ronase 1 of B. cinerea in grapevine (Poinssot et al. 2003).
In the present study, we report that laminarin is a potent
elicitor of defense reactions in grapevine cells. The early re-
sponses triggered by laminarin include calcium influx, an oxi-
dative burst, extracellular alkalinization of the culture medium,
and mitogen-activated protein kinase (MAPK) activation.
Laminarin induces the expression of defense genes associated
with the octadecanoid, phenylpropanoid, and stilbenoid path-
ways, and those corresponding to PR proteins. Moreover, lami-
narin induces a significant protection of grapevine leaves
against two pathogens, B. cinerea and P. viticola.
RESULTS
Early signaling events induced
by laminarin in grapevine cell suspensions.
The production of H
2
O
2
increased with increasing concentra-
tions of laminarin and became saturated at between 0.5 and 1
g/liter (Fig. 1A). The oxidative burst was detected approxi-
mately 10 min after laminarin application and the H
2
O
2
pro-
Fig. 1. Early signaling events activated in grapevine cells after elicitation with laminarin. A, Dose-
response curve of active oxygen species (AOS) production in
grapevine cell suspensions, 10 min after treatment with increasing concentrations of laminarin ( ). H
2
O
2
production was measured using chemiluminescence of
luminol. B, Time-course AOS production in grapevine cell suspensions treated with laminarin at 1 g/liter ( ) or in untreated cell suspensions ( ). C, Kinetic o
f
calcium influx in grapevine cells treated by laminarin at 1 g/liter (
) or in control cells ( ). Calcium influx was measured by
45
Ca
2+
incorporation, added 5 min
before treatment (0,033 MBq/g of fresh weight of cells). D, Extracellular pH shift of grapevine cell suspensions treated by laminarin at 1 g/liter (
) or untreated
(
). E and F, Kinetics of activation of two mitogen-activated protein kinases in control grapevine cells or treated by laminarin at 1 g/liter, E, analyzed by West-
ern blot using an antibody raised against human nonactivated ERK1/2 or F, raised against a phosphorylated peptide contained in the human ERK1/2. Protein
extracts (15 µg) were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis before tran
sfer onto nitrocellulose membrane and Western
blot analysis. Results are from one representative experiment out of three. Values represent the average data SD of triplicate assays.
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1120 / Molecular Plant-Microbe Interactions
duction was maximal after a further 10 min (Fig. 1B). Thereaf-
ter, H
2
O
2
concentration in the medium declined, reaching its
initial level 45 min after the beginning of the treatment.
Calcium influx has been shown to mediate elicitor-induced de-
fense responses (Blume et al. 2000; Jabs et al. 1997; Lecourieux
et al. 2002; Tavernier et al. 1995; Yang et al. 1997). Laminarin
induced a rapid uptake of
45
Ca
2+
in cultured grapevine cells
(Fig. 1C) in a dose-dependent manner, with 1 g/liter being the
most efficient concentration (data not shown). The increase in
Ca
2+
influx started within a few minutes, then reached a maxi-
mum value after 15 min corresponding to 60 nmol per gram of
fresh weight of cells (FWC). In control cells, the basal level of
Ca
2+
remained unchanged.
Alkalinization of the incubation medium has been reported
many times for elicitor-treated cell suspensions (Felix et al.
1993, 1998; Küpper et al. 2001; Mathieu et al. 1996). Treat-
ment of grapevine cells with laminarin at 1 g/liter caused an in-
crease in the pH of the medium from 5.12 to 5.35 within 30
min (Fig. 1D). Thereafter, the pH decreased slowly to 5.26 at
90 min after the beginning of the treatment.
A rapid activation of MAPKs has been described for differ-
ent plant systems in response to elicitors (Gomez-Gomez and
Boller 2002; Lebrun-Garcia et al. 1998; Romeis et al. 1999;
Zhang et al. 1998). In our control and laminarin (1 g/liter)-
treated grapevine cells, total MAPKs were quantified using an
antibody raised against human nonactivated ERK1/2. Western
blots performed with this antibody revealed two MAPKs with
relative molecular masses of 49 and 45 kDa, respectively, and
with comparable intensities in both control and laminarin-
treated cells (Fig. 1E). Thereafter, the time course of activation
of MAPKs in grapevine cells was analyzed using an antibody
raised against a phosphorylated peptide contained in the human
active ERK1/2. Immunodetection with this last antibody re-
vealed a rapid and transient activation of both MAPKs (Fig.
1F) only in laminarin-treated cells. Their activation was de-
tected within 5 min of treatment, peaked after 15 to 30 min,
and then fell to undetectable levels after 60 min.
Defense-related gene expression
in laminarin-treated grapevine cells and plants.
In grapevine, most elicitor- or pathogen-induced cDNA that
has been cloned corresponds to genes encoding PR proteins
(Busam et al. 1997; Davies and Robinson 2000; Jacobs et al.
1999; Robert et al. 2001, 2002) or enzymes involved in the
synthesis of stilbene phytoalexins (Melchior and Kindl 1990,
1991; Sparvoli et al. 1994; Wiese et al. 1994). To our knowl-
edge, many other putative defense genes, such as PR-1 and
PDF1.2, have not yet been identified in this species. For our
study, grapevine cells were challenged with laminarin at the
saturating concentration of 1 g/liter, and the expression pattern
of 11 selected defense-related genes was analyzed using real
time quantitative polymerase chain reaction (rtq-PCR) and spe-
cific primers (Table 1) with an actin gene as internal standard
(Bézier et al. 2002a). The rtq-PCR analysis gave reproducible
results that were confirmed using RNA gel blot analysis (data
not shown). In control cells, transcript accumulation of the dif-
ferent genes was always very low during the 25-h incubation
period (data not shown).
In grapevine cells treated with laminarin, 9-lipoxygenase
(LOX) and glutathione-S-transferase (GST) genes were rapidly
and transiently up-regulated (Fig. 2A). The steady state levels of
mRNA for LOX and GST increased 1 h after laminarin was sup-
plied, and were maximal (35- and 65-fold higher, respectively,
than in control cells at time zero) after 2 and 5 h of treatment,
respectively. mRNA levels subsequently decreased slowly.
Phenylalanine ammonia lyase (PAL) is a key enzyme of the
phenylpropanoid pathway which leads to various defense-
related compounds. Downstream of PAL, stilbene synthase 1
(STS1) is responsible for the synthesis of resveratrol, the main
phytoalexin produced by grapevine in response to biotic or
abiotic stresses (Adrian et al. 1997; Coutos-Thévenot et al. 2001;
Langcake and Pryce 1977a and b). In laminarin treated-cells,
PA L and STS1 mRNA were detected after 1 h of treatment, and
reached a 20-fold higher level than in control cells after 5 h.
Thereafter, transcripts of both slowly declined (Fig. 2A).
We also focused on a PGIP gene for which the product, a
polygalacturonase-inhibiting protein (PGIP), interacts with ex-
tracellular endo-a-1,4-polygalacturonases (PGs) secreted by
phytopathogenic fungi to inhibit their activity (Caprari et al.
1996). In laminarin-treated cells, PGIP transcript accumulation
was detectable after 10 h of treatment and reached a 40-fold
accumulation after 20 h (Fig. 2A).
The mRNA accumulation of three genes encoding chitinases
has been shown to be differentially regulated in grapevine
when challenged with U. necator, P. viticola, B. cinerea, or
Pseudomonas syringae pv. pisi (Busam et al. 1997; Jacobs et
al. 1999; Robert et al. 2002). Basic class I (CHIT1b), acidic
class III (CHIT3), and acidic class IV (CHIT4c) chitinase
cDNAs were cloned from grapevine cells (Busam et al. 1997)
or leaves (A. Bézier, personal communication) from various
cultivars. In laminarin-treated grapevine cells, the expression
of the three genes increased from 1 h of treatment and reached
a maximum between 5 and 10 h, with an approximately 140-
fold, 30-fold, and 700-fold increase for CHIT1b, CHIT3, and
CHIT4c, respectively. Thereafter, transcript accumulation re-
mained constant (CHIT3) or else decreased slowly (CHIT1b
and CHIT4c) until the end of the experiment (Fig. 2B).
The class I b-1,3-glucanases are antifungal vacuolar proteins
implicated in plant defense which exhibit developmental, hor-
monal, and pathogenesis-related regulation. The antifungal ac-
Table 1. Sequences of defense gene primers used for real time quantitative polymerase chain reaction
a
Names Forward primers Reverse primers
CHIT3 5 -AGATGGCATAGACTTCGACA-3 5-GTACTTTGACCACAGCATCA-3
CHIT4c 5 -GCAACCGATGTTGACATATCA-3 5-CTCACTTGCTAGGGCGACG-3
CHIT1b 5
-ATGCTGCAGCAAGTTTGGTT-3 5-CATCCTCCTGTGATGACATT-3
GLU1 5
-ATGCTGGGTGTCCCAAACTCG-3 5-CAGCCACTCTCCGACAGCAC-3
GST 5
-GCATGGGGTAAGAGGTGCATG-3 5-GCCTTGTTGTGATGTAATTGG-3
LOX 5
-CTGGGTGGCTTCTGCTCTC-3 5-GATAAGCCGCAGATTCATGC-3
PAL 5
-TGACCACTTGACTCACAAAT-3 5-ACTAGGTATGTGGTAGACAT-3
PIN 5
-AGTTCAGGGAGAGGTTGCTG-3 5-GCACTAGGGTCCGTGTTTGGGTCGACG-3
STS1 5
-TACGCCAAGAGATTATCACT-3 5-CTAAAGAGTCCAAAGCATCT-3
HSR 5
-GGACTACCGACATGCACCTG-3 5-GGTCATCACAAGCCTCTTGC-3
PGIP 5
-CCTAGACAATCCCTACATTC-3 5-GACATTGGGGTCGAATCCTC-3
a
CHIT1b and CHIT3 (Busam et al. 1997), PA L and STS1 (Sparvoli et al. 1994), HSR (Bézier et al. 2002b), and PGIP (Bézier et al. 2002a). Othe
r
sequences from A. Bézier and F. Bailleul, personal communication. Accession numbers: CHIT4c, no. AY137377; GLU1, no. AF239617; GST, no.
AY156048;
L
O
X
, no. AY159556; and PI
N
, no. AY156047.

Vol. 16, No. 12, 2003 / 1121
Fig. 2. Transcript accumulation of defense genes in grapevine cells after elicitation by laminarin. A, Expression profiles of defense-related genes encoding
a
lipoxygenase (LOX, accession no. AY159556), a glutathion-S-transferase (GST, accession no. AY156048), a polygalacturonase-inhibiting protein (PGIP; Bézie
r
et al. 2002a), a phenylalanine ammonia lyase (PA L ; Sparvoli et al. 1994), and a stilbene synthase (STS1; Sparvoli et al. 1994) in grapevine cells treated with
laminarin at 1 g/liter. B, Expression profiles of pathogenesis-related (PR) genes encoding a basic
-1,3-glucanase (GLU1, accession no. AF239617), a serine-
proteinase inhibitor (PIN, accession no. AY156047), a basic chitinase (CHIT1b; Busam et al. 1997), and two acidic chitinases of class III (CHIT3; Busam et al.
1997) and class IV (CHIT4c, accession no
. AY137377) in grapevine cells treated with laminarin at 1 g/liter. Analyses were performed by real time quantitative
polymerase chain reaction (rtq-PCR). Levels of transcripts were calculated using the standard curve method from triplicate data, with grapevine actin gene as
internal control and nontreated cells (zero time) as reference sample. Results represent the mean fold increase of mRNA level over nontreated cells, referred as
the 1× expression level. Absolute copy number of mRNA for each target gene in the reference sample was 10 (GLU1), 1,633 (PIN), 494 (CHIT1b), 1,194
(CHIT3), 735 (CHIT4c), 56 (LOX), 13 (GST), 60 (PGIP), 64 (PA L ), and 7 (STS1) × 10
3
molecules/µg of total RNA. Results presented are means of triplicate
data SD of one representative experiment out of three. C,
Expression of PR genes in grapevine detached leaves after 24 h of treatment with laminarin at 1
g/liter. Detached leaves were incubated for 24 h on aqueous solutions containing laminarin at 1 g/liter before inoculation with a conidial suspension (5 × 10
5
conidia/ml) of Botrytis cinerea, and rtq-
PCR was performed as described above. In control leaves, the transcript level of defense genes was very low. Values
represent the mean SD of triplicates of one representative experiment out of two.
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1122 / Molecular Plant-Microbe Interactions
tivity of plant b-1,3-glucanases is thought to hydrolyze the
structural b-1,3-glucan present in some fungal cell wall. Three
cDNA clones corresponding to different b-1,3-glucanase genes
from grapevine have been characterized previously (Jacobs et al.
1999). The rtq-PCR analysis (Fig. 2B) revealed that, in lami-
narin-treated grapevine cells, GLU1 transcripts accumulated
from 15 h of treatment with a 150-fold increase within 25 h.
Inhibitors of serine proteases (PIN) have emerged as a class
of antifungal PR-6 proteins which have potent activity against
plant and animal pathogens (Van Loon and Van Strien 1999).
Fig. 3. Phytoalexin synthesis, glucanase, and chitinase activities in grapevine cells in response to laminarin. Resveratrol (
l
) and -viniferin ( )
production measured in A, the cellular fraction or B,
the extracellular medium of cell suspensions treated with laminarin at 1 g/liter or in control cell
suspensions (resveratrol, and -viniferin, ). C, Glucanase and D, chitinase activities in cell ext
racts from cell suspensions treated with laminarin at 1
g/liter ( ) or in untreated cells ( ). Values represent the mean SD of duplicate assays of one representative experiment out of three.
Fig. 4. Laminarin-induced protection of grapevine leaves against Botrytis cinerea and Plasmopara viticola. A,
Effect of laminarin on the protection of
grapevine leaves against the fungus B. cinerea.
Detached leaves were incubated for 24 h on aqueous solutions containing laminarin at 1 g/liter before
inoculation with a conidial suspension (5 × 10
5
conidia/ml) of B. cinerea (40 leaves per condition). Disease assessment was determined measuring the
average diameter of lesions formed during 4 days post inoculation. Data represent the average diameter
SD of lesions spreading on 40 elicited leaves
(black bars) compared with untreated ones (open bars). Results are from one representative experiments out of four. Asterisk indicates that values are
significantly different (P < 0.05) according to c test. B, Development of downy mildew (P. viticola
) on grapevine plants treated with laminarin. Plants
were sprayed with increasing concentrations of laminarin and inoculated 10 days later by spraying a sporangial suspension (2 × 10
4
sporangia/ml). Disease
assessment was done 8 d
ays post inoculation and expressed as percent infected leaf surface. Fourteen plants were used per treatment. Results with
different letters are significantly different at 5% using Duncan’s multiple range test. The experiment was repeated twice with similar results.
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Vol. 16, No. 12, 2003 / 1123
The rtq-PCR analysis revealed that the PIN gene was up-regu-
lated in grapevine cells after 5 h of laminarin treatment (Fig.
2B), reaching approximately 600-fold within 20 to 25 h of
elicitation.
An increase of the expression of these genes also was moni-
tored in detached grapevine leaves treated with laminarin at 1
g/liter. Accumulation of mRNA corresponding to CHIT1b,
CHIT4c, GLU1, and PIN reached a 110-, 525-, 125-, and 410-
fold higher level, respectively, than in control detached leaves
after 24 h of treatment (Fig. 2C).
Laminarin did not induce
either HSR gene expression or cell death.
A cDNA showing significant homology with the tobacco
HSR203 cDNA has been isolated by differential display re-
verse transcriptase PCR from grapevine leaves infected with
B. cinerea (Bézier et al. 2002b). In tobacco, HSR203 mRNA
accumulation occurs during the HR and is considered to be a
marker of cell death (Pontier et al. 1998). Antisense expres-
sion of HSR203 in tobacco triggered an accelerated cell
death, suggesting that its product counteracts the cell death
process (Tronchet et al. 2001). Elsewhere, HSR203 is not ex-
pressed in response to various biotic stresses or elicitors
which did not induce HR (Brederode et al. 1991; Godiard et
al. 1991; Pontier et al. 1998). rtq-PCR results indicated that
this grapevine HSR homologue was not up-regulated by lami-
narin (data not shown). Furthermore, grapevine cell viability,
quantified by neutral red as a vital dye (Naton et al. 1996),
was not significantly altered in the presence of laminarin
even at a high concentration. The cells remained 95 to 97%
viable after a 24-h treatment with laminarin at 1 g/liter (data
not shown). These results clearly indicate that laminarin did
not induce any HR in grapevine cells. When applied on
whole plants, laminarin at 1 g/liter did not induce cell death
(data not shown).
Laminarin induces phytoalexin production
and increases chitinase and b-1,3-glucanase activities.
Resveratrol and its dimer, e-viniferin, are the major phy-
toalexins produced by grape (Vitis spp.) in response to micro-
bial attacks and UV (Adrian et al. 1997; Coutos-Thévenot et al.
2001; Langcake and Pryce 1977a and b; Pryce and Langcake
1977). Large amounts of both phytoalexins were produced by
grapevine cells in response to laminarin (Fig. 3A and B). Most
of the resveratrol accumulated in the extracellular medium,
where it peaked 8 h after treatment (65 µg/g FWC). e-viniferin
was present predominantly inside the cells, where it peaked
(130 µg/g FWC) after 20 h, though a significant amount of e-
viniferin (62 µg/g FWC) was detected after 12 h in the extra-
cellular medium. Subsequently, both compounds progressively
disappeared from both the cells and the extracellular medium
(Fig. 3A and B). The amounts of phytoalexins were very low in
control cells and the corresponding extracellular medium (2
µg/g FWC).
To determine if the accumulation of transcripts correspond-
ing to chitinase and b-1,3-glucanase genes correlates with an
increase of chitinase and b-1,3-glucanase activities, respec-
tively, crude enzyme extracts were prepared from laminarin-
treated as well as control grapevine cells. Results showed an
increase in both intracellular chitinase and b-1,3-glucanase ac-
tivities after 4 and 10 h, respectively, of laminarin treatment
(Fig. 3C and D). Both activities peaked after 25 h of treatment
and remained high throughout the experimental period. In con-
trol cells, both activities were very low. In detached grapevine
leaves treated for 24 h with laminarin at 1 g/liter, chitinase
activity increased from 0.23  0.1 (control) to 2.17  0.5 Unit/g
of FW leaves (treated).
Laminarin-induced protection
of grapevine against B. cinerea and P. viticola.
Leaves detached from in vitro cultivated grapevine plantlets
were preincubated on different concentrations of laminarin for
24 h, then inoculated with a B. cinerea conidial suspension of 5
× 10
5
conidia/ml. Under these conditions, the average diameter
of lesions measured 4 days post inoculation was reduced by
approximately 55% in the leaves treated with laminarin at 1
g/liter (Fig 4A). With laminarin at 0.5 and 0.2 g/liter, this re-
duction reached 40 and 15%, respectively (data not shown).
In a second series of assays, laminarin-triggered protection
against P. viticola was measured using whole plants (V. vinifera
cv. Gamay) sprayed on both leaf surfaces with different con-
centrations of laminarin. A first series of assays indicated that
laminarin-induced protection was evident after 8 days of treat-
ment. Thus, whole plants were used instead of detached leaves
and plants were inoculated 10 days after laminarin application
by spraying a sporangial suspension (2 × 10
4
sporangia/ml) on
the lower face of leaves. Disease intensity was estimated 8
days post inoculation by measuring the leaf area covered by the
oomycete and counting the sporangia. Our data (Fig. 4B) indi-
cate that laminarin at 0.5 to 1 g/liter reduced the percentage of
P. viticola-infected leaf surfaces from 28% in the control to 7%
in the laminarin-sprayed plants, corresponding to a 75% reduc-
tion in infection.
DISCUSSION
Laminarin triggers a typical
elicitor signal transduction network in grapevine cells.
When supplied to grapevine cell suspensions, laminarin trig-
gers a series of events, within minutes of treatment, typical of
those described for other, well-known elicitors (Fig. 1). In
grapevine cells, laminarin (as well as oligogalacturonides; data
not shown) induces a rapid calcium influx, similar to that ob-
tained in tobacco cells, which has been reported to trigger a bi-
phasic and transient free-calcium elevation in the cytosol
(Lecourieux et al. 2002). Calcium influx and increase in free
cytosolic calcium concentration have been reported to occur in
response to various abiotic and biotic stimuli, particularly to
elicitors (Blume et al. 2000; Knight et al. 1991; Lecourieux et
al. 2002; Mithöfer et al. 1999; Tavernier et al. 1995). In com-
mon with other elicitors, laminarin induces the alkalinization
of the extracellular medium of grapevine cells (Boller 1995).
As reported with laminarin-treated tobacco cells (Klarzynski et
al. 2000), this alkalinization is transient, which suggests the
activation of plasma membrane (PM) H
+
-ATPases in a second
step, to restore the pH gradient between the apoplast and the
cytosol. Laminarin also triggers, within moments of treatment,
a transient production of H
2
O
2
. AOS can have antimicrobial ef-
fects (Baker and Orlandi 1995) and are assumed to be involved
in lipid peroxidation (Rustérucci et al. 1996), oxidative cross
linking of cell wall proteins (Bradley et al. 1992; Brisson et al.
1994), phytoalexin production (Jabs et al. 1997), and defense
gene expression related to HR and SAR (Alvarez et al. 1998;
Chen et al. 1993; Costet et al. 2002; Levine et al. 1994). Nev-
ertheless, the role of AOS, as a signal, is not restricted to
plant–pathogen interactions, and their putative roles in SAR
and in HR-like cell death has been the subject of debate, with
the species and the level of AOS determining for the type of re-
sponse (Delledone et al. 2001; Rustérucci et al. 1996; Van
Breusegem et al. 2001). In plants, many MAPK modules par-
ticipate in the transduction of various biotic or abiotic stimuli
(Asai et al. 2002; Hirt 1997; Lee et al. 1998). In tobacco, sali-
cylic acid-induced protein kinase (SIPK) and wound-induced
protein kinase (WIPK) are two MAPKs activated in response
to various elicitors, including cryptogein and oligogalacturonides

1124 / Molecular Plant-Microbe Interactions
(Lebrun-Garcia et al. 1998; Zhang et al. 1998). In grapevine
cells, laminarin triggers a fast and transient phosphorylation-
dependent activation of two MAPKs with relative molecular
masses of 49 and 45 kDa, respectively, suggesting a conserved
phosphorylation cascade in this species.
Laminarin induces defense gene expression, accumulation
of phytoalexins, chitinase, and b-1,3-glucanase activities.
In elicitor- or pathogen-treated plants, important changes in
the plant transcriptome pattern have been reported (Baldwin et
al. 1999; Dangl and Jones 2001; Maleck et al. 2000; Schenk et
al. 2000). rtq-PCR has been used to analyze the expression of
11 defense-related genes in grapevine cell suspensions and
leaves treated with laminarin (Fig. 2). In cells, 10 of these
genes were up-regulated in response to the algal b-1,3-glucan,
whereas HSR, which is considered to be an HR-like marker
gene of cell death in tobacco (Pontier et al. 1998), was not in-
duced. This observation is consistent with our results, indicat-
ing that laminarin did not induce any cell death. Elsewhere,
some of these genes were activated rapidly after laminarin
treatment (LOX, GST, PAL, STS1, CHIT4c, and CHIT1b),
whereas others were up-regulated later (CHIT3, PIN, GLU1,
and PGIP). A 9-LOX could be essential for the resistance to
fungal infection, as it was demonstrated in tobacco using an
antisense strategy (Rancé et al. 1998). The gene expression of
GST, an enzyme which takes part in the detoxification of elici-
tor-generated oxidants, has been reported in response to the
oxidative burst (Levine et al. 1994; Mauch and Düdler 1993;
Vanacker et al. 2000). The induction of PA L and STS1 genes is
consistent with the production of resveratrol and e-viniferin,
two phytoalexins involved in the protection against pathogens
(Coutos-Thevenot et al. 2001; Hain et al. 1993; Stark-Lorenzen
et al. 1997). PGIPs are considered to favor plant defenses by
preventing plant cell wall degradation and modulating some
fungal polygalacturonase activities (De Lorenzo and Ferrari
2002), thus releasing elicitor-active long-chained oligogalactu-
ronides.
Laminarin-treated cells also exhibit an induction of genes
encoding different families of PR proteins with antimicrobial
properties (Fritig et al. 1998; Van Loon and Van Strien 1999).
The expression of the three chitinase genes occurred rapidly af-
ter laminarin treatment. Moreover, the CHIT4c transcripts were
very abundant, whereas CHIT3 amounts were low and CHIT1b
quantities were intermediate. The analysis of the expression of
some of these genes in grapevine leaves treated with laminarin
confirmed the transcript accumulation monitored in cells, and
particularly CHIT4c (Fig. 2C). In the same manner, Derckel
and associates (1998) showed that the class IV chitinase,
CHV5, is the most abundant chitinase produced in response to
fungal challenge in ripening grape berries. CHIT1b and CHIT3
were activated in grapevine treated with a yeast elicitor or sali-
cylic acid.
As expected, laminarin also triggers a large production of
both phytoalexins, resveratrol and e-viniferin. Interestingly,
resveratrol was detected mainly in the extracellular medium,
whereas its dimer, e-viniferin, was much more abundant in cell
extracts (Fig. 3A and B). In cells, both phytoalexins are not se-
questered in vacuoles (E. Martinoïa, personal communication)
and the presence of large amounts of e-viniferin suggests a
rapid dimerization of resveratrol. The presence of both com-
pounds in the extracellular medium could result from the trans-
port or diffusion of these compounds or their conjugates
through the PM to the apoplast, where, as previously reported,
they exert their antibiotic activities (Coutos-Thevenot et al.
2001). Alternatively, extracellular e-viniferin could result from
an oxidative coupling of resveratrol triggered by a cell-wall-lo-
calized peroxidase (Calderon et al. 1992, 1994; Langcake and
Pryce 1977c). Comparisons of kinetics of accumulation of both
compounds in cells and extracellular medium support this
process, though further experiments are required to verify this
assumption.
The activities of both chitinase and glucanase increased in
laminarin-treated cells of grapevine (Fig. 3C and D), correlat-
ing to the accumulation of the corresponding transcripts (Fig.
2B). In grapevine leaves treated for 24 h with laminarin at 1
g/liter, chitinase activity was 10-fold higher than in the control.
This result is in agreement with the induction of chitinase
genes in laminarin-treated leaves (Fig. 2C).
Both enzyme activities should participate in the plant de-
fense by hydrolyzing fungal cell wall components as previ-
ously reported (Van Loon and Van Strien 1999). They also
should amplify the plant defense by releasing b-1,3 glucans
and chitin fragments from the pathogen cell walls, both oli-
gosaccharides being well known elicitors (Côté et al. 1998).
Thus, laminarin triggers a variety of defense responses in
grapevine (our results) and in tobacco (Klarzynski et al.
2000).
Defense responses induced by laminarin lead
to grapevine protection against B. cinerea and P. viticola.
When applied on detached grapevine leaves or on intact
plantlets, laminarin 0.5 to 1 g/liter reduced the development of
B. cinerea and P. viticola by approximately 50 and 75%, re-
spectively (Fig. 4). Further protection is conceivable by im-
proving the penetration of the elicitor and by better characteriz-
ing the optimum time for defense activation. These assays are
in progress. Laminarin also confers protection of tobacco to the
soft rot disease agent, Erwinia carotovora (Klarzynski et al.
2000), indicating that this compound could be effective across
a large spectrum of plant species and against different patho-
gens, although laminarin probably will not be efficient against
all of them. Taken together, these data show that the activation
of plant defenses using elicitors is probably a valuable alterna-
tive strategy to restrict the pathogen spread.
MATERIALS AND METHODS
Biological materials.
Grapevine (V. vinifera cv. Gamay) cell suspensions were
cultivated in Nitsch-Nitsch medium (Nitsch and Nitsch 1969)
without added hormone on a rotary shaker (150 rpm, 25°C)
under continuous light (2,000 ergs/cm
2
). Cells were subcul-
tured every 7 days to be maintained in exponential phase and
1-day-prior assays. Vitro-plantlets of grapevine (V. vinifera
cv. Chardonnay 75) were grown in the Murashige and Skoog
(1962) medium at 26°C with a photoperiod of 14 h of light.
Grapevine plants (V. vinifera cv. Gamay) were obtained from
cuttings cultivated in a growth chamber at 25°C with a day
length of 18 h.
B. cinerea (Botryotinia fuckeliana p. f.) strain 630 (kind gift
of Y. Brygoo, INRA, Versailles, France) was grown on potato
dextrose in 250-ml flasks at 22°C.
A P. viticola isolate collected in Burgundy in 2001 was main-
tained on potted grapevine plants and subcultured every week.
Preparation of laminarin.
Laminarin was extracted and purified from the marine brown
algae L. digitata by the firm Goëmar as described by Klarzynski
and associates (2000). Thus, the same laminarin preparation
was tested for elicitor activity on both tobacco (Klarzynski et
al. 2000) and grapevine (this work) cells and plants. The aver-
age degree of polymerization (DP) of laminarin was estimated
by molecular size chromatography coupled with a refractomet-
ric detector. Purity, size, and structure were analyzed further by

Vol. 16, No. 12, 2003 / 1125
natural abundance
13
C nuclear magnetic resonance spectros-
copy and high performance anion exchange chromatography
and pulsed amperometric detection (Lépagnol-Descamps et al.
1998), confirming that laminarin is an essentially linear b-1,3-
glucan of a mean DP of 33.
Treatments.
Cells were collected during the exponential growth phase
and washed by filtration in a suspension buffer containing 175
mM mannitol, 0.5 mM K
2
SO
4
, 0.5 mM CaCl
2
, and 2 mM MES
(morpholineethanesulfonic acid) adjusted to pH 5.4. Cells were
resuspended at 0.1 g FW/ml with suspension buffer and equili-
brated for 2 h on a rotary shaker (150 rpm, 24°C). Grapevine
cells then were used for measurements of calcium influx, H
2
O
2
production, extracellular pH, protein kinase activation, and cell
death after treatment with laminarin. Control cells were incu-
bated under the same conditions without elicitor.
For gene expression, chitinase activity, and resveratrol pro-
duction, cells were collected during the exponential growth
phase and resuspended at 0.1 g FW/ml in a freshly prepared
cell culture medium and equilibrated for 2 h on a rotary shaker
(130 rpm, 24°C) before the addition of elicitor.
Measurement of medium alkalinization.
Grapevine cells in suspension buffer (pH 5.4) were equili-
brated for 1 h with continuous stirring until a steady pH value
was reached. The change in medium pH was monitored with a
glass combination electrode for 70 min after laminarin supply.
H
2
O
2
production measurement.
H
2
O
2
production was determined using chemiluminescence
of luminol as described previously (Poinssot et al. 2003).
Chemiluminescence, measured within a 10-s period with a lu-
minometer, (Lumat LB 9507, Berthold) was integrated and ex-
pressed in nanomole of H
2
O
2
per gram of FWC using a stan-
dard calibration curve obtained by H
2
O
2
addition in grapevine
cell suspension aliquots.
Ca
2+
influx measurement.
Five minutes before treatments, cells in the suspension
buffer (pH 5.4) were incubated with
45
Ca
2+
(0.033 MBq g
–1
FWC; Amersham Pharmacia Biotech). After different periods
of treatment, triplicate 1.5-ml aliquots were filtered under vac-
uum on glass microfiber (GF/A) filters and washed three times
with a total volume of 25 ml of buffer (175 mM mannitol, 0.5
mM K
2
SO
4
, 5 mM LaCl
3
, and 2 mM MES, pH 5.4) before
transferring the cells to scintillation vials. After a 12-h period
at 65°C, dry weight was determined and 10 ml of Ready Safe
cocktail (Beckman) were added to the vials before counting in
a scintillation counter (TRI-CARB 2100 TR, Packard).
MAP kinase activation monitored
by Western blotting assays.
Detection of nonactivated or activated MAPKs was per-
formed as described elsewhere (Poinssot et al. 2003).
Phytoalexin quantification.
At different times after treatment, aliquots (2 ml) of cells in
the culture medium were collected and filtered on GF/A glass
fiber filters. Filtrates were directly analyzed, whereas stilbenes
from the cell fraction were extracted in 2 ml of methanol over-
night at 4°C. Each sample (40 µl) was loaded onto a Lichrocart
C-18 inverse phase column (250 by 4 mm, 5 µm; Merck)
equilibrated with a 90/10 (vol/vol) H
2
O/acetonitrile mobile
phase. Phytoalexins were eluted with a linear gradient from 10
to 85% acetonitrile at a flow rate of 1 ml min
–1
. Quantification
of trans-resveratrol and e-viniferin was performed with stan-
dard calibration curves using peak areas of different amounts
of pure molecules fluorometrically detected (lex = 330 nm,
lem = 374 nm), as previously described (Jeandet et al. 1997).
Northern blot analysis and rtq-PCR.
Aliquots of grapevine cell suspensions (2 ml) in the culture
medium were filtered and subsequently frozen in liquid N
2
. To-
tal RNA isolation was obtained by adding 1 ml of trizol
(Gibco-BRL) following the manufacturer’s procedure.
For Northern blot analysis, 10 µg of total RNA were sepa-
rated on a 1.2% agarose gel containing 1.1% formaldehyde,
then transferred to a Hybond N
+
membrane (Amersham Phar-
macia Biotech) and crosslinked by UV. The blot was hybrid-
ized at 65°C with cDNA probes labeled with 50 µCi [a-
32
P]-
dCTP using the Ready-To-Go labeling kit (Amersham Phar-
macia Biotech). Blots were exposed for 24 h in a phos-
phorimager screen or to X-Omat AR film (Kodak).
For rtq-PCR, total RNA were incubated with 15 units of
RNase-free DNase I (Promega Corp.) for 30 min at 37°C and
stopped with a phenol:chloroform:isoamylic alcohol mixture
(25:24:1, vol/vol/vol). DNase-treated RNA (2 µg) was reverse
transcribed with 5 µM of oligo(dT) following the manufac-
turer’s instructions (Life Technologies/Gibco-BRL). The tran-
script levels were determined by rtq-PCR using the GeneAmp
5700 Sequence Detector (Applied Biosystems) using the
SYBR Green Master Mix PCR kit as recommended by the
manufacturer (Applied Biosystems). PCR reactions were car-
ried out in triplicates in 96-well plates (25 µl per well) in a
reaction buffer containing 1× SYBR Green I mix (including
Taq polymerase, dNTPs, SYBR Green dye), 300 nM forward
and reverse primers, and a 1:250 dilution of reverse-transcribed
RNA. After denaturation at 95°C for 10 min, amplification oc-
curred in a two-step procedure: 15 s of denaturation at 95°C
and 1 min of annealing and extension at 60°C, with a total of
40 cycles. Identical thermal cycling conditions were used for
all targets. The gene-specific primers are indicated in Table 1.
The absence of primer-dimer formation, which could interfere
with specific amplification, was checked in controls lacking
templates. Transcript level was calculated using the standard
curve method (User Bulletin #2; ABI PRISM 7700 Sequence
Detection System, Applied Biosystems). Standard curves were
generated by performing rtq-PCR on serial dilutions of specific
purified DNA. This latter consisted of a PCR product prepared
by “classical” PCR from plasmid harboring the target gene as
template. The copy number (CN) for these PCR products used
as standards was calculated from their concentration measured
by absorbance at 260 nm. Standard curves were constructed by
plotting the threshold cycle (Ct values, PCR cycle at which the
reporter fluorescence of SYBR Green above the baseline signal
can be detected) versus the logarithm of the CN of specific
purified PCR products. The absolute CN for each sample was
calculated from standard curves using their Ct value and nor-
malized against grapevine actin gene as internal control (Bézier
et al. 2002a) and nontreated cells as reference sample. Subse-
quently, for each gene, the reference sample was referred as the
1× expression level and results were expressed as the fold in-
crease of mRNA level over the reference sample.
Chitinase and glucanase activity assays.
Chitinase activity was measured according to the procedure
described by Wirth and Wolf (1992) using carboxy-
methyl/chitin/Remazol/brilliant violet 5R as a substrate. For b-
1,3-glucanase assays, proteins from the crude extract (0.5 ml)
were precipitated with 80% ammonium sulfate and redissolved
in 0.5 ml of 50 mM sodium acetate buffer, pH 5.0. b-1,3-Glu-
canase activity was assayed according to Derckel and associ-
ates (1998) using laminarin (Flucka) as a substrate.

1126 / Molecular Plant-Microbe Interactions
Cell death determination.
The vital dye neutral red was used to test for cell death. Ac-
cumulation of the dye within the vacuole was observed by light
microscopy. Cells not stained by neutral red were considered
dead (Naton et al. 1996). A neutral red aqueous stock solution
of 1 mg ml
–1
was diluted for staining to a final concentration of
0.01% (wt/vol) in suspension buffer, pH 7.5. Aliquots of 1 ml
of cell suspension were examined in triplicates for each lami-
narin concentration.
Protection assays.
For B. cinerea infections, conidia were collected with 10 ml
of sterile water on a 10-day-old potato dextrose liquid culture,
filtered to remove mycelia and counted. For each treatment, 40
leaves were excised from 10-week-old grapevine vitro-plant-
lets and pre-incubated on a standard buffer (2 mM MES, 0.5
mM CaCl
2
, 0.5 mM K
2
SO
4
, pH 5.9) containing various con-
centrations of laminarin (0.01, 0.1, 0.2, 0.5, and 1.0 g/liter). Af-
ter 24 h, the leaves were wrapped in wet absorbing paper and
placed on plastic petri dishes. One needle-prick wound was ap-
plied to each leaf, and the fresh wounds were covered with 5-µl
drops of a suspension of 5 × 10
5
conidia/ml. Quantification of
disease development in grapevine leaves after inoculation with
B. cinerea was measured as average diameter of lesions formed
during infection.
For Plasmopara viticola infections, plants carrying 12 to 15
expanded leaves were first sprayed on both leaf surfaces with
solutions of laminarin at various concentrations (0.01, 0.1, 0.,5
and 1.0 g/liter). Héliosol (Samabiol), a tensio-active additive,
was added at 0.1% (vol/vol) in all sprayed solutions, including
water control. Fourteen plants were used per treatment. Treated
plants were kept in a growth chamber (25°C, 18 h of daylight,
60 to 70% relative humidity [RH]) for 10 days, then inoculated
by spraying a sporangial suspension of 2 × 10
4
sporangia/ml
onto the lower face of leaves. Inoculated plants were trans-
ferred to a dew chamber (100% RH, 20°C, darkness) for one
night and then in a growth chamber (23°C, 18-h photoperiod,
60 to 70% RH) for 8 days. Sporulation was induced afterward
by placing plants in the dew chamber as described above. Dis-
ease intensity was estimated 8 days post inoculation by meas-
uring the leaf area covered with mycelia and counting the spo-
rangia. In the latter method, leaves were washed with 50%
ethanol and sporangia were counted with a hematocytometer.
Both methods gave similar results.
Statistical analysis was performed by analysis of variance
and Duncan’s multiple range test.
ACKNOWLEDGMENTS
This work was supported by the Bureau Interprofessionnel des Vins
de Bourgogne and the Regional Councils of Burgundy and Cham-
pagne. We thank F. Baillieul for providing the sequences of primers
used for real time quantitative PCR and helpful discussions, K. Gould
for reviewing the English manuscript, and A. Klinguer for excellent
technical assistance.
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