Download Oligosaccharide signalling for defense responses in plant

Physiological and Molecular Plant Pathology (2001) 59, 223±233
doi:10.1006/pmpp.2001.0364, available online at http://www.idealibrary.com on
MINI-REVIEW
Oligosaccharide signalling for defence responses in plant
N . S H IB U YA and E . M IN A M I
Biochemistry Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan
(Accepted for publication October 2001)
Keywords: elicitor; receptor; OGA; chitin; chitosan; beta-glucan; signal transduction; ion ¯ux; protein
phosphorylation; defence response.
INTRODUCTION
Higher plants have the ability to initiate various defence
reactions such as the production of phytoalexins,
antimicrobial proteins, reactive oxygen species, and
reinforcement of cell walls when they are infected by
pathogens such as fungi, bacteria and viruses. If these
reactions occur in a timely manner, the infection will not
proceed further. However, if the defence reactions occur
too late or are suppressed, the infection process will
proceed successfully [89]. Thus, it is critically important
for plants to detect infecting pathogens e€ectively and
deliver such information intracellularly/intercellularly to
activate their defence machinery.
It is believed that the detection of pathogens is
mediated by chemical substances secreted/generated by
the pathogens. Various types of such compounds (elicitor
molecules) including oligosaccharides, (glyco)proteins,
(glyco)peptides and lipids, have been shown to induce
defence responses in plant cells and their involvement in
the detection of ( potential) pathogens in plant has been
discussed [5, 24, 34]. Oligosaccharides derived from
fungal and plant cell wall polysaccharides are one class of
well characterized elicitors that, in some cases, can induce
defence responses at a very low concentration e.g. nM. In
view of their well-de®ned chemical nature and the
presence of highly sensitive perception systems in plants,
some of these elicitors have provided good model systems
to study how plant cells recognize such chemical signals
and transduce them for activation of the defence
machinery. These studies include structure±activity
relationships of the elicitor molecules, characterization
of the corresponding receptors and analysis of signal
Abbreviations used in text: A9-C, anthracene 9-carboxylic
acid; MBP, mannan binding protein; OGAs, oligogalacturonides; TLR, toll-like receptors; 2-D PAGE, two-dimensional gel
electrophoresis.
0885-5765/01/110223+11 $35.00/00
transduction cascades and elicitor-responsive genes.
Several reviews [5, 25, 34, 40] have already been
published on these subjects and in this review the authors
try to focus mostly on recent progress.
CHARACTERISTICS OF OLIGOSACCHARIDE
ELICITORS
b-glucan oligosaccharides
Oligosaccharide elicitors derived from the b-glucans of
pathogenic Oomycetes, such as Phytophthora sojae, have
been very well characterized. A doubly-branched heptab-glucoside that was generated from P. sojae glucan by
partial acid hydrolysis was shown to be a very active
elicitor for glyceollin biosynthesis in soybean cotyledon
cells [86]. From detailed studies with a set of naturally
obtained as well as synthetic oligosaccharides, it has been
shown that soybean cells recognize speci®c features of the
hepta-b-glucoside structure, including all three nonreducing terminal glucosyl residues and their spacing
along the backbone of the molecule [10]. A partial
hydrolysate of the P. sojae b-glucan also acts as an active
elicitor on various plant cells belonging to the family
Fabaceae, indicating the presence of similar perception
systems in these plants [14]. In the case of tobacco cells,
however, the hepta-b-glucoside did not act as an elicitor,
but linear b-1,3-linked glucooligosaccharides (laminarioligosaccharides) were active elicitors [47]. Interestingly,
a cyclic 1,3- 1,6-linked b-glucan secreted by a symbiotic
bacterium, Bradyrhizobioum japonicum, was reported to act
as a suppressor for the induction of phytoalexin
biosynthesis in soybean by b-glucan elicitor [62].
There has been little information on the b-glucan
fragment elicitor that acts on monocots. Inui et al. [38]
reported the induction of chitinase and PAL activity in
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cultured rice cells by laminarihexaose. Recently Yamaguchi et al. [104] reported that a pentasaccharide (in the
reduced form) puri®ed from an enzymatic digest of the bglucan from the rice blast fungus, Magnaporthe grisea
(Pyricularia oryzae), shows potent elicitor activity to induce
phytoalexin biosynthesis in suspension-cultured rice cells.
The elicitor-active glucopentaose induced phytoalexin
biosynthesis in rice cells at 10 nM concentrations. Structural elucidation of the elicitor-active glucopentaose
showed a quite contrasting structure compared to the
hepta-b-glucoside that is active on soybean. In the case of
the hepta-b-glucoside elicitor from P. sojae, the backbone
is a 1,6-linked b-glucooligosaccharide with branches at
the 3-position of two 6-linked glucosyl residues. In the case
of the elicitor-active b-glucopentaose from M. grisea, the
backbone oligosaccharide is 1,3-linked and branched at
the 6-position of a 3-linked residue. Two related
glucopentaoses that di€er only in the position of the
glucosyl stub showed little elicitor activity, indicating a
strict recognition of the active oligosaccharide in rice cells.
Comparison of the elicitor activity of the b-glucan
fragments from M. grisea and synthetic hexa-b-glucoside,
the minimum structural unit for activity of the hepta-bglucoside in soybean, using both suspension-cultured rice
cells and soybean cotyledons, indicated that each glucan
fragment can only be recognized by the corresponding
host plants [104]. Although the structural requirement for
the recognition by the rice cells must be examined in more
detail, these results indicate clear di€erences in the speci®city of the corresponding receptors in rice and soybean.
It will be interesting from an evolutionary viewpoint to see
whether various plants can be grouped depending on
their abilities to recognize di€erent b-glucan elicitors.
Chitin and chitosan oligosaccharides
Chitin (b-1,4-linked polymer of N-acetylglucosamine) is
a common component of fungal cell walls and its
fragments, N-acetylchitooligosaccharides, have been
shown to act as potent elicitor signals in several plant
systems. These include the induction of ligni®cation in
wheat [3], ion ¯ux and protein phosphorylation in
cultured tomato cells [27], chitinase activity in melon
[79], and gene expression of glucanase in cultured barley
cells [42]. In suspension-cultured rice cells, the action of
chitin fragments has been extensively studied. These
studies showed the induction by chitin fragments of
enzyme activities involved in the biosynthesis of terpenoid
phytoalexins [75, 103], membrane depolarization [45,
50], Cl ÿ and K ‡ e‚ux, cytoplasmic acidi®cation [52],
generation of reactive oxygen species [51], biosynthesis of
jasmonic acid [69] and expression of unique early
responsive genes and typical defence related genes
[60, 68, 92] by chitin fragments.
The requirements for the size and structure of active
chitin fragments are di€erent depending on the experimental systems. In the case of rice and wheat, larger
oligosaccharides such as heptamer or octamer are
preferentially recognized and showed detectable activity
even at nM concentrations [3, 103]. On the other hand, in
the case of tomato cells, the elicitor activity was almost the
same for oligosaccharides larger than tetramer [27]. Such
di€erences in the speci®city probably re¯ect the characteristics of the corresponding receptors and will be
explained in future studies of the interaction of these
oligosaccharides with these receptor molecules. In these
plant cells, deacetylated oligosaccharides, chitosan fragments, did not show elicitor activity.
Chitosan or its fragments can induce defence responses
in several plant systems, mostly dicots. Chitosan induces
callose formation in soybean and parsley cells [13, 49],
proteinase inhibitors in tomato leaves [102] and phytoalexin biosynthesis in pea [31]. However, the concentrations required for these responses are usually much higher
than those necessary for activity of chitin oligosaccharides
in other plant systems. The e€ects of the degree of
acetylation as well as the molecular size of chitosan and its
oligosaccharides were examined with respect to callose
formation, phytoalexin induction and ligni®cation [2, 43,
99]. The elicitor activity of chitosan seems to be mediated
through the interaction of this polycationic molecule with
negatively charged phospholipids, rather than a speci®c
interaction with a receptor-like molecule [43].
Oligogalacturonides
Oligogalacturonides (OGAs) derived from pectic polysaccharides of plant cell walls have also been known to act
as elicitor molecules. OGAs have been shown to induce
biosynthesis of phytoalexins in the cotyledons of soybean
[70] and kidney bean [23], proteinase inhibitors in
tomato leaves [26], and ligni®cation of cucumber
cotyledons [78] and cultured castor bean cells [7]. So
far, there has been no report of elicitor activity of OGAs
in monocot plants, which might be reasonable in the light
of the much lower content of pectic polysaccharides in
monocot cell walls. OGAs can be generated from pectic
polysaccharides by partial acid hydrolysis or by the
action of pectinase or pectate lyase. Regardless of the
method of generation, OGAs of DP ˆ 10±12 appear to be
the most active component. However, Reymond et al.
[77] showed that the ability of OGAs to induce protein
phosphorylation in vitro in tomato increased with the size
of the OGAs even to a DP more than 20. This result
indicates that the optimum size of OGAs for elicitor
activity observed with plant cells or tissues might re¯ect
the penetratability of the OGAs, rather than the
speci®city of the recognition. OGAs usually require
higher concentrations to show elicitor activity compared
Oligosaccharide signalling for defence responses
to chitin or glucan fragment elicitor. Clear synergism,
which might be important to amplify the information on
site, was observed for OGAs and the hepta-b-glucoside
elicitor for the induction of phytoalexin biosynthesis in
soybean cotyledons [21].
Receptors for oligosaccharide elicitors
Receptor for b-glucan oligosaccharide elicitor. The search for
receptors for glucan oligosaccharide elicitors was initiated
by Yoshikawa et al. [106] with the use of a radio-labelled
elicitor-active polysaccharide, mycolaminaran. Binding
assays with 125I-labelled, elicitor-active hepta-b-glucoside
or a mixture of elicitor-active b-glucan fragments has
provided a wealth of information on the high-anity
binding site present in membrane preparations from
soybean roots. The binding speci®city of this site was most
extensively studied by Cheong and Hahn [11] by using
various synthetic oligosaccharides that are structurally
related to the hepta-b-glucoside. These studies demonstrated the presence in membrane preparations from root
and other parts of soybean of a single class of high-anity
binding sites for this elicitor, whose speci®city correlated
very well with the elicitor activity of various b-glucan
oligosaccharides in the soybean cotyledon assay [11, 17,
18]. Density gradient centrifugation indicated the enrichment of the high-anity binding sites mostly in the
plasma membrane fraction [9, 84]. Similar high-anity
binding sites for b-glucan elicitor were also detected in
the membrane preparation from the plant cells belonging
to the family Fabaceae that responded to the elicitor [14].
CoÃte et al. [20] recently showed that two model legumes,
Medicago truncatula and Lotus japonicus, both possess heptab-glucoside binding sites in their membranes, but the
speci®city of these sites seems to be di€erent from the
soybean site.
Photoanity labelling experiments using the solubilized microsomal proteins from soybean roots detected a
major band of approx. 70 kDa as a candidate polypeptide for the binding protein [16].
Various attempts have been made to solubilize and
purify the binding protein. These studies resulted in the
identi®cation of e€ective detergents such as ZW 3±12
and n-dodecanoylsucrose for the solubilization of the
elicitor binding activity [9, 15]. From the analysis of the
behavior of the solubilized fraction on gel ®ltration, it was
assumed that the binding protein is present as a kind of
multimeric complex [9]. By using the anity chromatography with the immobilized elicitor-active b-glucan
fragments, MithoÈfer et al. [66] succeeded in purifying the
75 kDa protein that behaved as 240 kDa protein under
native conditions.
Umemoto et al. [97] also puri®ed a glucan elicitorbinding protein from soybean roots using a similar
approach, but used a mixture of oligosaccharides released
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by enzymatic digestion of the P. sojae glucan (average
molecular mass was 10 kDa) for preparing the anity
matrix. Using partial amino acid sequence information
from this polypeptide, they obtained a cDNA encoding a
polypeptide of 667 amino acids and a calculated mass of
74 424 Da. The predicted sequence had no homology
with any known proteins, except some homology with
three gene products from yeast, whose function is
unknown. An 125I-labelled b-glucan oligosaccharide
mixture from P. sojae bound to the crude extracts from
Escherichia coli and suspension-cultured tobacco cells, both
of which expressed the cDNA. An antibody to the
recombinant protein inhibited half of the binding of the
labelled ligand to the membrane preparation from
soybean cotyledon cells and also inhibited half of the
phytoalexin production elicited by the b-glucan fragments. From these results, the cDNA was concluded to
encode a receptor for b-glucan elicitor. Closely related
cDNAs for glucan elicitor binding protein were recently
cloned from French bean and soybean by another group
and reported to contain a single transmembrane domain
[64, 65]. These recent studies showed that the soybean
protein expressed in trans in tomato cells, bound the
hepta-b-glucoside elicitor with a Kd of 4.5 nM and showed
binding characteristics roughly similar to those observed
for the microsomal fraction from soybean. However, the
soybean protein was inactive when expressed in trans in
insect cells [65]. An antibody generated against the insect
cell-expressed protein could eliminate over 80 % of heptab-glucoside binding activity from the solubilized membrane protein preparation. From these results the authors
suggested that the cDNA encodes the hepta-b-glucoside
binding protein identi®ed previously.
The progress achieved to date provides an important
clue to clarify the function of the 75 kDa elicitor-binding
protein in the signal transduction pathway activated by
the b-glucan oligosaccharide elicitor. To assess the
biological function of this protein further, it will be
necessary to answer the following questions. Firstly, the
biological function of the gene/protein should be more
clearly demonstrated, hopefully with the use of transgenic
plants or mutants in this gene. Secondly, it would also be
very interesting to know how the 75 kDa protein can
function as a receptor molecule, because this protein
seems to have no domains/motifs typical for known
receptor families. Further, it would be desirable to
examine the binding speci®city of the transgenically
expressed protein in more detail to assure that its glucan
elicitor binding characteristics coincide with those determined previously for the site in soybean membranes.
No information is available for the glucan elicitorbinding protein in rice, although the inability of the
elicitor-active glucan fragments to inhibit binding competitively of the radio-labelled chitin oligosaccharide to
plasma membranes of rice cells indicates the presence of
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di€erent binding sites/proteins for these two oligosaccharide elicitors (Yamaguchi et al., unpublished work).
Comparison of the characteristics of the glucan elicitorbinding proteins of soybean and rice should be of great
interest because of the di€erences in the responses of these
plants to di€erent glucan fragments from corresponding
pathogenic fungi.
Receptor for chitin oligosaccharide elicitor
A single class of high-anity binding site for chitin
oligosaccharide elicitor was detected in the microsomal/
plasma membrane preparation from suspension-cultured
rice cells [87, 88], tomato cells [4] and soybean root and
cultured cells [22] by using radio-labelled, elicitor-active
derivatives of the oligosaccharide. The binding speci®city
of these sites corresponded well with the elicitor activity of
chitin oligosaccharides in these systems, re¯ecting the
di€erent preferences of these plants for the size of the
oligosaccharides, as discussed already. The binding sites
in rice and soybean cells were shown to be mostly
concentrated in the plasma membrane [22, 87].
Photoanity labelling as well as anity cross-linking
experiments with 125I-labelled derivatives of N-acetylchitooctaose revealed the presence of a high-anity
binding protein with the size of 75 kDa in the plasma
membrane from suspension-cultured rice cells and also
from rice leaves and roots [39, 72]. The speci®city of the
labelling of this protein again coincided with the binding
speci®city of the membrane preparation and also with the
elicitor activity of the chitin oligosaccharides. The
coincidence of the binding speci®city with the biological
activity of the oligosaccharides as well as its localization in
the plasma membrane strongly suggest that the 75 kDa
protein is the receptor molecule, or a part of the receptor
complex, for this elicitor.
Binding experiments as well as anity cross-linking
with these radio-labelled oligochitin ligands detected the
presence of high-anity binding sites as well as the
corresponding binding proteins in plasma membrane
preparations from several plants including soybean [22],
cultured barley and carrot cells and wheat leaves (Okada
et al., unpublished work). Interestingly, these plant cells
were also shown to respond to the chitin oligosaccharide
elicitor through the generation of reactive oxygen species
or the initiation of ligni®cation. In contrast, binding sites
or binding proteins for oligochitins were not detected in
the membrane preparations from a cell line of tobacco
BY-2, which also did not respond to this elicitor in the
assay system. These results, along with the results for rice
and tomato, indicate the distribution of similar oligochitin perception systems among various plants belonging
to both monocots and dicots. The parallelism between
the presence of the oligochitin, binding protein and the
responsiveness of these plant cells to this elicitor again
supports a functional role of these binding proteins in the
perception of the oligochitin elicitor signal. Detection of
the oligochitin binding protein in soybean is especially
interesting because oligochitin perception, in addition to
the perception of lipochitin oligosaccharides (Nod
factors), has been suggested to play a role in the initiation
of nodulation in legume plants [22, 59].
Cloning of the gene encoding the oligochitin elicitorbinding protein will not only reveal the structure of this
protein but also make it possible to analyse the biological/
biochemical function of this protein by the use of a
suitable expression system. It will also be interesting to
compare the characteristics of the glucan and chitin
elicitor-binding proteins.
Signal transduction
Most of the defence reactions induced by oligosaccharide
elicitors are also observed when plant cells are treated with
non-saccharide elicitors or infected with pathogens. One of
the major di€erences is that oligosaccharide elicitors, in
contrast to at least some other elicitors, do not induce the
hypersensitive reaction (HR) leading to cell death. For
example, fungal xylanase from Tricoderma viride [105] or a
secreted proteinaceous elicitor, INF1 elicitin from P. infestans [82], induces the HR in suspension-cultured tobacco
cells, whereas OGAs do not. In suspension-cultured rice
cells, ¯agellin from Pseudomonas avena induces the HR [8],
but N-acetylchitooligosaccharides do not (Tanabe et al.,
unpublished work). In plant±pathogen interactions, the
HR requires a correct combination of an avirulence gene
of the pathogen and a corresponding resistance gene of the
host plant. Until now, a number of pathogen-derived
elicitors causing HR have been characterized but no
oligosaccharide elicitors are included in this group.
The earliest events after the recognition of elicitor
molecules are membrane depolarization and ion ¯uxes. In
tobacco suspension cells, a rapid change in the membrane
potential, in¯ux of Ca2‡ and H ‡ e‚ux of K ‡ , which
resulted in cytoplasmic acidi®cation and extracellular
alkalinization, were induced by OGAs [57]. The relationships between these events are unclear, except that the K ‡
e‚ux was dependent on the extracellular Ca2‡ , which can
be replaced with Mg2‡ , indicating that the K ‡ e‚ux is
the downstream event of Ca2‡ in¯ux. OGA-induced
cytoplasmic acidi®cation was prevented by pre-treatment
with the protein kinase inhibitors, staurosporine and
dimethylaminopurine, while a protein phosphatase
inhibitor, calyculin A, induced cytoplasmic acidi®cation
by itself. These results suggest that protein kinase(s)/
phosphatase(s) sensitive to these inhibitors regulate the
H ‡ ¯ux across the plasma membrane [58].
N-acetylchitooligosaccharides of DP 4 or higher
induced extracellular alkalinization and the rapid phosphorylation of proteins in suspension-cultured tomato
Oligosaccharide signalling for defence responses
cells [27]. The extracellular alkalinization was transient
and ®nished within 10 min. It was signi®cantly inhibited
by the addition of the protein kinase inhibitor, K-252a,
and induced by calyculin A, suggesting again that the
H ‡ in¯ux is dependent on protein phosphorylation/
dephosphorylation events. Interestingly, tomato cells
treated with N-acetylchitooligosaccharides did not
respond to a second treatment with the same elicitor for
up to 8 h, i.e. stayed in a refractory stage, although the
cells were still able to respond to another elicitor, T. viride
xylanase, and alkalinized the medium. Another interesting observation is that the addition of xylanase induced
later cellular responses such as the activation of PAL and
biosynthesis of ethylene in tomato cells [29], while Nacetylchitooligosaccharides seem to induce only early
cellular responses, e.g. alkalinization of the medium and
protein phosphorylation, in tomato cells [27].
In contrast, N-acetylchitooligosaccharides of DP 7 and
8 induced a variety of defence responses in suspensioncultured rice cells as already described. N-acetylchitoheptaose induced a transient depolarization of the plasma
membrane with a lag time of 20±60 s [45]. The
depolarization required the presence of extracellular
Ca2‡ that could not be replaced with Mg2‡ . The
depolarization was not a€ected by anaerobic treatment
or the addition of azide, indicating that ion channels, but
not energy-dependent ion pumps, have a central role in
this process. However, none of the tested inhibitors of
Ca2‡ channels and anion channels completely cancelled
the depolarization. Thus, it was dicult to specify which
ion channels are responsible for the depolarization,
though the involvement of ion channel(s) that have rather
broad speci®city, as demonstrated in cultured parsley cells
[108], was suggested. Recently, pore-forming activity
within plasma membranes was shown for yeast elicitor
fractions in parsley cells [48]. It is tempting to speculate
that other elicitors including oligosaccharide elicitors
might have similar activities. In conclusion, Ca2‡ appears
essential for the elicitor-induced depolarization in rice
cells. However, its mode of action is not clear at present. In
this experimental system, N-acetylchitoheptaose also
induced transient e‚ux of K ‡ and Cl ÿ , and in¯ux of
H ‡ , leading to the acidi®cation of the cytoplasm and
concomitant alkalinization of the culture medium [45,
52]. The H ‡ in¯ux was inhibited by K-252a and induced
by calyculin A in the absence of the elicitor, analogous to
the response of tobacco cells to treatment with OGAs [58],
indicating the involvement of protein phosphorylation/
dephosphorylation in the regulation of this response [36].
Similar ion ¯uxes were observed in soybean cells
treated with the b-glucan elicitor [24]. The dynamics of
the cytosolic Ca2‡ levels were analysed using soybean
cells transformed with a Ca2‡ monitoring protein,
aequorin [63]. The change of Ca2‡ level induced by
the elicitor was biphasic, dependent on the extracellular
227
Ca2‡ and abolished in the presence of La3‡ , an inhibitor
of Ca2‡ channel.
What are the roles of these early events in the elicitoractivated signal-transduction pathway? Or how is the
signal transmitted to later defence reactions? It has been
well described that Ca2‡ plays important roles as a
second messenger in a variety of biochemical processes in
plants [81] including defence reactions [83]. An inhibitor
of anion channels, A9-C (anthracene 9-carboxylic acid),
has also been shown to inhibit many defence reactions
[36, 41, 68]. It is likely that rapid changes in the ionic
environment of the cytoplasm of the plant cells could
modify enzyme activities, leading to the metabolic
changes. In this context, it is interesting that production
of secondary metabolites is enhanced in response to
changes in cytoplasmic pH induced by the addition of
orthovanadate [33, 35, 90], an inhibitor of H ‡ -ATPase,
H ‡ -speci®c ionophores [32], or elicitor [80].
In tobacco cells, it was shown that the level of mRNAs
for two key enzymes of secondary metabolism, PAL and
3-hydroxy-3-methylglutaryl CoA reductase, increased by
treatment with propionic acid, inorganic phosphate, or a
H ‡ -ATPase inhibitor, erythrosine B, all of which induced
cytoplasmic acidi®cation [53]. In rice cells, activation of a
speci®c set of elicitor-responsive genes was observed in
response to the addition of propionic acid or butyric acid,
both of which signi®cantly acidi®ed the cytoplasm [36].
These results indicate that cytoplasmic acidi®cation or
modi®cation of the electrochemical potential, such as the
H ‡ gradient across the plasma membrane, could be a
signal for the induction of defence reactions. However,
some reports do not support this hypothesis. In soybean
cells, cytoplasmic acidi®cation was not observed for at
least the ®rst 30 min when the pH of the OGAs was
adjusted to that of the media, although whether or not
later defence reactions occurred in the same experimental
system were not examined [37]. In tomato cells carrying
the host resistance gene, CF5, which confers resistance to
Cladosporium fulvum carrying an avirulence gene, avr5, the
plasma membrane H ‡ -ATPase was activated by addition
of a crude elicitor fraction prepared from the fungus
including the avr5 product and the pH of the culture
medium decreased, implying the occurence of the
cytoplasmic alkalinization, although this was not con®rmed [100]. The di€erences in the various reports on
elicitor-induced cytoplasmic acidi®cation could be
ascribed to di€erences in the experimental conditions as
well as to the plant species used for these experiments.
In tobacco cells, cytoplasmic acidi®cation was transiently induced when cells were treated with puri®ed
OGAs, whereas it was sustained for hours when cells were
treated with a crude preparation of the culture media of
P. megasperma. Regardless of the di€erent time pro®les of
cytoplasmic acidi®cation, both OGA and the crude
elicitor fraction induced PAL and o-diphenolmethyl
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N. Shibuya and E. Minami
transferase [30]. In general, the cytoplasmic acidi®cation
induced by addition of acid or inhibitors of H ‡ -ATPase is
persistent, whereas it is transient when induced by OGA
in tobacco cells [57] or by N-acetylchitooligosacharides in
rice cells [52]. These observations lead us to speculate
that the rapid decrease in pH is the signal for the
induction of defence reactions.
At present, the molecular basis of how the pH signal is
transmitted further within the cell has not been revealed.
In tobacco cells, activation of a MAP kinase was observed
following cytoplasmic acidi®cation induced by acetic acid
or butyric acid [94]. MAP kinases play essential roles in a
wide range of biological processes including stress
responses in plant cells [55, 85, 91, 98, 107]. In tobacco
cells treated with an elicitor fraction from the cell wall of
P. infestans, a rapid and transient activation of a MAP
kinase was shown [91]. In cultured parsley cells treated
with an oligopeptide elicitor, Pep-13, similar activation of
a MAP kinase and its re-location to the nucleus were
observed [55]. In the signalling pathway(s) activated by
oligosaccharide elicitors, similar activation of MAP kinase
is likely but has not been demonstrated yet.
Many reports using di€erent elicitors and cells support
the hypothesis that elicitor-signalling pathways involve
protein phosphorylation/dephosphorylation steps [5]. In
these works, the e€ects of protein kinase inhibitors, such
as K-252a or staurosporin, or protein phosphatase
inhibitors, such as calyculin A or okadaic acid, have
been examined, and, in most plant systems with some
exceptions, protein kinase inhibitors blocked and protein
phosphatase inhibitors stimulated the defence reactions.
Inhibitors of protein phosphatase 1 and 2A have been
reported to be the most e€ective in mimicing some part of
plant defence reactions [28, 56]. The most e€ective
inhibitors often vary depending on the plant species. For
example, calyculin A, okadaic acid (both speci®c to 1 and
2A type) and cantharidin (speci®c to 2A type) are active
in soybean cells [56], whereas only calyculin A is active in
rice cells (Minami and Shibuya, unpublished work). In
soybean cotyledons, many kinds of phosphatase 1 and 2A
inhibitors potentially induced the biosynthesis of a
¯avonoid compound, daidzein [56], which is a precursor
of the soybean phytoalexin, glyceollin. Since the hepta-bglucoside elicitor also induces the biosynthesis of glyceollin in soybean cotyledons, it could imply that these
inhibitors act on signalling steps that are involved in the
signal transduction pathway activated by the hepta-bglucoside.
It should be noted that some results are not consistent
with the e€ects of protein kinase/phosphatase inhibitors.
In rice cells, activation of a class II chitinase gene by an
elicitor fraction from the rice blast fungus, Magnaporthe
grisea, was inhibited by the protein phosphatase inhibitors, odakaic acid or calyculin A, but not by the protein
kinase inhibitors, K-252a or staurosporin [46], whereas
class I chitinase gene activation by N-acetylchitoheptaose
was inhibited by K-252a [68]. In cultured parsley cells
and protoplasts, addition of K-252a activated the
synthesis of a phenylpropanoid phytoalexin, ¯anocoumarin, and okadaic acid inhibited the synthesis
induced by a proteinaceous elicitor [76]. Irrespective of
the direction of the regulation, positive or negative,
protein phosphorylation/dephosphorylation appear to
play critical roles in elicitor signalling.
In order to understand the molecular mechanisms of
the elicitor-signal transduction, it is essential to identify
which proteins are phosphorylated or dephosphorylated
in the cell. In tobacco cells, a MAP kinase was phosphorylated at its tyrosin residue after treatment with a
crude elicitor-fraction from the fungal cell wall [91].
Similar results were shown in soybean cells treated with
OGA7±12 [93]. These phosphorylations were essential to
the kinase activities. A proteinaceous elicitor, cryptogein,
was shown to induce phosphorylations of at least
19 proteins by in vivo 32P-labelling of tobacco cells followed
by two-dimensional gel electrophoresis (2-D PAGE) [54].
By combination of 2-D PAGE and mass spectrometoric
analysis, a 43 kDa polypeptide, AtPhos43, which was
phosphorylated in Arabidopsis cells immediately after
addition of a ¯agellin-derived peptide elicitor, ¯g22, was
shown to contain ankyrin-repeat. Two orthologous
proteins were found to be rapidly phosphorylated in rice
cells treated with N-acetylchitohexaose [73]. These
proteomic approaches can be a powerful tool for both
the detection and identi®cation of the phosphorylated (or
dephosphorylated) proteins in response to environmental
stimuli including elicitors.
Potential role of oligosaccharide elicitor signalling the defence
machinery in plants
What is the function of oligosaccharide signalling in
plants? It is dicult to answer this question thoroughly at
this stage. However, it has been shown at a minimum that
many plants are equipped with sophisticated systems by
which they can respond to certain oligosaccharide
elicitors and activate a set of defence reactions. Concerning the possibility of the generation of such oligosaccharides at the site of infection, many plants have been shown
to produce chitinases and glucanases constitutively or
inducibly as a part of their defence machinery [19]. These
enzymes have been indicated to act on the ( potential)
pathogens and inhibit their growth, but they can also
generate various fragments that in turn can act as elicitors
in the host plants, which then results in the induction/
ampli®cation of defence reactions. In addition, invading
microbes themselves may generate such cell wall
fragments, as during spore germination of P. sojae [101].
For OGA, many pathogenic microbes are known to
Oligosaccharide signalling for defence responses
produce polygalacturonase or pectate lyase that can
release OGA from the cell walls of host plants [19].
These observations indicate that oligosaccharide signalling has the potential to function in plant defence.
Evolutionary conservation of oligosaccharide signalling
pathways in various types of plants also supports the
biological importance of these systems. Interestingly,
the use of di€erences in cell surface carbohydrates for
the detection of self and non-self, and for further defence
reactions, are a common strategy for various living
systems. For example, chitin or fungal b-glucans, and
their fragments, have been known to induce defence
responses in vertebrates [61, 96] as well as non-vertebrates
[67]. Chitinase and glucanase, which degrade polysaccharides not existing on the cell surfaces of plants and
vertebrates, have been considered to be important
components for defence systems. Recent identi®cation of
a chitinase in human serum has also been interpreted in a
similar context [6]. Various types of lectin-like molecules
which bind to chitin or its fragments are also distributed
among higher plants [12, 74], non-vertebrates [44], and
even bacteria [71] and have also been discussed for their
involvement in defence machinery.
Plant resistance mechanisms against ( potential) pathogens are often classi®ed into two categories. That is, a
very speci®c resistance response between a speci®c race
carrying an avirulence gene and a cultivar carrying the
corresponding resistance gene, and a more general one
that gives resistance to the majority of potential
pathogens [5, 25, 34, 40, 89]. Potential coverage of
wide varieties of pathogens by oligosaccharide elicitors
indicates the involvement of this system, as well as other
``non-speci®c elicitors'', in the latter type of mechanism,
which is often called general resistance, basic incompatibility, or non-host resistance. The phrase ``non-speci®c
elicitors'' may lead to misunderstandings about the
features of this group of elicitors. These elicitors seem to
be classi®ed into several groups depending on the
potential coverage of the target organisms. For example,
by using chitin oligosaccharides, plants may detect a
broad range of fungi, but may not distinguish their types.
However, in the case of glucan oligosaccharide elicitors,
it may be possible to distinguish and detect a speci®c
group of fungi based on their structural di€erence, as
indicated by the detection of speci®c glucan fragments
from P. sojae and M. grisea by soybean and rice cells,
respectively. A peptide elicitor derived from P. megasperma,
Pep-13, seems to serve as a non-host elicitor that is
recognized by non-host plants for the pathogen but not
by the host plant [41, 108]. Thus, even for so-called
``non-speci®c elicitors'', speci®city of one sort or another
is observed.
What relationships might exist between such ``speci®c''
and ``non-speci®c'' defence systems? An interesting
example for the cooperative use of an evolutionarily
229
old, broader detection system in combination with a more
developed, speci®c system in a whole defence array can be
seen in vertebrates. Mannan-binding protein (MBP) is a
circulating component of mammalian serum and can
bind to potential pathogens such as yeast. When MBP
binds to such microbes or cell surface macromolecules,
the complex can activate the complement system in the
absence of an antigen±antibody complex that is required
for the activation through the ``classical pathway'' [95].
Thus, MBP seems to function as a complement to the
immune system, which takes time for full activation.
Another example is the close connection between innate
and acquired immunity through toll-like receptors
(TLRs). Rapidly growing information on this ®eld
indicates that activation of the innate immune system
by various molecules from ( potential) pathogenic
microbes, such as LPS, ¯agellin, zymosan etc. plays an
important role for the activation of acquired immune
system [1]. Somewhat analogous relationships between
race cultivar speci®c resistance and basic resistance in
plant may be imagined.
Apart from their biological function in vivo, oligosaccharide elicitors provide an excellent model system to
study the perception and transduction of external signals
to induce defence responses in plant cells. The knowledge
gained by such studies also provides a base for the
development of novel agrochemicals for disease control
and also for the development of disease-resistant crops by
regulating the defence system in plant through genetic
manipulation.
c 2001 Government of Japan
*
REFERENCES
1. Akira S, Takeda K, Kaisho T. 2001. Toll-like receptors:
critical proteins linking innate and acquired immunity.
Nature Immunology 2: 675±680.
2. Akiyama K, Kawazu K, Kobayashi A. 1995. Partially
N-deacetylated chitin oligomers ( pentamer to heptamer) are potential elicitors for (‡)-pisatin induction in
pea epicotyls. Zeitschrift fuÈr Naturforschung 50c: 391±397.
3. Barber MS, Bertram RE, Ride JP. 1989. Chitin oligosaccharides elicit ligni®cation in wounded wheat leaves.
Physiological and Molecular Plant Pathology 34: 3±12.
4. Baureithel K, Felix G, Boller T. 1994. Speci®c, high
anity binding of chitin fragments to tomato cells and
membranes, competitive inhibition of binding by
derivatives of chitin fragments and a nod factor of
Rhizobium. Journal of Biological Chemistry 269:
17931±17938.
5. Boller T. 1995. Chemoperception of microbial signals in
plant cells. Annual Review of Plant Physiology and Molecular
Biology 46: 189±214.
6. Boot RG, Renkema H, Strijland A, Jan van Zonneveld
A, Aerts JMFG. 1995. Cloning of a cDNA encoding
chitotriosidase, a human chitinase produced by macrophages. Journal of Biological Chemistry 270: 26252±26256.
230
N. Shibuya and E. Minami
7. Bruce RJ, West CA. 1989. Elicitation of lignin biosynthesis and isoperoxidase activity by pectic fragments in
suspension-cultures of castor bean. Plant Physiology 91:
889±897.
8. Che F-S, Nakajima Y, Tanaka N, Iwano M, Yoshida T,
Takayama S, Kadota I, Isogai A. 2000. Flagellin from
an incompatible strain of Pseudomonas avenae induces a
resistance response in cultured rice cells. Journal of
Biological Chemistry 275: 32347±32356.
9. Cheong JJ, Alba R, CoÃte F, Enkerli J, Hahn MG. 1993.
Solubilization of functional plasma membrane-localized
hepta-b-glucoside elicitor binding proteins from soybean. Plant Physiology 103: 1173±1182.
10. Cheong JJ, Birberg W, Fugedi P, Pilotti A, Garegg PJ,
Hong N, Ogawa T, Hahn MG. 1991. Structure±
activity relationships of oligo-b-glucoside elicitors of
phytoalexin accumulation in soybean. The Plant Cell 3:
127±136.
11. Cheong JJ, Hahn MG. 1991. A speci®c, high-anity
binding site for the hepta-b-glucoside elicitor exists in
soybean membranes. The Plant Cell 3: 137±147.
12. Chrispeels MJ, Raikhel NV. 1991. Lectins, lectin genes,
and their role in plant defense. The Plant Cell 3: 1±9.
13. Conrath U, Domard A, Kauss H. 1989. Chitosan-elicited
synthesis of callose and of coumarin derivatives in
parsley cell suspension cultures. Plant Cell Report 8:
152±155.
14. Cosio EG, Feger M, Miller CJ, Antelo L, Ebel J. 1996.
High-anity binding of fungal b-glucan elicitors to cell
membranes of species of the plant family Fabaceae. Planta
200: 92±99.
15. Cosio EG, Frey T, Ebel J. 1990. Solubilization of
soybean membrane binding site for fungal b-glucans
that elicits phytoalexin accumulation. FEBS Letters 264:
1115±1123.
16. Cosio EG, Frey T, Ebel J. 1992. Identi®cation of a highanity binding protein for a hepta-b-glucoside phytoalexin elicitor in soybean. European Journal of Biochemistry
204: 235±238.
17. Cosio EG, Frey T, Verduyn R, Boom J, Ebel J. 1990.
High-anity binding of a synthetic heptaglucoside and
fungal glucan phytoalexin elicitors to soybean membranes. FEBS Letters 271: 223±226.
18. Cosio EG, PõÃpperl H, Schmidt WE, Ebel J. 1988. Highanity binding of fungal b-glucan fragments to soybean
(Glycine max. L.) microsomal fractions and protoplasts.
European Journal of Biochemistry 175: 309±315.
19. CoÃte F, Hahn MG. 1994. Oligosaccharins: structure
and signal transduction. Plant Molecular Biology 26:
1397±1411.
20. CoÃte F, Roberts KA, Hahn MG. 2000. Identi®cation of
high-anity binding sites for the hepta-b-glucoside
elicitor in membranes of the model legumes Medicago
truncatula and Lotus japonicus. Planta 211: 596±605.
21. Davis K, Darvill A, Albersheim P. 1986. Several biotic
and abiotic elicitors act synergistically in the induction
of phytoalexin accumulation in soybean. Plant Molecular
Biology 6: 23±32.
22. Day RB, Okada M, Ito Y, Tsukada K, Zaghouani H,
Shibuya N, Stacey G. 2001. Binding site for chitin
oligosaccharides in the soybean plasma membrane. Plant
Physiology 126: 1162±1173.
23. Dixon RA, Jennings AC, Davies IA, Gerrish C,
Murphy DL. 1989. Elicitor-active components from
French bean hypocotyls. Physiological and Molecular Plant
Pathology 34: 99±115.
24. Ebel J, Bhagwat AA, Cosio EG, Feger M, Kissel U,
MithoÈfer A, WaldmuÈller T. 1994. Elicitor-binding
proteins and signal transduction in the activation of a
phytoalexin defense response. Canadian Journal of Botany
73: S506±S510.
25. Ebel J, Cosio EG. 1994. Elicitors of plant defense
responses. International Review of Cytology 148: 1±36.
26. Farmer EE, Moloshok TD, Saxton MJ, Ryan CA. 1991.
Oligosaccharide signaling in plants. Speci®city of oligouronide-enhanced plasma membrane protein phosphorylation. Journal of Biological Chemistry 266:
3140±3145.
27. Felix G, Regenass M, Boller T. 1993. Speci®c perception of subnanomolar concentrations of chitin fragments by tomato cells: induction of extracellular
alkalinization, changes in protein phospholylation, and
establishment of a refractory state. The Plant Journal 4:
307±316.
28. Felix G, Regenass M, Spanu P, Boller T. 1994. The
protein phosphatase inhibitor caluculin A mimics
elicitor action in plant cells and induces rapid hyperphosphorylation speci®c proteins as revealed by pulse
labelling with [33P] phosphate. Proceedings of the National
Academy of Sciences, USA 91: 952±956.
29. Grosskopf DG, Felix G, Boller T. 1990. K-252a inhibits
the response of tomato cells to fungal elicitors in vivo and
their microsomal protein kinase in vitro. FEBS Letters 275:
177±180.
30. Guern J, Mathieu Y, Thomine S, Jouanneau J-P, Beloeil
J-C. 1992. Plant cells counteract cytoplasmic pH
changes but likely use these pH changes as secondary
messages in signal perception. Current Topics in Plant
Biochemistry and Physiology 11: 249±269.
31. Hadwiger LA, Beckman J. 1980. Chitosan as a
component of pea±Fusarium solani interactions. Plant
Physiology 66: 205±211.
32. Hagendoorn MJM, Poortinga AM, Wong Fong Sang
HW, van der Plas LHW, Walraven HS. 1991. E€ect of
elicitors on the plasmamembrane of Petunia hybrida cell
suspensions. Role of DpH in signal transduction. Plant
Physiology 96: 1261±1267.
33. Hagendoorn MJM, Traas TP, Boon JJ, van der Plas
HW. 1990. Orthovanadate induced lignin production, in
batch and continuous cultures of Petunia hybrida. Journal
of Plant Physiology 137: 72±80.
34. Hahn MG. 1996. Microbial elicitors and their receptors in
plants. Annual Review of Phytopathology 34: 387±412.
35. Hattori T, Ohta Y. 1985. Induction of phenylalanine
ammonia-lyase activation and iso¯avone glucoside
accumulation in suspension-cultured cells of red bean,
Vigna angularis, by phytoalexin elicitors, vanadate, and
elevation of medium pH. Plant and Cell Physiology 26:
1101±1110.
36. He D-Y, Yazaki Y, Nishizawa Y, Takai R, Yamada K,
Sakano K, Shibuya N, Minami E. 1998. Gene
activation by cytoplasmic acidi®cation in suspensioncultured rice cells in response to the potent elicitor, Nacetylchitoheptaose. Molecular Plant±Microbe Interactions
12: 1167±1174.
37. Horn MA, Meadows R, Apostol I, Jones CR,
Gorenstein DG, Heinstein PF, Low PS. 1992. E€ect
of elicitation and changes in extracellular pH on the
cytoplasmic and vacuolar pH of suspension-cultured
soybean cells. Plant Physiology 98: 680±686.
38. Inui H, Yamaguchi Y, Hirano S. 1997. Elicitor actions of
N-acetylchitooligosaccharides and laminarioligosac-
Oligosaccharide signalling for defence responses
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
charides for chitinase and L-phenylalanine ammonialyase induction in rice suspension culture. Bioscience
Biotechnology and Biochemistry 61: 975±978.
Ito Y, Kaku H, Shibuya N. 1997. Identi®cation of a high
anity binding protein for N-acetylchitooligosaccharide
elicitor in the plasma membrane of suspension cultured
rice cells by anity labelling. The Plant Journal 12:
347±356.
Ito Y, Shibuya N. 2000. Receptors for the microbial
elicitors of plant defense responses. In: Stacey G, Keen
N, eds. Plant-Microbe Interactions. St. Paul, MN, U.S.A.:
APS Press, 269±295.
Jabs T, TschoÈpe M, Colling C, Hahlblock K, Scheel D.
1997. Elicitor-stimulated ion ¯uxes and O2 ÿ from the
oxidative burst are essential components in triggering
defense gene activation and phytoalexin synthesis in
parsley. Proceedings of the National Academy of Sciences, USA
94: 4800±4805.
Kaku H, Shibuya N, Xu P, Aryan AP, Fincher GB. 1997.
N-acetylchitooligosaccharide elicitor expression of a
single (1±3)-b glucanase gene in suspension-cultured
cells from barley (Hordeum vulgare). Physiologia Plantarum
100: 111±118.
Kauss H, Jeblick W, Domard A. 1989. The degree of
polymerization and N-acetylation of chitosan determine its ability to elicit callose formation in suspension
cells and protoplasts of Cathalanthus roseus. Planta 178:
385±392.
Kawabata S, Nagayama R, Hirata M, Shigenaga T,
Agarwala KL, Saito T, Cho J, Nakajima H, Takagi T,
Iwanaga S. 1996. Tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial
protein with chitin-binding activity. Journal of Biological
Chemistry 120: 1253±1260.
Kikuyama M, Kuchitsu K, Shibuya N. 1997. Membrane
depolarization induced by N-acetylchitooligosaccharide
elicitor in suspension-cultured rice cells. Plant and Cell
Physiology 38: 902±909.
Kim CY, Gal SW, Choe MS, Jeong SY, Lee SI, Cheong
YH, Lee SH, Choi YJ, Han C-D, Kang KY, Cho MJ.
1998. A new class II chitinase, Rcht2, whose induction by
fungal elicitor is abolished by protein phosphatase 1 and
2A inhibitor. Plant Molecular Biology 37: 523±534.
Klarzynski O, Plesse B, Joubert JM, Yvin JC, Kopp M,
Kloareg B, Fritig B. 2000. Linear b-1,3 glucans are
elicitors of defense responses in tobacco. Plant Physiology
124: 1027±1037.
KluÈsener B, Weiler EW. 1999. Pore-forming properties of
elicitors of plant defense reactions and cellulolytic
enzymes. FEBS Letters 459: 263±266.
KoÈhle H, Jeblick W, Poten F, Blaschek W, Kauss H.
1985. Chitosan-elicited callose synthesis in soybean cells
as a Ca2‡ -dependent process. Plant Physiology 77:
544±551.
Kuchitsu K, Kikuyama M, Shibuya N. 1993. Nacetylchitooligosaccharides, biotic elicitor for phytoalexin production, induce transient membrane
depolarization in suspension-cultured rice cells. Protoplasma 174: 79±81.
Kuchitsu K, Kosaka H, Shiga T, Shibuya N. 1995. EPR
evidence for generation of hydroxyl radical triggered by
N-acetylchitooligosaccharide elicitor and a protein
phosphatase inhibitor in suspension-cultured rice cells.
Protoplasma 188: 138±142.
Kuchitsu K, Yazaki Y, Sakano K, Shibuya N. 1997.
Transient cytoplasmic pH change and ion ¯uxes
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
231
through the plasma membrane in suspension cultured
rice cells triggered by N-acetylchitooligosaccharide
elicitor. Plant and Cell Physiology 38: 1012±1018.
Lapous D, Mathieu Y, Guern J, LaurieÁre C. 1998.
Increase of defense gene transcripts by cytoplasmic
acidi®cation in tobacco cell suspensions. Planta 205:
452±458.
Lecourieux-Ouaked F, Pugin A, Lebrun-Garcia A.
2000. Phosphoproteins involved in the signal transduction of cryptogein, an elicitor of defense reactions in
tobacco. Molecular Plant±Microbe Interactions 13: 812±829.
Ligterink W, Kroj T, zur Nieden U, Hirt H, Scheel D.
1997. Receptor-mediated activation of a MAP kinase in
pathogen defense of plants. Science 276: 2054±2057.
MacIntosh C, Lyon GD, MacIntosh RW. 1994. Protein
phosphatase inhibitors activate anti-fungal defence
responses of soybean cotyledons and cell cultures. The
Plant Journal 5: 137±147.
Mathieu Y, Kurkdjian A, Xia H, Guern J, Koller A,
Spiro MD, O'Neil M, Albersheim P, Darvill A. 1991.
Membrane responses induced by oligogalacturonides in
suspension-cultured tobacco cells. The Plant Journal 1:
333±343.
Mathieu Y, Lapous D, Thomine S, LaurieÁre C, Guern
J. 1996. Cytoplasmic acidi®cation as an early
phosphorylation-dependent response of tobacco cells to
elicitors. Planta 199: 416±424.
Minami E, Kouchi H, Cohn JR, Ogawa T, Stacey G.
1996. Expression of the early nodulin, ENOD40, in
soybean roots in response to various lipo-chitin signal
molecules. The Plant Journal 10: 23±32.
Minami E, Kuchitsu K, He DY, Kouchi H, Midoh N,
Ohtsuki Y, Shibuya N. 1996. Two novel genes rapidly
and transiently activated in suspension-cultured rice
cells by treatment with N-acetylchitoheptaose, a biotic
elicitor for phytoalexin production. Plant and Cell
Physiology 37: 563±567.
Misaki A, Kakuta M. 1997. Fungal (1±3)-b-D-glucans:
chemistry and antitumor activity. In: Witezak ZJ,
Nieforth KA, eds. Carbohydrates in Drug Design. New
York, NY, U.S.A.: Marcel Dekker, 655±689.
MithoÈfer A, Bhagwat AA, Feger M, Ebel J. 1996.
Suppression of fungal b-glucan-induced plant defence in
soybean (Glycine max L.) by cyclic 1,3- 1,6-b-glucan from
the symbiont Bradyrhizobium japonicum. Planta 199:
270±275.
MithoÈfer A, Ebel J, Bhagwat AA, Boller T, NeuhausUrl G. 1999. Trasgenic aequorin monitors cytosolic
calcium transients in soybean cells challenged with bglucan or chitin elicitors. Planta 207: 566±574.
MithoÈfer A, Fliegmann J, Ebel J. 1999. Isolation of a
French bean (Phaseolus vulgaris L.) homolog to the
b-glucan elicitor-binding protein of soybean (Glycine max
L.). Biochimica et Biophysica Acta 1418: 127±132.
MithoÈfer A, Fliegmann J, Neuhaus-Url G, Schwarz H,
Ebel J. 2000. The hepta-b-glucoside elicitor-binding
proteins from legumes represent a putative receptor
family. Biological Chemistry 381: 705±713.
MithoÈfer A, Lottspeich F, Ebel J. 1996. One-step
puri®cation of the b-glucan elicitor-binding protein
from soybean (Glycine max L.) root and characterization
of an anti-peptide antiserum. FEBS Letters 381:
203±207.
Muta T, Seki N, Takaki Y, Hashimoto R, Oda T,
Iwanaga A, Tokunaga F, Iwanaga S. 1995. Puri®ed
horseshoe crab factor G: reconstitution and characteriz-
232
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
N. Shibuya and E. Minami
ation of the (1±3)-b-D-glucan-sensitive serine protease
cascade. Journal of Biological Chemistry 270: 892±897.
Nishizawa Y, Kawakami A, Hibi T, He DY, Shibuya N,
Minami E. 1999. Regulation of the chitinase gene
expression in suspension-cultured rice cells by Nacetylchitooligosaccharides: di€erences in the signal
transduction pathways leading to the activation of
elicitor-responsive genes. Plant Molecular Biology 39:
907±914.
Nojiri H, Sugimori M, Yamane H, Nishimura Y,
Yamada A, Shibuya N, Kodama O, Murofushi N,
Ohmori T. 1996. Involvement of jasmonic acid in
elicitor-induced phytoalexin production in suspensioncultured rice cells. Plant Physiology 110: 387±392.
Nothnagel EA, McNeil M, Albersheim P, Dell A. 1983.
Host±pathogen interactions. XXII A galacturonic acid
oligosaccharide from plant cell walls elicits phytoalexins.
Plant Physiology 71: 916±926.
Oita S, Horita M, Yanagi SO. 1996. Puri®cation and
properties of a new chitin-binding antifungal CB-1 from
Bacillus licheniformis. Bioscience Biotechnology and Biochemistry 60: 481±483.
Okada M, Matsumura M, Shibuya N. 2001. Identi®cation of a high-anity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane
from rice leaf and root cells. Journal of Plant Physiology
158: 121±124.
Peck S, NuÈhse TS, Hess D, Iglesias A, Meins F, Boller
T. 2001. Directed proteomics identi®es a plant-speci®c
protein rapidly phosphorylated in response to bacterial
and fungal elicitors. The Plant Cell 13: 1467±1475.
Peumans WJ, Van Damme EJM. 1995. Lectins as plant
defense proteins. Plant Physiology 109: 347±352.
Ren YY, West CA. 1992. Elicitation of diterpene biosynthesis in rice (Olyza sativa L.) by chitin. Plant
Physiology 99: 1169±1178.
Renelt A, Colling C, Hahlbrock K, NuÈrnberger T,
Parker JE, Sacks WR, Scheel D. 1993. Studies on
elicitor recognition and signal transduction in plant
defence. Journal of Experimental Botany 44: 257±268.
Reymond P, GruÈnberger S, Paul K, MuÈller M, Farmer
EE. 1995. Oligogalacturonide defense signals in plants:
large fragments interact with the plasma membrane in
vitro. Proceedings of the National Academy of Sciences, USA 92:
4145±4149.
Robertsen B. 1986. Elicitors of the production of ligninlike compounds in cucumber hypocotyls. Physiological and
Molecular Plant Pathology 28: 137±148.
Roby D, Gadelle A, Toppan A. 1987. Chitin oligosaccharides as elicitors of chitinase activity in melon
plants. Biochemical and Biophysical Research Communications
143: 885±892.
Roos W, Evers S, Hieke M, TschoÈpe M, Schumann B.
1998. Shifts of intracellular pH distribution as a part of
the signal mechanism leading to the elicitation of
benzophenanthridine alkaloids. Phytoalexin biosynthesis in cultured cells of Eschscholtzia californica. Plant
Physiology 118: 349±364.
Rudd JJ, Franklin-Tong VE. 1999. Calcium signaling in
plants. Cellular and Molecular Life Sciences 55: 214±232.
Sasabe M, Takeuchi K, Kamoun S, Ichinose Y, Govers
F, Toyoda K, Shiraishi T, Yamada T. 2000. European
Journal of Biochemistry 267: 5005±5013.
Scheel D. 1998. Resistance response physiology and signal
transduction. Current Opinion in Plant Biology 1: 305±310.
84. Schmidt WE, Ebel J. 1987. Speci®c binding of a fungal
glucan phytoalexin elicitor to membrane fraction from
soybean Glycine max. Proceedings of the National Academy of
Sciences, USA 84: 4117±4121.
85. Seo S, Okamoto M, Seto H, Ishizuka K, Sano H,
Ohashi Y. 1995. Tobacco MAP kinase: a possible
mediator in wound signal transduction pathways. Science
270: 1988±1992.
86. Sharp JK, McNeil M, Albersheim P. 1984. The primary
structures of one elicitor active and seven elicitor inactive
hexa-b-glucopyranosyl-D-glucitol isolated from the
mycelial cell walls of Phytophtra megasperma f. sp. glycinea.
Journal of Biological Chemistry 259: 11321±11336.
87. Shibuya N, Ebisu N, Kamada Y, Kaku H, Cohn J, Ito Y.
1996. Localization and binding characteristics of a
high-anity binding site for N-acetylchitooligosaccharide elicitor in the plasma membrane from suspensioncultured rice cells suggest a role as a receptor for
the elicitor signal at the cell surface. Plant and Cell
Physiology 37: 894±898.
88. Shibuya N, Kaku H, Kuchitsu K, Maliarik MJ. 1993.
Identi®cation of a novel high-anity binding site for Nacetylchitooligosaccharide elicitor in the plasma membrane fraction from suspension-cultured rice cells. FEBS
Letters 329: 75±78.
89. Somssich IE, Hahlbrock K. 1998. Pathogen defense in
plantsÐa praradigm of biological complexity. Trends in
Plant Science 3: 86±90.
90. Steffens M, Ettl F, Kranz D, Kindl H. 1989. Vanadate
mimics e€ects of fungal cell wall in eliciting gene
activation in plant cell cultures. Planta 177: 160±168.
91. Suzuki K, Shinshi H. 1995. Transient activation and
tyrosine phosphorylation of a protein kinase in tobacco
cells treated with a fungal elicitor. The Plant Cell 7:
639±647.
92. Takai R, Hasegawa K, Kaku K, Shibuya N, Minami E.
2001. Isolation and analysis of expression mechanisms
of a rice gene, EL5, which shows structural similarity
to ATL family from Arabidopsis, in response to Nacetylchitooligosaccharide elicitor. Plant Science 160:
577±583.
93. Taylor ATS, Kim J, Low PS. 2001. Involvement of
mitogen activated protein kinase activation in the signaltransduction pathways of the soya bean oxidative burst.
Biochemical Journal 355: 795±803.
94. Tena G, Renaudin J-P. 1998. Cytosolic acidi®cation but
not auxin at physiological concentration is an activator
of MAP kinases in tobacco cells. The Plant Journal 16:
173±182.
95. Thiel S, Vorup-Jensen T, Stover CM, Schwaeble W,
Laursen SB, Poulsen K, Willis AC, Eggleton P,
Hansen S, Holmskov U, Reid KBM, Jensenius JC.
1997. A second serine protease associated with mannanbinding lectin that activates complement. Nature 386:
506±510.
96. Tokoro A, Tatewaki N, Suzuki K, Mikami T, Suzuki S,
Suzuki M. 1988. Growth-inhibitory e€ect of hexaN-acetylchitohexaose and chitohexaose against Meth-A
solid tumor. Chemical and Pharmaceutical Bulletin 36:
784±790.
97. Umemoto N, Kakitani M, Iwamatsu A, Yoshikawa M,
Yamaoka N, Ishida I. 1997. The structure and function
of a soybean b-glucan-elicitor-binding protein. Proceedings of the National Academy of Sciences, USA 94:
1029±1034.
Oligosaccharide signalling for defence responses
98. Usami S, Banno H, Nishihama R, Machida Y. 1995.
Cutting activates a 46-kilodalton protein kinase in
plants. Proceedings of the National Academy of Sciences,
USA 92: 8660±8664.
99. Vander P, VaÊrum KM, Domard A, El Gueddari NE,
Moerschbacher BM. 1998. Comparison of the ability
of partially N-acetylated chitosans and chitooligosaccharides to elicit resistance reactions in wheat leaves.
Plant Physiology 118: 1353±1359.
100. Vera-Estrella R, Barkla BJ, Higgins VJ, Blumward E.
1994. Plant defense response to fungal pathogens.
Activation of host-plasma membrane H ‡ -ATPase by
elicitor-induced enzyme dephosphorylation. Plant
Physiology 104: 209±215.
101. WaldmuÈller T, Cosio EG, Grisebach H, Ebel J. 1992.
Release of highly elicitor-active glucans by germinating
zoospores of Phytophthora megasperma f. sp. Glycinea. Planta
188: 498±505.
102. Walker-Simmons M, Ryan CA. 1984. Proteinase inhibitor synthesis in tomato leaves. Plant Physiology 76:
787±790.
103. Yamada A, Shibuya N, Kodama O, Akatsuka T. 1993.
Induction of phytoalexin formation in suspensioncultured rice cells by N-acetylchitooligosaccharides.
Bioscience Biotechnology and Biochemistry 57: 405±409.
233
104. Yamaguchi T, Yamada A, Hong N, Ogawa T, Ishii T,
Shibuya N. 2000. Di€erences in the recognition of
glucan elicitor signals between rice and soybean: bglucan fragments from the rice blast disease fungus
Pyricularia oryzae that elicit phytoalexin biosynthesis in
suspension-cultured rice cells. The Plant Cell 12:
817±826.
105. Yano A, Suzuki K, Uchimiya H, Shinshi H. 1998.
Induction of hypersensitive cell death by a fungal
protein in cultures of tobacco cells. Molecular Plant±
Microbe Interactions 11: 115±123.
106. Yoshikawa M, Keen NT, Wang MC. 1983. A receptor on
soybean membranes for a fungal elicitor of phytoalexin
accumulation. Plant Physiology 73: 497±506.
107. Zhang S, Du H, Klessig DF. 1998. Activation of the
tobacco SIP kinase by both a cell wall-derived
carbohydrate elicitor and puri®ed poteinaceous elicitins
from Phytophthora spp. The Plant Cell 10: 435±449.
108. Zimmermann S, NuÈrnberger T, Frachisse J-M, Wirtz
W, Guern J, Hedrich R, Scheel D. 1997. Receptormediated activation of a plant Ca2‡ -permeable
ion channel involved in pathogen defense. Proceedings
of the National Academy of Sciences, USA 94: 2751±2755.