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15
MOLECULAR ASPECTS OF GRAPEVINE-
PATHOGENIC FUNGI INTERACTIONS
E. Gomès
1
& P. Coutos-Thévenot
2
1
Institut des Sciences de la Vigne et du Vin,
UMR INRA 1287 Ecophysiologie et Génomique Fonctionnelle de la Vigne,
Domaine de la Grande Ferrade, 33883 Villenave d’Ornon, FRANCE
2
FRE CNRS 3091 Physiologie Moléculaire des Transports de sucres
Université de Poitiers, Bâtiment de Botanique,
40 Av. du Recteur Pineau, 86022 Poitiers Cedex, FRANCE
1. INTRODUCTION
Grapevine is a major and highly valuable fruit crop with roughly 2.25 mil ha
grown worldwide in 2007 (source: U.S. Food and Agriculture Organization).
Unfortunately, most of the premium cultivars used for winemaking, including
2. MAIN GRAPEVINE FUNGAL OR OOMYCETE-
INDUCED DISEASES
2.1. Foliage and berry diseases
2.1.1. Powdery mildew
Powdery mildew, caused by the ascomycete Uncinula necator (syn. Erysiphe
necator), an obligate biotrophic parasite of grapevine, is considered to be one of
407
the widely used European Vitis vinifera cultivars, are highly susceptible to several
pathogenic microorganisms including fungi, oomycetes, bacteria, phytoplasma
and viruses. In the past 15 years, the understanding of grapevine-pathogen in-
teractions has entered the molecular era and will most certainly constitute a
basis for future improvement of grapevine disease tolerance. After a brief pres-
entation of the main fungal- or oomycete-induced diseases, this chapter aims to
give an overview of some aspects of grapevine-pathogenic fungi or oomycete
interactions, at the molecular level. It includes an overview of resistance
genes
analogs, elicitors that induce defense reactions in grapevine, signalling path-
ways and gene activation.
K.A. Roubelakis-Angelakis (ed.), Grapevine Molecular Physiology & Biotechnology, 2nd edn.,
DOI 10.1007/978-90-481-2305-6_15, © Springer Science+Business Media B.V. 2009
E. Gomès & P. Coutos-Thévenot
408
the most important fungal diseases in viticulture worldwide. Symptoms appear
as grayish powdery or dusty patches of fungus growth on the upper side of the
leaves and on other green parts of the vines, leading to a decrease in photosyn-
thetic activity. In infected clusters, berries turn hard, brown, are smaller than
uninfected ones, and may split open. Besides direct loss of yield, infected ber-
ries fail to properly mature and significantly alter wine quality (Calonnec et al.
2004). Almost no V. vinifera cultivar is immune to U. necator, but other grape-
vine species such as Vitis labrusca, Vitis aestivalis or Vitis berlandieri as well
as Muscadinia rotundifolia possess various levels of resistance (Mullins et al.
1992).
2.1.2. Downy mildew
Downy mildew is caused by the oomycete Plasmopara viticola, also an obligate
biotrophic parasite of grapevine. It still is one of the most destructive grapevine
diseases in Europe and in the eastern half of the United States. Downy mildew
affects the leaves, fruit, and shoots of grapevines. First symptoms occur as yel-
lowish oily lesions on the leave upper surfaces; they rapidly give rise to white,
felt-like “downy” fungal mass on the corresponding lower sides of the leaves.
Infected berries first appear grayish then turn “downy” during pathogen sporula-
tion. Yield losses occur through death of leaf tissue, low-quality fruit, and
weakened young shoots.
2.1.3. Grey mould
Grey mould is the third major fungal disease affecting grapevine foliage and
berries, particularly severe in areas where wet weather occurs between véraison
and harvest. It is due to the broad host-range necrotrophic ascomycete Botrytis
cinerea, which causes necrotic spots on leaves, total or partial destruction of the
bunches before flowering and later on, rotting of berry clusters. Besides losses
of fruit yield, infection of berries by B. cinerea also deteriorates wine quality by
inducing the appearance of mushroom earthy off-odors (La Guerche et al.
2006).
Grey mould, powdery and downy mildews are controlled at the vineyard
mostly by chemical spraying, sterol demethylation inhibitors or quinone outside
inhibiting fungicides. However, besides negative environmental impacts, patho-
gens develop resistances towards these pesticides (Délye et al. 1997, Leroux
et al. 1998, Chen et al. 2007).
GRAPEVINE-PATHOGENIC FUNGI INTERACTIONS
409
2.2. Wood decay diseases
2.2.1. Eutypa dieback
Eutypa dieback is a wood decay disease caused by the ascomycete Eutypa lata.
Symptoms do not usually appear until vines are at least six years old. Shoot
symptoms are most evident during the beginning of the spring, with shoot aris-
ing from infected trunks being stunted with small chlorotic leaves (Moller and
Kasimatis 1978). Berries fail to develop or develop very poorly, inducing yield
losses ranging from 30 to 60% on highly susceptible cultivars (Munkvold and
Marois 1994). Eutypa dieback shoot symptoms are always accompanied by a
canker, which often appears V-shaped in a cross-section of the perennial wood.
Cankers progress toward the trunk, killing the distal portions of the vine, and
eventually, the entire vine may die in an average period of 10 years after the ini-
tial infection (Pascoe 1999). Currently, there is no cure for Eutypa dieback.
2.2.2. Esca
Esca, a.k.a. ‘apoplexy’ or ‘lack measles’ is a complex trunk disease involving at
least five fungi, Fomitiporia punctata, Stereum hirsutum, Phaeoacremonium
aleophilum, Phaeomoniella chlamydospora, and E. lata, that obstruct the vascu-
lar system (Larignon and Dubos 1997). It affects both young and older vines.
Cross section in infected trunks shows a central soft, white necrosis (touch-
wood), surrounded by a brownish hard zone. Esca develops slowly in the
grapevine until the plant exhibits a sudden apoplectic decline, eventually killing
the vine within a few days. No chemical is currently available to control esca.
3. GRAPEVINE RESISTANCE GENES ANALOGS
The European grape V. vinifera, which accounts for about 90% of worldwide
grape production for wine making, shows very low disease resistance. On the
other hand, wild American species, such as Muscadinia rotundifolia, are resis-
tant to various pathogens, including U. necator (powdery mildew), P. viticola
(downy mildew) or Xylella fastidiosa (Pierce’s disease; Olmo 1986). Enhancing
the resistance of cultivated grapevine to diseases therefore constitutes a major
goal for breeders (Bisson et al. 2002).
3.1. R-genes and the plant immune system
In the past decade, our understanding of the molecular basis of plant disease re-
sistance has increased steadily. Plants lack the adaptive immune system, which
is the privilege of the vertebrates. To detect and successfully ward off pathogens,
E. Gomès & P. Coutos-Thévenot
410
plants rely solely on innate immunity of each cell and on systemic signals ema-
nating from infected sites (Dangl and Jones 2001). It is now widely admitted
that the plant immune system uses two distinct defense systems (Chisholm et al.
2006). A first line of defense uses transmembrane pattern recognition receptors
to recognize slowly evolving pathogen-associated molecular patterns (PAMPs),
such as bacterial flagellin, lipopolysaccharide or fungal/oomycete cellulose
binding elicitor proteins to activate basal defenses (Ausubel 2005). Pathogens,
however, have evolved to acquire mechanisms that help them to by-pass
PAMP-triggered immunity. Plants struck back by setting up a second line of de-
fense that relies on proteins which recognize pathogen effectors or modifica-
tions of their cellular targets, a mechanism called effector-triggered immunity.
Both PAMP- and effector-triggered immunity rely on the so-called resistance
genes (R-genes).
R-genes can be grouped into 5 classes (Ellis et al. 2000, Dangl and Jones
2001) encoding for: (i) cytoplasmic serine/threonine kinases such as the Pto
gene in tomato (Fig. 1A); (ii) extracellular leucine-rich re-peats (LRRs) proteins
anchored to a transmembrane domain, as exemplified by the tomato Cf-9 gene
(Fig. 1B); (iii) receptor-like kinases (RLKs) with an extracellular LRR and an
intracellular serine/threonine kinase (i.e. the rice gene Xa21, Fig. 1C), (iv) pro-
teins with an N-terminal transmembrane anchor and a cytoplasmic coiled-coil
(CC) domain encoded by the Arabidopsis RPW8 (Resistance to Powdery Mil-
dew 8, Fig. 1D) genes; and (v) proteins with a nucleotide binding site (NBS),
Fig. 1. Schematic representation of the R-genes products. (A) Intracellular Pto-like serine/
threonine kinases (Kin). (B) Cf-9-like trans-membrane-anchored Leucine-Rich repeats (LRR).
(C) Xa21-like proteins with an intracellular serine/threonine kinase domain and an extracellular
LRR domain. (D) RPW8 protein with a transmembrane-anchored coiled-coil (CC) domain.
(E) Intracellular NBS-LRR proteins with an LRR domain in their C-terminus, a nucleotide bind-
ing site (NBS) and either a toll/interleucine-1 receptor (TIR) or a CC domain in their N-terminus.
Adapted from Dangl and Jones (2001), with permission.
Kin
LRR
LRR
Kin
CC
CC TIR
NBS NBS
LRR
LRR
AB C D E
GRAPEVINE-PATHOGENIC FUNGI INTERACTIONS
411
and a LRR domain in their C-terminus (NBS-LRR proteins, Fig. 1E). That later
class of R-genes can be sub-divided in two sub-classes based on their
N-terminal domain (Bai et al. 2002), which can either be a toll/interleukine-1
receptor (TIR-NBS-LRR, specific to dicotyledonous species) or a coiled-coil
domain (CC-NBS-LRR, present in all angiosperms).
3.2. R-genes in grapevine
NBS-LRR genes are the most largely represented R-genes in plant genomes, as
exemplified by the 149 genes found in the Arabidopsis genome (Meyers et al.
2003), or the 480 in the rice genome (Zhou et al. 2004). Grapevine is no excep-
tion and in a survey of the grape cv Pinot Noir draft genome Velasco et al.
(2007) detected 233 genes encoding for proteins containing both NBS and LRR
domains (InterPro IPR001611 and IPR002182, respectively). Among them, 84
genes belong to the CC-NBS-LRR subfamily while the TIR-NBS-LRR subfam-
ily includes 37 genes. Additionally, 112 truncated NBS-LRR encoding genes
are also present in the grape genome.
A complete inventory of defense-related RLKs genes is not easy to make,
because these proteins are also implicated in a wide range of developmentally-
related signalling pathways (Shiu and Bleecker 2001). Nevertheless, 53 genes
encoding putative RLKs have been identified in grapevine (Di Gaspero and
Cipriani 2003), eight of them being closely related to the Pto cytosolic protein
from tomato and 3 to the products of the R-genes Xa21 from rice and its Arabi-
dopsis homolog FLS2.
To date, no true homolog of the Arabidopsis RPW8 resistance genes have
been identified in grapevine. Two genes, however, VRP1-1 and VRP1-2 (for
Vitis Resistance to Plasmopara 1-1 and 1-2) encode CC-NBS-LRR with a
RPW8 domain in their N-termini (Kortekamp et al. 2008). Such chimeric resis-
tance proteins could link the pathogen effector-triggered (gene-for-gene) re-
sponses attributed to NBS-LRR proteins with the basal general resistance re-
sponses credited to RPW8 proteins
( Xiao et al. 2005, Wang et al. 2007).
Interestingly, VRP1-1 and VRP1-2 sequences show nucleotide polymorphism that
led to amino acid substitutions at several positions when compared in the downy
mildew resistant Vitis accession Regent and the susceptible Pinot noir (Kortekamp
et al. 2008), making them potentially interesting to breed resistance.
3.3. Cluster of R-genes map to chromosomal region of grapevine
genetic disease resistance
R-genes, particularly the NBS-LRR class, are arranged in clusters in plant
genomes, a physical disposition that generates sequence variation and gene
family expansion at a high rate, a point that is crucial to generate new resistance
E. Gomès & P. Coutos-Thévenot
412
specificities (Bergelson et al. 2001, Meyers et al. 2003, Zhou et al. 2004). In
grapevine, TIR-NBS-LRR gene clusters are preferentially located on linkage
group (LG) 18, CC-NBS-LRR gene clusters on LG 9 and 14 and truncated NBS-
LRR on LG 12 and 13
( Velasco et al. 2007, Moroldo et al. 2008). RLKs are
more evenly dispersed in the grape genome, with LG 14 scoring the highest
number of RLK coding genes (Moroldo et al. 2008).
In agreement with the role of R-genes in plant innate immunity, several
clusters of NBS genes map to chromosomal regions where genetic resistance to
bacterial, fungal or oomycete-induced diseases were previously assigned (Di
Gaspero et al. 2007, Velasco et al. 2007). The Run1 locus (Resistant to Un-
cinula necator 1), originating from M. rotundifolia (Pauquet et al. 2001), which
confers resistance to powdery mildew, has a counterpart in the Vitis genome
physically located on LG 12, in a region which contains several copies of R-
genes (Barker et al. 2005). Additional loci for powdery mildew resistance have
been reported on LG 15 and 14 in Vitis hybrids (Dalbo et al. 2001, Fischer et al.
2004). In the same region of LG 14, a primary locus for resistance to Pierce’s
disease causal agent, Xylella fastidiosa, was identified in the wild grape Vitis
arizonicai (Krivanek et al. 2006). Quantitative trait loci for downy mildew re-
sistance have been mapped with SSR markers to the distal part of LG 18
(Fischer et al. 2004), and in the middle of LG 7 (Grando et al. 2003) in Vitis re-
sistant accessions, nearby regions where NBS-LRR genes are clustered. Another
major determinant responsible for resistance to P. viticola has been identified
on LG 12 (Merdinoglu et al. 2003). In conjunction with the knowledge of the
grape genome sequence, the availability of linkage maps based on transferable
molecular markers (reviewed by Doligez et al. 2006) will constitute valuable
tools for pathogen resistance breeding in premium Vitis cultivars.
4. ELICITORS ACTIVE ON GRAPEVINE
Several molecules coming from microorganisms, plants or algae have been
characterized as elicitors. These molecules, which encompass lipids, oligosac-
charides and proteins, trigger defense responses in plants, such as the hypersen-
sitive response (HR), the localized acquired resistance (LAR) or the systemic
acquired resistance (SAR). Besides, some molecules coming from non-
pathogenic microorganisms potentiate ISR (Induced Systemic Resistance) in
plants, leading to tolerance against many pathogens. These signal molecules, of-
ten recognized by a receptor (see R-genes, § 3), offer several possible applica-
tions as natural inducers of defense and tolerance in plants.
GRAPEVINE-PATHOGENIC FUNGI INTERACTIONS
413
4.1. Oligosaccharide elicitors
Several elicitors such as β-1,3-glucans or α-1,4-oligogalacturonides are known
to be active in many plant species. In grapevine some oligosaccharides appear
to be efficient, like β-1,4-cellodextrins (Aziz et al. 2007), cyclodextrins
(Morales et al. 1998, Bru et al. 2006), laminarin extracted from algae (Aziz
et al. 2003) and induce tolerance against B. cinerea or P. viticola. Sulfated glu-
cans like β-1,3-glucan sulfate enhance tolerance to P. viticola (Trouvelot et al.
2008). In addition, two novel oligosaccharidic elicitors were purified from B.
cinerea. These molecules, obtained from crude mycelium cell wall extracts and
4.2. Lipid elicitors
If several lipid molecules are known to act as elicitors in plants, in grapevine the
main lipidic elicitor described up to now is the ergosterol molecule. This sterol,
which is typical of fungi, was described as an inducer of a specific set of de-
fense-related genes in tobacco and associated signal transduction pathways
(Kasparovsky et al. 2004, Lochman and Mikes 2006, Rossard et al. 2006).
Some PR-proteins (PR-14) and enzymes of the stilbene biosynthesis pathway
are highly induced in grapevine by ergosterol treatment, most probably through
the activation of WRKY trans-activator factors (Gomès et al. 2003, Laquitaine
et al. 2006, Marchive et al. 2007). The putative specific receptors of ergosterol
remain to be identified.
4.3. Proteinaceous elicitors
In terms of proteinaceous elicitors, two major lines of work have emerged these
last years. Poinssot et al. (2003) described an endo-polygalacturonase secreted
in the culture medium of B. cinerea, which is able to trigger full-scale early de-
fense reactions in grapevine cell suspension cultures. Apparently, elicitor effect
is not due to the enzyme activity. More recently, it was demonstrated that oli-
culture filtrate preparations, were named Botrycin and Cinerein, respectively
(Repka et al. 2001a, Repka 2002, 2006). In all cases, treatment with these elici-
tors triggered the classical PR (Pathogenesis Related) proteins accumulation,
reactive oxygen species (ROS) production, as well as Ca
2+
, jasmonic acid (JA)
and salicylic acid (SA) signalling pathways.
gandrin, an elicitin of Pithium oligandrum, enhances Vitis tolerance towards
B. cinerea (Mohamed et al. 2006). These results are of interest because elicitins
were previously described to be active on tobacco but not on other plants
(Ponchet et al. 1999). The fact that an elicitin could induce protection against
E. Gomès & P. Coutos-Thévenot
414
fungal pathogens without HR response, but with modifications of the redox
status of the cells, is indeed a very innovative concept
.
5. EARLY CELLULAR EVENTS IN DEFENSE REACTIONS
Numerous studies of early plant defense reactions have been made in the last
ten years. In Fig. 2 the principal steps, keys of the knowledge about this signal
transduction pathway, are summarized. In model plants such as tobacco or
Arabidopsis all these steps including perception, calcium flux activation, ROS
synthesis, MAPKs (mitogen-activated protein kinases) or phosphatases activa-
tion are well characterized. In grapevine, the amount of information available,
regarding early events of defense reactions, was less developed until recently
(Busam et al. 1997, Jacobs et al. 1999). In the past few years, several publica-
tions aimed to decipher defense-related early signalling events in
grape-vine
using model cell suspension cultures or entire plants (Repka 2006, Vandelle
et al. 2006).
Fig. 2. Early cellular events triggered by pathogen recognition, as exemplified by the case of the
signalling cascades induced on tobacco cells by cryptogein, an elicitin from Phytophthora crypto-
gea. Reprinted from Garcia-Brugger et al. (2006), with permission.
GRAPEVINE-PATHOGENIC FUNGI INTERACTIONS
415
As described in paragraph 4, many elicitors have been characterized and
some putative receptors identified in the Pinot Noir genome sequence. After the
classical step of pathogen (or elicitor) perception, most of the crucial signalling
events have been identified in grapevine. It seems that Ca
2+
influx is the first
event occurring after elicitation, by modulation of plasmamembrane
Ca
2+
channels. Later on, NO
y
is synthesized and mobilizes internal stores of Ca
2+
.
Then come ROS (O
2
y
-
, H
2
O
2
) production, MAPK and phosphatase activities that
have also been evidenced in elicited plants.
A second class of signalling compounds (JA, SA and ethylene) are pro-
duced as endogenous signalling molecules and elicit pathogen protection proc-
ess. Finally, a pool of defense genes gets activated, including PR proteins en-
coding genes, as well as phenylpropanoid and stilbene biosynthesis genes
(Repka 2006, Vandelle et al. 2006). A particular class of PR-proteins is in-
duced, the lipid transfer proteins (LTP, PR-14). These secreted proteins are able
In conclusion, it seems that grapevine species possess all the signalling
elements to respond to a pathogen attack. Nevertheless, it is clear that there are
some differences between wild grapevine species and the European V. vinifera.
For example, some R genes are absent from V. vinifera (Run1) and could ex-
plain the sensitivity of premium cultivars to pathogenic fungi (Barker et al.
2005).
6. GRAPEVINE PR-PROTEIN GENES
As already mentioned, one of the major steps in plant defense reactions is the
synthesis of a particular class of proteins classically termed as ‘PR’ proteins.
These proteins were distributed in 1999 in 11 classes. Recently, Van Loon et al.
(2006) published a broader classification with 17 classes. The PR-proteins are
defined as proteins that are not expressed in plants without pathogen interaction
or largely induced during infection. For a protein to be classified as a PR-
protein, it is necessary that its induction is described for two different
plant/pathogen systems in two different laboratories. Not all the PR-proteins
classes have been described in grapevine so far.
A putative sequence of a V. vinifera PR-1 protein was identified and
cloned by Bertsch et al. (2003). The expression kinetics of a PR-1 defense-
related gene is strongly dependent on the nature of elicitor used (Repka 2001b).
Comparison of PR-1 expression in grapevine cell cultures after inoculation with
a host and a non-host pathogen revealed a high PR-1 expression rate 3 weeks
post-inoculation in V. vinifera cv Riesling and Vitis riparia cv Gloire de Mont-
pellier, even if pathogens development was not blocked. Thus, the role of PR-1
to bind JA, at least in vitro and trigger protection against B. cinerea (Girault
et al. 2008). LTP-JA complexes could be one element of SAR signalling, lead-
ing to global protection of the plant against pathogens (Grant and Lamb 2006).
E. Gomès & P. Coutos-Thévenot
416
expression in impeding the downy mildew pathogen remains equivocal. It
seems that expression of PR-1 genes is a general stress response in some grape-
vine culture systems (Wielgoss and Kortekamp 2006). Three PR-1-like proteins
were found to accumulate in grapevine leaves after infection by U. necator. Ex-
pression of these proteins was also induced by elicitor treatments in grapevine
cell suspension culture (Repka et al. 2000).
U. necator, the causal agent of grapevine powdery mildew, induces ex-
pression of chitinases and ß-1,3-glucanases in leaves and berries in various
grapevine cultivars, including susceptible ones. Indeed, Jacobs et al. (1999)
showed that the hydrolytic activity was directly related to the severity of infec-
tion at the pathogen location. PR-2, -3 and -5 were also observed in infected
berries at pre-véraison stage and were highly induced by ethephon treatment.
These results demonstrate a paradox: even if these classes of PR-proteins are
expressed during pathogen invasion this does not offer complete protection
against U. necator. Probably, some other more specific proteins are necessary.
Alternatively, the key of the protection might be more determined by the pres-
ence or the absence of R-genes (resistance genes).
Furthermore, the diversity of PR proteins expressed decreases during
grape maturation (Monteiro et al. 2007) and could explain the enhanced suscep-
tibility of the berries during the last stages of ripening. Accordingly, it was
demonstrated that constitutive expression in transgenic V. vinifera of thaumatin-
like protein protects grapevine plants against anthracnose (Jaysankar et al.
2003). These plants, however, were not protected against other fungi. In addi-
tion, induction of chitinase genes in V. vinifera depends on the infecting patho-
gen, but also the type of chitinase is different in a compatible or incompatible
interaction (Robert et al. 2002). The selective expression of specific chitinases
might be a reliable indicator of the SAR response in V. vinifera (Busam et al.
1997).
A grapevine class 10 PR-protein was cloned from V. vinifera leaves infil-
trated with the incompatible bacterial pathogen Pseudomonas syringae pv Pisi
(Robert et al. 2001). To our knowledge, it is the only example of PR-10 gene
characterized in grapevine. The accumulation of the corresponding mRNA was
The PR-2, -3, -4 and -5 classes are better documented in grapevine. In a
susceptible V. vinifera cv, such as Riesling, inoculated with Pseudoperonospora
cubensis (downy mildew of cucumber), a non-host pathogen in grapevine,
ß-1,3-glucanases (PR-2) and chitinases (PR-3 and -4) are largely accumulated in
comparison with a host situation (P. viticola). Following treatment with
P. cubensis, sporulation intensity was significantly reduced in Riesling after
subsequent inoculation with P. viticola (Kortekamp 2006). Several PR-proteins
are expressed in berries at maturity and liquid chromatography-mass spectrometry
analysis of grape juice revealed the presence of several PR-3 and PR-5 (thaumatin-
like) isoforms with different molecular masses, as a function of the varieties
(Hayasaka et al. 2001).
GRAPEVINE-PATHOGENIC FUNGI INTERACTIONS
417
observed from 3 to 96 h post-inoculation and was followed by the accumula-
tion, between 24 and, at least, 96 h after inoculation, of the encoded polypep-
tide, detected by immunoblotting.
The story of the PR-14 family is more complicated. These proteins (Lipid
Transfer Proteins) were shown to be involved in several physiological proc-
esses. It is now quite clear that some isoforms are clearly involved in defense
reaction signalling process (Maldonado et al. 2002, Blein et al. 2002, Grant and
Lamb 2006). Several isoforms of LTP have been described in grapevine
(Coutos-Thévenot et al. 1993). Some of them were induced by fungal elicitor
treatments (Gomès et al. 2003). Ergosterol-induced protection of grape against
B. cinerea relies on the expression of a type I lipid transfer protein, which is
mediated by a WRKY trans-activating protein (Laquitaine et al. 2006). In addi-
tion, Girault et al. (2008) demonstrated that some grapevine LTP were able to
bind JA, and that exogenous application of a LTP-JA complex induces protec-
tion of grapevine towards infection by B. cinerea. All LTPs, however, are not
defense-related. Other isoforms seem to be involved in other physiological
process, like somatic embryo development and epidermal layer formation. Ac-
cordingly, over-expression of the VvLTP1 gene interferes with somatic embryo
development in grapevine and abolishes the bilateral symmetry of embryos
(François et al. 2008).
The last classes of PR-proteins that have been described in grapevine are
the germin and germin-like proteins (PR-15 and -16). Recently, 7 members of
the grapevine germin-like multigenic family were cloned in V. vinifera
According to the literature, PR-6, -7, -8, -9, -11, -12, and -13, have not
been described in grapevine yet. Nevertheless, using the sequences of the first
member of each class to be published (Van Loon et al. 2006), a BLAST analy-
sis indicate the presence of putative homolog genes in the Pinot Noir genome
(http://www.genoscope.cns.fr/externe/
GenomeBrowser /Vitis/) for all these PR-
protein classes, with the noticeable exception of PR-13 (thionins). Table 1
summarizes the BLAST results, by presenting for each PR-protein class the first
6 transcripts detected in the database (only 3 where detected for PR-12, de-
fensins). Such an analysis is of course far from being exhaustive and is just in-
tended to point the need of additional studies in the future to better characterize
PR-protein families in grapevine.
(Godfrey et al. 2007). Among them, one gene, VvGLP3 (V. vinifera germin-like 3),
have no basal expression level and is strongly induced by powdery mildew infec-
tion. Another member of the family, VvGLP7, responds to both P. viticola and
B. cinerea infection. Some germin-like proteins exhibit oxalate oxidase or
superoxide dismutase activities, but their exact role in plant defense reactions is
far from being elucidated.
E. Gomès & P. Coutos-Thévenot
418
Table 1. Putative homolog genes for PR-6, 7, 8, 9, 11, 12 and 13 in the Pinot noir genome. For
each PR-protein class, the grapevine genome database was probed with the sequence a typical
member, as described by Van loon et al. (2006).
PR-protein
class
Typical member Properties Putative grape homolog
genes*
Linkage
group
6 Tomato inhibitor I Proteinase inhibitors
GSVIVT00020160001
GSVIVT00020161001
GSVIVT00029370001
5
5
13
7 Tomato P
69
Endoproteases
GSVIVT00001054001
GSVIVT00001055001
GSVIVT00001051001
GSVIVT00001053001
GSVIVT00001034001
GSVIVT00001056001
2
2
2
2
2
2
8
Cucumber chiti-
nase
Type III Chitinases
GSVIVT00006464001
GSVIVT00026961001
GSVIVT00026949001
GSVIVT00026950001
GSVIVT00006463001
GSVIVT00020672001
Unknown
15
15
15
Unknown
14
9
Tobacco lignin-
forming peroxi-
dase
Peroxidase
GSVIVT00024722001
GSVIVT00025396001
GSVIVT00024724001
GSVIVT00024717001
GSVIVT00037460001
GSVIVP00018771001
6
8
6
6
8
12
11 Parsley PR-1 Ribonuclease-like
GSVIVP00012304001
GSVIVP00012300001
GSVIVP00012296001
GSVIVP00005601001
GSVIVP00005604001
GSVIVP00005606001
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
12 Radish Rs-AFP3 Defensins
GSVIVT00035146001
GSVIVT00014577001
GSVIVT00002075001
1
18
19
13
Arabidopsis
THI2.1
Thionins
None detected
* Putative grape PR-proteins transcripts are designated by their Genoscope annotation number
(http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/).
GRAPEVINE-PATHOGENIC FUNGI INTERACTIONS
419
7. GRAPEVINE PHYTOALEXINS BIOSYNTHESIS AND
METABOLISM
7.1. Stilbene synthase genes
The knowledge of grapevine phytoalexin metabolism (Fig. 3) has increased
vastly in the past decade. In woody plants, the classical phenylpropanoid path-
way is split at the coumaroyl-CoA step by the stilbene synthase activity, which
synthesizes resveratrol by condensation of 3-malonyl-CoA with one molecule
of coumaroyl-CoA (Langcake and Pryce 1976, Langcake and Pryce 1977, Pont
and Pezet 1990, Pezet and Pont 1995). This enzyme activity belongs to a multi-
genic family and has been already characterized by Wiese et al. (1994).
The first genes coding a Vitis stilbene synthase (VST) was cloned by
Melchior and Kindl (1990), and afterwards several other genes were character-
ized (Richter et al. 2005). The recent publication of the grapevine Pinot Noir
genome revealed the presence of 21 putative stilbene synthase genes, essentially
on LG 10 and 16 (Velasco et al. 2007). This number fits nicely with a recent
stilbene synthase gene expression analysis in infected grapes leaves (Richter
et al. 2005). Stilbene synthases exhibit a high degree of homology with chal-
cone synthase and are known to be induced by several stimuli, including UV
light, which is the major abiotic inducer (Adrian et al. 2000, Bonomelli et al.
2004).
Stilbene synthase genes are down-regulated during grape berry ripening,
due to a competition between stilbene and chalcone synthase activities; that lat-
ter enzyme being activated after véraison for anthocyan and flavonoid accumu-
lation in pellicula. A large group of biotic inducers are able to promote stilbene
synthase expression and microorganisms were shown to have a direct effect on
resveratrol accumulation. B. cinerea is the most common fungus able to act on
phenylpropanoid pathway ( Liswidowati et al. 1991, Bais et al. 2000, Keller et
al. 2003). The level of the response, however, depends largely on the cultivar
(Gabler et al. 2003), and it is possible to class the level of tolerance of cultivars
in the field in regard to the capacity to accumulate resveratrol after UV light in-
duction, as summarized in the Table 2 (Coutos-Thevenot et al. 2001).
Several other fungi were found to induce resveratrol production. Asper-
gillus induces expression of VST genes (Jean-Denis et al. 2006) and more prob-
lematic pathogens like three of the fungi associated with Esca, promote stilbene
production (Bruno and Sparapano 2006a, 2006b). Bacteria-like Pseudomonas
syringae pv Pisi seem also to act as inducers during HR of grapevine (Robert
et al. 2001). Various studies have demonstrated the ability of purified elicitors
E. Gomès & P. Coutos-Thévenot
420
to induce VST gene activation or resveratrol synthesis. Ergosterol, a specific
sterolof fungi and a non-specific elicitor, is highly efficient (Laquitaine et al.
2006). The endo-polygalacturonase BcPG1 produced by B. cinerea induces VST1
mRNA accumulation 4 hours after treatment of grapevine cell suspension and is
Fig. 3. Biosynthesis pathway of grapevine stilbenes Reprinted from Coutos-Thevenot et al. (2001)
with permission
GRAPEVINE-PATHOGENIC FUNGI INTERACTIONS
421
more active than oligogalacturonate elicitors (Poinssot et al. 2003). Several other
molecules are also effective inducers of resveratrol production in grapevine (Borie
et al. 2004, Laura et al. 2007), like oligogalacturonates (Aziz et al. 2004), β-1-3
glucane sulfate (Trouvelot et al. 2008), as well as chemicals like aluminium chlo-
ride (Adrian et al. 1996) or benzothiazole (Iriti et al. 2004, see also Chapter 12 in
this book). Even if its mechanism of action is not well understood, resveratrol in-
hibits the growth of several fungi or oomycetes. Microbiologic tests revealed a
good effect on Botrytis and Eutypa lata mycelium growth (Adrian et al. 1997,
Coutos-Thevenot et al. 2001), P. viticola (Pezet et al. 2004) and Venturia in-
aequalis growth (Schulze et al. 2005). If resveratrol is active, its metabolites like
pterostilbene (methylated form) and viniferins (oligomer metabolites) seem to be
much more efficient (Pezet et al. 2004). On the other hand, the glycosylated form
(piceid) seems to be less active and could be a soluble storage form (Pezet et al.
2004). It is suspected that enzymes, like methyl transferases and oxidases, could be
involved in these mechanisms.
Table 2. Effects of Botrytis infection or UV treatment on resveratrol accumulation by various
grapevine cultivars. Data are means of four independent experiments ± standard error. Nd: not de-
tected. Reprinted from Coutos-Thévenot et al. (2001), with permission.
Variety / cultivars
Control
(not induced)
Botrytis
Resveratrol
(µg.g
-1
DW)
UV light
Resveratrol
(µg.g
-1
DW)
Rupestris nd nd 350 (± 115)
41B Rootstock nd 112 (± 30) 240 (± 120)
Ugni blanc 479 nd 86 (± 45) 210 (± 74)
Pinot Noir 386 nd 103 (± 31) 87 (± 49)
Folle blanche nd 101 (± 16) 38 (± 11)
7.2. Hormones and signalling
Signalling pathways involved in stilbenes accumulation in grapevine are less
clear-cut. It seems that stilbene synthase activation is probably due to a cross-
talk between several pathways. The role of JA and methyl-JA are well docu-
mented with a very efficient induction ( Zhang et al. 2002, Curtin et al. 2003,
Larronde et al. 2003, Repka et al. 2004, Tassoni et al. 2005, Vezzulli et al.
2007), but the one of SA is less understood. It seems that SA acts on the
phenylpropanoid pathway by inducing phenylalanine ammonia lyase and stil-
bene synthase genes expression (Chen et al. 2006, Wen et al. 2005, Wen et al.
2008). Ethylene also appears be involved (Grimmig et al. 2002).
E. Gomès & P. Coutos-Thévenot
422
7.3. Use in transgenic plants
After the fist evidence that tobacco plants, which do not naturally produce stil-
benes, become tolerant to B. cinerea when transformed with p35S-VST1 chi-
meric gene (Hain et al. 1993), the idea of generating transgenic grapevine that
over-express stilbene synthase rapidly gained ground. The 41B rootstock was
transformed with the PR10 promoter, which is highly inducible by B. cinerea
infection, fused to the VST1 coding sequence. Some transgenic clones showed a
high level of in vitro tolerance to Botrytis and could be also tolerant to E. lata
(Coutos-Thevenot et al. 2001). More recently, these results were confirmed by
several groups on various plant species, like grapevine (Fan et al. 2008), hop
8. CONCLUSIONS
In the last 15 years, our understanding of molecular aspects of grapevine-fungi
interactions has increased largely. The recent publication of the Pinot Noir ge-
nome will undoubtedly be a valuable tool for future studies. However, a fair
deal of work remains to be done, to precisely decipher and finely characterize
the different steps of the pathogen detection by the plant and the subsequent ac-
tivation and establishment of defense reactions.
This applies, for example, to the characterization of the not yet described,
but present in the grapevine genome, PR-protein families; or to the comparative
studies of the genetic diversity of resistance genes and other defense-related
genes, in the various cultivated and wild grapevines. The global outcome of the
knowledge of grapevine defense reaction studies at the molecular level has al-
ready started to be integrated to breeding experiments, either through genetic
transformation or through marker-assisted selection. It is reasonably safe to bet
that this tendency will increase in the future.
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