Báo cáo khoa học: Novel b-1,3-, 1,6-oligoglucan elicitor from Alternaria alternata 102 for defense responses in tobacco docx

Novel b-1,3-, 1,6-oligoglucan elicitor from Alternaria
alternata 102 for defense responses in tobacco
Tomonori Shinya
1,2
, Rozenn Me
´
nard
3
, Ikuko Kozone
1
, Hideaki Matsuoka
1
, Naoto Shibuya
4
,
Serge Kauffmann
3
, Ken Matsuoka
2
and Mikako Saito
1
1 Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Japan
2 RIKEN Plant Science Center, Yokohama, Japan
3 Institut de Biologie Mole
´
culaire des Plantes du Centre National de la Recherche Scientifique, Universite
´
Louis Pasteur, Strasbourg, France
4 Department of Life Sciences, Faculty of Agriculture, Meiji University, Kawasaki, Japan
In plant–microbe interactions, pathogenic microorgan-
isms produce elicitors that trigger the induction of def-

ense responses in plants. The major defense responses
induced by these elicitors involve the production of
antimicrobial enzymes, cell-wall fortification, produc-
tion of reactive oxygen, and programmed cell death.
Typical pathogen-derived elicitors derived by a wide
range of microbes or pathogen groups involve oligo-
saccharides [1–3] and peptides [4–6]. Many of the elici-
tors so far characterized serve as general or nonhost
elicitors inducing defense responses in a wide range of
plant cells. Recently, it has emerged that many of these
Keywords
Alternaria alternata; BY-2 cells; chitinase;
elicitor; b-1,3-, 1,6-glucan
Correspondence
M. Saito, Department of Biotechnology and
Life Science, Faculty of Technology, Tokyo
University of Agriculture and Technology,
Koganei, Tokyo 184–8588, Japan
Fax: +81 42 387 1503
Tel.: +81 42 388 7400
E-mail:
(Received 13 January 2006, revised 23
March 2006, accepted 28 March 2006)
doi:10.1111/j.1742-4658.2006.05249.x
A novel elicitor that induces chitinases in tobacco BY-2 cells was isolated
from Alternaria alternata 102. Six other fungi, including A. alternata IFO
6587, could not induce, or weakly induce chitinase activity. The purified
elicitor was soluble in 75% methanol and showed the chitinase-inducing
activity when applied at concentrations of as low as 25 ngÆmL
)1

. Structural
determination by methylation analysis, reducing-end analysis, MALDI-
TOF ⁄MS, and NMR spectroscopy indicated that the elicitor was a mixture
of b-1,3-, 1,6-oligoglucans mostly with a degree of polymerization of
between 8 and 17. Periodate oxidation of the elicitor suggested that the
1,6-linked and nonreducing terminal residues are essential for the elicitor
activity. Further analysis of the elicitor responses in BY-2 cells indicated
that the activity of this b-1,3-, 1,6-glucan elicitor was about 1000 times
more potent than that of laminarin, which is a known elicitor of defense
responses in tobacco. Analyzing the expression of defense-related genes
indicated that a phenylalanine ammonia-lyase gene and a coumaroyl-CoA
O-methyltransferase gene were transiently expressed by this b-1,3-,
1,6-glucan elicitor. The elicitor induced a weak oxidative burst but did not
induce cell death in the BY-2 cells. In the tissue of tobacco plants, this
b-1,3-, 1,6-glucan elicitor induced the expression of basic PR-3 genes, the
phenylpropanoid pathway genes, and the sesquiterpenoid pathway genes.
In comparison with laminarin and laminarin sulfate, which are reported to
be potent elicitors of defense responses in tobacco, the expression pattern
of genes induced by the purified b-1,3-, 1,6-glucan elicitor was more similar
to that induced by laminarin than to that induced by laminarin sulfate.
Abbreviations
CCoAOMT, coumaroyl-CoA O-methyltransferase; CL, chemiluminescence; DP, degree of polymerization; HMGR, 3-hydroxy-3-methylglutaryl-
CoA reductase; HPAEC-PAD, high-performance anion-exchange chromatography with pulsed amperometric detection; MeJA, methyl
jasmonate; MeOH, methanol; OMT, O-methyltransferase; PAL, phenylalanine ammonia-lyase; PR protein, pathogenesis-related protein;
SA, salicylic acid; STC, sesquiterpene cyclase; TBC, tobacco BY-2 chitinase.
FEBS Journal 273 (2006) 2421–2431 ª 2006 The Authors Journal compilation ª 2006 FEBS 2421
elicitors are derived from molecular sequences con-
served among various microorganisms, which are
known as pathogen-associated molecular patterns
(PAMPs) [4,6,7]. These PAMPs are thought to be

recognized by pattern-recognition receptors in plants
and to trigger the expression of defense responses in
the plant cells. On the other hand, so-called gene-for-
gene resistance, which is governed by the presence of
corresponding resistance (R) and avirulence (avr) genes
in the plant and the pathogen, confers much more spe-
cific recognition [7–10].
Glucans are among the best studied oligosaccharide
elicitors. Active glucans can derive from fungal patho-
gen as well as from algal cell walls. Laminarin is an
algal b-1,3-glucan with b-1,6 glucose branches, triggers
defense responses in a noncultivar-specific manner in
many plants including tobacco. The best known glucan
elicitor is a heptaglucoside, which is a penta b-1,6 glu-
cose backbone and two b-1,3 glucose side chains, was
isolated from Phytophthora megasperma cell walls. It
elicits defense responses in soybean but not in tobacco
or rice cells [11–13]. A b-1,6 b-1,3 glucan (b-1,3 back-
bone with a b-1,6 side chain) isolate from Pyricularia
oryzae induces phytoalexin production in rice but not in
soybean [12]. Linear glucans are active in tobacco [11],
but not in rice [12], or soybean [13]. These observations
allowed us to form the hypothesis that different plants
have developed the ability to react to structurally
different, but related, b-glucans.
During the course of a search for a potent elicitor of
defense responses in a model plant cell (tobacco BY-2
cells), we found that the fungal strain Alternaria alter-
nata 102 produces a substance that induces chitinase
activity when applied to BY-2 cells, whereas cell wall

extracts from six other fungi, including A. alternata
IFO 6587, remained almost inactive [14]. From these
findings, we suspected the existence of a specific elicitor
produced by A. alternata 102 and active in tobacco
BY-2 cells. In this paper, we describe the isolation and
characterization of this elicitor, which is a novel b-1,3-,
1,6-glucan acting as a potent elicitor of defense
responses in tobacco BY-2 cells. We also discuss re-
sponses triggered by this elicitor in comparison with
responses triggered by other elicitors.
Results
Purification of the elicitor-active component
isolated from A. alternata 102
Our previous work has indicated that the filtrate
obtained from autoclaved A. alternata 102 culture was
able to induce marked chitinase activity in tobacco
BY-2 cells without inducing cytotoxic effect [14]. We
hypothesized that the active component was different
from peptide-like substances, which are known to be
produced by A. alternata 102 and displaying cytotoxic-
ity against tobacco BY-2 cells [15]. Preliminary experi-
ments indicated that the elicitor-active fraction could
not be extracted with organic solvents, such as meth-
anol, ethanol, n-butanol, ethyl acetate, and n-hexane.
The elicitor activity was stable after heat treatment at
50 °C and also after freezed at )30 °C for 4 days. The
elicitor activity was also not affected by treatment with
proteases such as trypsin and proteinase K. The
molecular weight of the elicitor-active component was
estimated to be larger than 4000 by gel filtration

chromatography. These results suggested that the elici-
tor-active component could be a polysaccharide. The
purification scheme is shown in Fig. 1. The elicitor-act-
ive compound was extracted with 75% methanol from
methanol-washed lyophilized powder of the autoclaved
extract of the fungal culture. The extracted elicitor was
recovered by precipitation with trichloroacetic acid,
then the supernatant was separated by reversed phase
chromatography (Fig. 2A), and gel filtration chroma-
tography (Fig. 2B). After the separation by reversed
phase column chromatography, the elicitor-active frac-
tion was shown to be composed mainly of carbohy-
drates (Fig. 2B). We obtained 2.6 mg of purified
A. alternata 102 culture medium
MeOH extraction
75% MeOH extraction
Treatment with trichloroacetic acid
Dialysis
Lyophilization
ODS chromatography
ODS HPLC
Gel filtration HPLC
0%
MeOH
25%
MeOH
50%
MeOH
100%
MeOH

Autoclaving
Filtration
Fig. 1. Purification scheme of elicitor-active fraction form A. alternata
102 culture medium.
Novel b-glucan elicitor from A. alternata 102 T. Shinya et al.
2422 FEBS Journal 273 (2006) 2421–2431 ª 2006 The Authors Journal compilation ª 2006 FEBS
elicitor from 10.2 L of A. alternata 102 culture. The
recovery of the elicitor activity was 28% from
the starting material, the methanol-insoluble fraction.
The specific activity of the purified elicitor was
increased 1200 times compared to the starting material.
Figure 3 shows the dose dependent response analyzing
the chitinase induction by the purified elicitor. Chi-
tinase activity was induced when applying an elicitor
dose as low as 25 ngÆmL
)1
.
Structural analysis of the A. alternata elicitor
The purified elicitor contained about 97% carbohy-
drate and consisted almost solely of glucose (data not
shown). Methylation analysis of the elicitor indicated
the presence of terminal, 3-linked, 6-linked, and 3,6-
linked glucosyl residues (Table 1). The molar ratios of
terminal, 3-linked, 6-linked, and 3,6-linked glucosyl
residues were 1 : 2 : 1 : 1. These results indicated that
the elicitor is a branched 1,3-, 1,6-linked glucan. The
elicitor was thus called AaGlucan.
To investigate whether the AaGlucan has a redu-
cing-end, in other words, to examine the possibility of
a cyclic glucan, the AaGlucan was reduced with

sodium borohydride and then hydrolyzed (Fig. 4). Sor-
bitol was recovered from the hydrolysis of the AaGlu-
can. As a control, we applied the same procedure to a
cyclic b-1,2-glucan from Agrobacterium radiobacter
IFO 12664 [16], and no sorbitol was produced, thus
excluding the possibility of a cyclic structure for the
AaGlucan. These results also indicated that the
AaGlucan has a reducing-end glucose.
We used
1
H- and
13
C-NMR spectrometry to identify
the anomeric configuration of the glucose residue. In
the anomeric region of the
1
H-NMR spectrum, a sig-
nal was found at 4.4 p.p.m. with a coupling constant
J
1,2
¼ 8.5.
13
C-NMR analysis of the elicitor showed a
C1 resonance at 103 p.p.m. These results indicate that
Retention time (min)
0
)


(tne

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r
a
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e
v
i
t
aleR
1.0
0.5
1.0
0.5
10 20
100
50
25
75
0
)



,IR(ytis
ne
t
ni

evitaleR
) ( ytivitca roticilE
B
15 30 450
Retention time (min)
,%( lonahteM)
100
50
25
75
0
)





(t
n
et
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rag
us
evita
l
e
R
1.0
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1.0

0.5
1.0
0.5
0
) ,mn 002(ytisnetni evitaleR
) ( ytivitca roticilE
A
Fig. 2. Purification of elicitor-active fraction by HPLC on an ODS
column (A) and by gel filtration HPLC (B). Relative elicitor activity
was determined by the induction of TBC-1. Relative sugar content
was measured by the phenol–sulfuric acid procedure.
TBC-1
TBC-2
TBC-3
0200100502512.5 150
(ng/ml)
Fig. 3. Dose dependency of TBC-1 induction by the purified elicitor.
Chitinase activity was measured 3 h after treatment with the
respective samples. The induction of TBC-1 with distilled water
was used as a control. Protein extracts (100 lg per lane) from elici-
tor- and water-treated BY-2 cells were separated by native PAGE,
and the chitinase activities were measured by the activity staining
method using Calcofluor White.
Table 1. Methylation analysis of AaGlucan, the purified glucan elicitor. Laminarin was also methylated and used to identify partially methyl-
ated alditol acetates.
Retention time (min)
Major mass
fragments (m ⁄z)
Position of
O-methyl groups

Glucosyl
residue Composition
9.85 205,161,117 2,3,4,6 Terminal-Glc 0.97
10.49 233,161,117 2,4,6 3-linked-Glc 1.94
10.69 233,161,189,117 2,3,4 6-linked-Glc 1.16
11.31 189,117 2,4 3,6-linked-Glc 1.00
T. Shinya et al. Novel b-glucan elicitor from A. alternata 102
FEBS Journal 273 (2006) 2421–2431 ª 2006 The Authors Journal compilation ª 2006 FEBS 2423
all glucose residues in the elicitor have a b configur-
ation.
The molecular mass of the b-1,3, 1,6-glucan elicitor
was determined by MALDI-TOF ⁄MS. We detected a
series of molecular ions with masses corresponding to
the masses of reducing oligosaccharides consisting of
hexose units within the experimental error (Fig. 5).
These results in combination with the results of the
methylation analysis and the reducing-sugar assay,
suggested that the AaGlucan is a mixture of branched,
noncyclic, b-1,3-, 1,6-glucan oligosaccharides with DP
between 8 and 17.
Effects of laminarinase treatment and periodate
oxidation on the elicitor activity of the AaGlucan
To investigate which structure of the AaGlucan was
responsible for eliciting the defense responses in the
BY-2 cells, the AaGlucan was treated with laminari-
nase, which hydrolyzes the b-1,3-linkages, or with
periodate. Laminarinase dramatically decreased the eli-
citor activity, providing further evidence that the elici-
tor activity is carried by a polysaccharidic molecule
with b-1,3-linkages (Fig. 6A). Periodate oxidation of

0 5 10 15 20 25
0 5 10 15 20 25
Time (min) Time (min)
Glucose Glucose
Sorbitol
Sorbitol
esnopseR rotceteD
esnopseR rotceteD
AB
Fig. 4. Analysis of reducing-end sugar. Hydrolysate of the reduced
purified-glucan elicitor (AaGlucan) was analyzed by HPAEC-PAD (A).
As a control, the cyclic b-1,2-glucan from Agrobacterium radiobacter
was analyzed under the same conditions (B). Glucose and sorbitol
were identified by comparing the retention times with those of
authentic samples.
1000
100
2000 3000 4000
20
80
60
40
0
Mass (m/z)
ytisnetnI %
G[ 8331
8=PD
]aN+
+
G[ 0051

9=
P
D
]aN+
+
G[ 3661
0
1
=
P
D
]aN+
+
G[ 5281
1
1=
P
D
]aN+
+
G[ 7891
21
=
P
D
]aN+
+
G[ 9412
31
=

P
D
]aN+
+
G
[
2132
41=PD
]aN+
+
G[ 4742
5
1=
P
D
]aN+
+
G[ 6362
61
=PD
]aN+
+
G[ 997
2
71=
P
D
]
a
N+

+
Fig. 5. MALDI-TOF mass spectra of the purified glucan elicitor. The
m ⁄z-values are shown as the nominal masses of the pseudomolec-
ular ions [M + Na]
+
.
TBC-1
W
a
e
t
r
A
a
G
l
u
c
a
n
A
a
G
l
cu
a
n
+
L
a

m
i
n
a
r
i
n
a
e
s
A
B
Aa
G
l
cu
a
n
Co
n
t
r
o
l
Aa
G
l
cu
a
n

(
p
e
r
i
do
t
ae
o
x
i
d
ta
i
o
n
)
L
ma
i
n
a
r
i
n
(
p
e
ri
o

d
a
t
e
o
x
i
d
a
t
i
on
)
L
ma
i
n
a
r
i
n
TBC-1
Fig. 6. Effects of laminarinase treatment and periodate oxidation on
the induction of chitinase by AaGlucan. (A) Effect of laminarinase
treatment on the elicitor activity of AaGlucan (100 ngÆmL
)1
final
concentration). Lane 1: Negative control without the enzyme or
AaGlucan. Lane 2: AaGlucan without laminarinase treatment. Lane
3: Laminarinase-treated AaGlucan. (B) Effect of periodate oxidation

on the elicitor activities of AaGlucan (100 ngÆmL
)1
final concentra-
tion) and laminarin (100 lgÆmL
)1
final concentration). Water without
glucan was treated under the same conditions and used as a control.
The induction of TBC-1 was measured 3 h after the addition of the
elicitor. Protein extracts (100 lg per lane) from the elicitor- and
water-treated BY-2 cells were separated by native PAGE, and the
chitinase activities were measured by the activity staining method.
Novel b-glucan elicitor from A. alternata 102 T. Shinya et al.
2424 FEBS Journal 273 (2006) 2421–2431 ª 2006 The Authors Journal compilation ª 2006 FEBS
the AaGlucan and of laminarin dramatically decreased
their elicitor activities (Fig. 6B). Periodate oxidizes the
1,6-linked and the nonreducing terminal residues but
not the 1,3-linked glucose residues. Thus, the results of
both the laminarinase and periodate treatments indi-
cate that the b-1,3-linked and the periodate-sensitive
residues are essential for the chitinase inducing activity
of AaGlucan.
Cellular responses induced by the AaGlucan
elicitor
The chitinase inducing activity of the AaGlucan was
first compared with that of other polysaccharidic elici-
tor fractions (Fig. 7). Laminaripentaose, N-acetylchito-
octaose and glycolchitin remained inactive. Laminarin
and the AaGlucan triggered a similar level of chitinase
activity. However, laminarin was applied at a concen-
tration that was 1000 times that of the AaGlucan, indi-

cating that the AaGlucan has a much higher activity
to induce TBC-1 activity in BY-2 cells.
We then investigated whether the AaGlucan would
induce cell death measured using propidium iodide
assay. Less than 3% of the BY-2 cells were stained
with propidium iodide 48 h after treatment with
AaGlucan, a value that is comparable to the 3% for a
control experiment without the elicitor (Fig. 8A). As
negative and positive controls, we used laminarin and
benzyladenine, respectively. It is reported previously
that laminarin does not induce cell death of tobacco
BY-2 cells [11], whereas benzyladenine is cytotoxic to
the same cells [17]. As expected, laminarin application
did not induce cell death, whereas a treatment with
benzyladenine resulted in staining of 76% of the BY-2
cells. These results indicate that the AaGlucan does
not induce cell death in the BY-2 cells under the condi-
tions employed.
The production of reactive oxygen intermediates
through an oxidative burst is a hallmark of plant def-
ense responses. A transient and dose-dependent induc-
tion of an oxidative burst was detected in BY-2 cells
treated with the AaGlucan (Fig. 8B).
As the AaGlucan induces the expression of tobacco
BY-2 chitinase (TBC)-1 in the BY-2 cells, we further
investigated whether other defense-related genes would
TBC-1
W
a
et

r
A
a
G
l
u
c
a
n
5(0
n
g
/
m
l
)
L
ma
i
n
a
r
i
n
(
5
0
µ
g
/

m
)l
L
ma
i
n
a
r
i
p
e
n
t
a
o
s
e
5(
0 µ
g
/
m
)l
N-
cA
e
yt
cl
h
i

ot
o
c
t
ao
s
e
5(
0
µ
g
m/
l
)
G
l
y
c
o
l
c
h
i
it
n
5
(0
µg
/
ml

)
F
i
l
r
ta
t
e
o
f
A
.
a
l
nret
a
t
a
c
u
l
ut
r
e
(
1m
g
/
m
l

)
Fig. 7. Comparison of elicitor activities of several poly and oligosac-
charides in BY-2 cells. The induction of TBC-1 was measured 3 h
after the elicitor treatment. Protein extracts (100 lg per lane) from
the elicitor-treated BY-2 cells were separated by native PAGE, and
chitinase activities were measured by the activity staining method.
∆
∆
SOR ( tnuoma SOR
)roticilE(
SOR-
)lortnoC(
)
H
2
O
2
M)n(
-10
0
10
20
30
40
0246810
Incubation time (h)
B
A
100
0

20
40
60
80
ret
a
W
culGa
A
a
n
ni
r
a
nimaL
B
eninedal
y
zne
)%( sllec deniats-IP
Fig. 8. Evaluation of oxidative burst and cell death induced by
AaGlucan. (A) Cell viability was measured using PI (propidium iod-
ide) 48 h after treatment with AaGlucan (200 ngÆmL
)1
), laminarin
(200 lgÆmL
)1
), or benzyladenine (150 lM). Values are the
means ± standard deviation from 3 to 7 independent experiments.
(B) AaGlucan-induced oxidative burst was determined by luminol

assay. Untreated cells were used as a control. d,50lgÆmL
)1
AaGlucan; m,100lgÆmL
)1
AaGlucan; n, 200 lgÆmL
)1
AaGlucan.
ROS accumulation in the medium was measured by the same
method as described in Experimental procedures and expressed as
the corresponding amount of H
2
O
2
. Means ± standard deviation
from three independent measurements are shown.
T. Shinya et al. Novel b-glucan elicitor from A. alternata 102
FEBS Journal 273 (2006) 2421–2431 ª 2006 The Authors Journal compilation ª 2006 FEBS 2425
be expressed in response to the AaGlucan in both BY-
2 cell line and intact tobacco plant. Expression of def-
ense-related genes was monitored by semiquantitative
RT-PCR. We analyzed the expression of known def-
ense-related genes, which are typical of three different
defense pathways: acidic and basic PR3 encoding chi-
tinase enzymes of the PR protein family, PAL and
CCoAOMT from the phenylpropanoid pathway, and
STC and HMGR from the sesquiterpenoid pathway
(Fig. 9). In AaGlucan-treated BY-2 cells (Fig. 9A),
PAL and CCoAOMT genes were transiently induced,
and the STC gene remained expressed during the
course of the experiment compared to the control. No

induction of the PR3 genes was observed. For the ana-
lysis of the AaGlucan eliciting activity in tobacco
plants, we included treatments with laminarin and
sulphated laminarin (Fig. 9B). Expression of the basic
PR3, PAL, CCoAOMT, OMT, HMGR and STC genes
was induced upon treatment with the AaGlucan as
well as with laminarin and sulphated laminarin, the
latter being the most efficient. Of note, as for lamina-
rin, the AaGlucan did not induce the expression of the
acidic PR3 as well as of the acidic PR1 and PR2 (data
not shown), while sulphated laminarin did, as expected
[18]. These results indicated that AaGlucan was active
in tobacco tissue and that the responses of defense-
related genes toward AaGlucan were more similar to
the responses toward laminarin than toward PS3.
Discussion
In previous papers, we reported that the filtrate of
autoclaved A. alternata 102 culture could induce mul-
tiple defense responses, such as increases in chitinase
activity and glucanase activity, in tobacco BY-2 cells
[14,19,20]. The filtrate from A. alternata 102 showed
the highest activity among the preparations obtained
from the 7 fungi that we tested [14]. As an index of eli-
citor activity, we measured the induction of TBC-1, a
tobacco class IV chitinase homolog [14]. Interestingly,
the activity of this isozyme increased remarkably
following treatment with the autoclaved extract of
A. alternata 102, but was not affected by other stimuli
such as UV-irradiation, heat ⁄ cold treatment, or heavy
metal application. Therefore, we regarded the induc-

tion of TBC-1 as an excellent indicator for use in the
purification of the elicitor-active compound. By using
this criterion, we obtained a single elicitor-active frac-
tion after several purification steps. Because there was
no other elicitor-active fraction, the purified com-
pound, mainly consisting of carbohydrate, was thought
to be responsible for the elicitor activity seen in the
autoclaved extract. Structural analysis indicated that
AaGlucan, the elicitor from A. alternata 102, is most
likely a mixture of branched b-1,3-, 1,6-glucan oligo-
saccharides with a DP of 8–17.
The oligosaccharide nature of AaGlucan raises an
interesting question regarding its generation and func-
tion. Usually, oligosaccharide elicitors, such as frag-
ments of chitin and b-glucan, have been postulated to
be generated from the corresponding polysaccharides
in the fungal cell walls by hydrolases secreted by the
host plants as well as by the invading fungus itself
[21]. However, the structure of AaGlucan raises the
A
B
CCoAOMT
PAL
STC
OMT
HMGR
EF1α
α
acidic PR3
basic PR3

03120312
Control AaGlucan
(h)
reffu
B
ma
L
3
S
P
l
G
a
A
u
nac
CCoAOMT
PAL
STC
OMT
HMGR
EF1α
acidic PR3
basic PR3
Fig. 9. Gene expression induced by AaGlucan in BY-2 cells and
tobacco leaves. (A) BY-2 cells were treated with AaGlucan at
200 ngÆmL
)1
. Total RNA was extracted 0, 3, and 12 h after the
treatments and used for semiquantitative PCR. (B) Leaves of

tobacco plants were infiltrated with laminarin (Lam) at 200 lgÆmL
)1
,
laminarin sulfate (PS3) at 200 lgÆmL
)1
, or AaGlucan at 30 lgÆmL
)1
.
Total RNA was extracted 24 h after the treatments and analyzed by
semiquantitative PCR.
Novel b-glucan elicitor from A. alternata 102 T. Shinya et al.
2426 FEBS Journal 273 (2006) 2421–2431 ª 2006 The Authors Journal compilation ª 2006 FEBS
possibility that the oligosaccharides are synthesized as
they are and secreted outside of the cells of the fungus,
similarly to some cyclic glucans and nod-factors secre-
ted by rhizobial bacteria [16,22]. If so, the function of
these b-glucan oligomers for the fungus might be very
different from that of the known cell-wall glucans.
Clarifying the origin of these oligosaccharides and
their function for the fungus itself remains an interest-
ing question for future studies.
The branched oligosaccharide structure of AaGlucan
might explain its solubility in 75% methanol or 75%
ethanol, in which most polysaccharides are insoluble.
Cyclic b-1,6, 1,3-glucans with a DP of 12 isolated from
several Bradyrhizobium sp. are also reported to be sol-
uble in 75% ethanol [22,23]. Although AaGlucan is a
reducing oligosaccharide and does not have a cyclic
structure, the fact that the purification procedure for
AaGlucan was somewhat similar to the purification

procedure for those cyclic glucans is indicative of a
common basis for their solubility. The unique solubil-
ity of AaGlucan in alcoholic solvents may be an
advantage for the future agricultural use of this elicitor
to induce defense responses in intact plants, with the
aim of protecting against pathogen infection, because
the water-repellent characteristics of leaf surfaces
seems to be a potential problem for such applications.
Several carbohydrate and protein elicitors have been
reported to induce defense responses in tobacco plants
[18,24]. Laminarin, oligogalacturonides, fucan, and chi-
tin are carbohydrates which act as elicitors in tobacco
[11,18,25,26], and recently, PS3 was reported to be a
potent elicitor in tobacco and Arabidopsis thaliana [18].
In the present study, we found that the elicitor activity
of AaGlucan compared to these known elicitors was
very high in tobacco BY-2 cells, showing elicitor activ-
ity even at concentrations as low as 25 ngÆmL
)1
. The
higher activity of AaGlucan was especially evident
when the induction of TBC-1 in BY-2 cells was used
to measure the elicitor activity: the elicitor activity of
AaGlucan was 1000 times as high as that of laminarin
(Fig. 7). These results suggest that AaGlucan might
contain some structural unit specifically recognized by
tobacco BY-2 cells. AaGlucan also showed elicitor
activity in intact tobacco plants, although the elicitor
activity was somewhat lower than that in the tobacco
BY-2 cells (Fig. 9). In addition to the difference

between the penetrability of the elicitor into BY-2 cells
and into intact plant tissue, this difference in elicitor
activity may reflect differences in the specificity of the
receptors expressed in these cells. As the responses
induced by AaGlucan in the intact tobacco plant
seems similar to those induced by laminarin, which
was reported to reduce pathogen infection [11], it
might be expected that AaGlucan also has a potential
to protect the plant from pathogenic diseases.
Protein elicitors, such as INF-elicitin, harpin, and
cryptogein, are reported to induce the production of
PR proteins, oxidative burst, and cell death in tobacco
BY-2 cells [24,27,28]. On the other hand, AaGlucan
induced a defense response without cell death
(Fig. 8A). Therefore, the defense signaling pathways
induced by AaGlucan seems to be somewhat different
from the pathways induced by protein elicitors. SA
and MeJA are well-known plant hormones involved in
plant defense signaling. When applied to BY-2 cells,
however, neither of them induced TBC-1 chitinase
(data not shown), suggesting that the signaling path-
way of TBC-1 induction by AaGlucan is not depend-
ent on the SA and MeJA pathways. Because laminarin
has been reported to induce ethylene-dependent PR
proteins but not SA-dependent PR proteins in tobacco
[18], and because the responses induced by AaGlucan
in tobacco tissue were quite similar to those induced
by laminarin, it seems possible that AaGlucan induces
TBC-1 induction through the ethylene-dependent sign-
aling pathway; however, experimental evidence for this

should be obtained in future studies.
Experimental procedures
Fungi and plant materials
The fungus Alternaria alternata 102 was kindly provided by
K Takatori (National Institute of Health Sciences, Tokyo,
Japan). A. alternata 102 maintained on a PDA slant
(potato dextrose agar; Franklin Lakes, NJ, USA) was
inoculated into a 30-mL LSD medium (Linsmaier–Skoog
medium supplemented with 2,4-dichlorophenoxyacetic acid)
[14]. After incubation for a week, the entire culture medium
was transferred into 900 mL of fresh LSD medium and
incubated for another week before use.
The cultured tobacco cell line BY-2 derived from Nicoti-
ana tabacum L. cv. Bright Yellow-2 was maintained in an
LSD medium at 28 °C in the dark with rotation at
130 r.p.m. BY-2 cells were transferred into fresh medium
every week. Cells cultured for 4 days into this weekly per-
iod were used in the experiments. Tobacco plants (Nicoti-
ana tabacum cv Samsun H) used for the experiments were
grown in a greenhouse under controlled conditions (16-h
light period at 22 °C).
Bioassay of elicitor activity
The elicitor activity was evaluated from the induction of
TBC-1 [14]. TBC-1 is the most abundant chitinase isozyme
induced in BY-2 cells by the filtrate of autoclaved
T. Shinya et al. Novel b-glucan elicitor from A. alternata 102
FEBS Journal 273 (2006) 2421–2431 ª 2006 The Authors Journal compilation ª 2006 FEBS 2427
A. alternata 102 culture. Twenty ml aliquots of BY-2 cells
were used for the analysis of elicitor activity. The amount of
elicitor used for each experiment was shown in figures or fig-

ure legends. The chitinase activity was assayed by the activ-
ity staining method following native polyacrylamide gel
electrophoresis (native PAGE) [14,29].
Isolation of elicitor-active fraction
The culture of A. alternata 102 was autoclaved twice at
121 °C for 15 min and then separated by filtration into a fil-
trate and mycelia. The filtrate was lyophilized and then
washed four times with methanol. The methanol-insoluble
residue was dissolved in distilled water. The water-insoluble
matter was removed by centrifugation (1500 g, 5 min). The
obtained supernatant was mixed with 3 volumes of meth-
anol and then centrifuged (1500 g, 5 min). The supernatant
was recovered, and the precipitate was further extracted 3
times with 75% methanol. All 75% methanol fractions were
collected and dried in vacuo. This dried matter was
dissolved in distilled water, to which trichloroacetic acid had
been added to a final concentration of 10% (v ⁄v). The
resulting solution was allowed to stand for 12 h at 4 °C.
After removing the precipitate by centrifugation, the trichlo-
roacetic acid was removed by ether extraction. The water
layer was neutralized to pH 6.0 with 1 m NaOH and dried
in vacuo. The solid matter was dissolved in 75% methanol.
The 75% methanol-soluble fraction was dried in vacuo and
dissolved in distilled water. The solution was dialyzed
(molecular weight cutoff, 12 000 Da) against distilled water.
The dialyzed solution was put on a C18 silica reversed phase
chromatography column that was pre-equilibrated with
water. The column was eluted with 0%, 25%, 50%, and
100% methanol, in that order. The fraction eluted with
25% methanol showed elicitor activity. This fraction was

purified by HPLC on an Inertsil ODS-3 column
(20 · 250 mm; GL Sciences, Tokyo, Japan). The column
was eluted with a linear gradient of methanol solution, from
0% methanol at 0 min to 75% methanol at 45 min, which
was then followed by elution with 100% methanol for
15 min. The flow rate was 5 mLÆmin
)1
throughout. Elicitor
activity and sugar concentration were measured for each
fraction. Sugar concentration was measured by the phenol–
sulfuric acid procedure using glucose as a standard [30].
After the elicitor-active fractions were separated again on
the Inertsil ODS-3 column under these conditions, the elici-
tor-active fractions thus obtained were collected and further
fractionated by gel filtration HPLC on a KW-802.5 column
(8 · 300 mm; Showa Denko, Tokyo, Japan) with water.
Each fraction was monitored with a refractometer.
Structural analysis
The composition of the monosaccharide in each fraction
was analyzed according to the following steps. The fraction
purified by gel filtration HPLC was hydrolyzed in 1 N sulf-
uric acid for 12 h at 105 °C. The resulting hydrolysates
were neutralized with Dowex (OH
–
) (Muromachi Chemicals
Inc., Omuta, Japan) and reduced with 3 mgÆmL
)1
NaBH
4
in 0.5 m NH

3
. Glacial acetic acid was added to the solu-
tion, and the solution was neutralized with Dowex (H
+
).
Alditol acetates were synthesized by the reaction of acetic
anhydrate and pyridine for 2 h at 105 °C, and were ana-
lyzed by GC-MS using a JEOL (Akishima, Japan) mass
spectrometer (SX-102 A) equipped with a Hewlett-Packard
5890 II (Palo Alto, CA, USA) gas chromatograph (DB-1
MS; 0.25 mm · 15 m capillary column; J & W Scientific,
Folsom, CA, USA).
Glycosyl linkages were analyzed by converting the sugars
to partially methylated alditol acetates. Each fraction was
methylated according to the method of Hakomori [31,32],
and the partially methylated sugars were converted to the
corresponding alditol acetates. The partially methylated
alditol acetates were dissolved in chloroform and analyzed
by GC-MS.
Analysis of reducing-end sugar
The purified elicitor was reduced with 3 mgÆmL
)1
NaBH
4
in 0.5 m NH
3
for 12 h. Glacial acetic acid was added to the
solution, and the solution was neutralized with Dowex
(H
+

). Methanol was added to the solution and evaporated.
After the same procedure was performed an additional 3
times to remove borate, the sample was hydrolyzed in 1 N
sulfuric acid for 4 h at 105 °C. The resulting hydrolysates
were neutralized with Dowex (OH
–
) and analyzed by
HPAEC-PAD. HPAEC was carried out with the DX-300
system (Dionex, Sunnyvale, CA, USA) equipped with a
pulsed amperometric detector by using a CarboPac PA-1
column (4 · 250 mm). For analysis of reducing-end sugar,
the column was eluted with 8 mm NaOH at a flow rate of
0.8 mLÆmin
)1
. A cyclic b-1,2-glucan was treated and ana-
lyzed under the same conditions. The cyclic b-1,2-glucan
was kindly donated by M Hisamatsu (Mie University, Mie,
Japan).
NMR spectroscopy and MALDI-TOF/MS
1
H- and
13
C-NMR spectroscopy experiments were per-
formed with an Alpha-500 spectrometer (JEOL). The sam-
ples of purified elicitor were dissolved in D
2
O, and acetone
was used as an internal standard (d
H
¼ 2.225 p.p.m., d

C
¼
31.45 p.p.m).
MALDI-TOF ⁄MS was performed with a Voyager-DE
PRO (Applied Biosystems, Foster City, CA, USA) instru-
ment operated at an acceleration energy of 20 kV, in reflec-
tor mode, and with positive-ion detection. The samples of
purified elicitor were prepared for analysis according to the
method used for analysis of glucans [33]. The purified
elicitor was dissolved in water at a concentration of
Novel b-glucan elicitor from A. alternata 102 T. Shinya et al.
2428 FEBS Journal 273 (2006) 2421–2431 ª 2006 The Authors Journal compilation ª 2006 FEBS
0.4 lgÆlL
)1
. A matrix of 2,5-DHBA (2,5-dihydroxybenzon-
ic acid) acid in 60% acetonitrile at a concentration of
20 lgÆlL
)1
was used. The elicitor and DHBA solutions
were mixed 1 : 1 (v ⁄v), and then a 1 ⁄10 volume of 0.1%
NaCl was added to the solution. A 1.0-lL quantity of the
solution of matrix and analyte was applied to the sample
plate and dried under ambient conditions.
Treatment of BY-2 cells and plants with poly and
oligosaccharides
BY-2 cell suspensions were treated with the following com-
pounds: laminarin, laminaripentaose, N-acetylchitooctaose,
glycol chitin, filtrate of autoclaved A. alternata culture, and
the purified fraction. Laminarin was purchased from Sigma
(St Louis, USA), and laminaripentaose was purchased from

Seikagaku (Tokyo, Japan). N-Acetylchitooctaose was
obtained from Yaizu Suisankagaku Industry (Shizuoka,
Japan). Glycol chitin was synthesized from glycol chitosan
according to the protocol reported previously [29]. After
the addition of each stressor compound, BY-2 cells were
incubated for 3 h at 28 °C in the dark. Then the chitinase
activity of BY-2 cells was measured.
Tobacco plants were infiltrated with the following com-
pounds: laminarin and PS3 obtained as described previ-
ously [18] at a concentration of 200 lgÆmL
)1
and AaGlucan
at a concentration of 30 lgÆmL
)1
. They were dissolved in
Mes-NaOH buffer (2 mm, pH 6). Plant treatments were
performed by infiltration into the mesophyll of fully devel-
oped leaves of the elicitor solutions using a 1 mL syringe.
Laminarinase treatment and periodate oxidation
AaGlucan was treated with laminarinase (b-1,3-glu-
canohydrolase) purchased from MP Biomedicals (CA,
USA). After 2 lg of purified fraction was treated with 0.5
units of laminarinase in 0.1 m phosphate buffer (pH 5.8)
for 24 h at 37 °C, the reaction mixture was heated for
5 min at 90 °C. The elicitor activity was then evaluated
from the induction of TBC-1. To further analyze the elici-
tor structure, AaGlucan solution (2 lg), laminarin solution
(200 lg), or water without glucan was mixed with sodium
periodate solution (40 lmol) and placed in a cold room at
4 °C for 18 h. At the end of this time, excess periodate was

destroyed by addition of 150 lL of ethylene glycol. BY-2
cells were treated with these reaction mixtures (AaGlucan
at 100 ngÆmL
)1
and laminarin at 100 lgÆmL
)1
), and the
induction of TBC-1 was measured.
Detection of cell viability and chemiluminescence
assay
Cell viability was detected using propidium iodide (PI)
which stains nonviable cells [15]. PI-stained cells were coun-
ted under a fluorescence microscope. Generation of reactive
oxygen species induced by the elicitor in the medium of the
suspension-cultured cells was monitored in terms of chemi-
luminescence due to the ferricyanide-catalyzed oxidation of
luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) [34]. A
50 lL aliquot of the elicitor solution was added to 950 lL
of suspension-cultured cells, which contained about 50 mg
fresh weight cells. After incubation, the suspension-cultured
cells were allowed to stand for precipitation, and then
10 lL samples of the supernatant were collected and used
for the assay. A standard solution of H
2
O
2
was used to
make a calibration curve.
RT-PCR gene expression analysis in BY-2 cells
and tobacco plants

Total RNA from tobacco plants was extracted using the
RNeasy plant mini kit (Qiagen, Courtaboeuf, France)
according to the protocol supplied by the manufacturer.
First-strand cDNA synthesis was made from 2 lg of RNA
using the Superscript III reverse transcriptase (Invitrogen,
Cergy Pontoise, France). Total RNA from BY-2 cells was
extracted using Trizol (Invitrogen, CA, USA) according to
the protocol supplied by the manufacturer. First-strand
cDNA synthesis was made from 1 lg of RNA using the with
M-MLV reverse transcriptase (Promega, WI, USA). The
synthesized cDNA was used as template for PCR. Using the
cDNA from plant, PCR was performed with denaturing,
annealing and extension temperatures of 94 ° C for 1 min,
55–58 °C for 1 min and 72 °C for 1 min, respectively, for
Table 2. Primer sequences used in RT-PCR gene expression analysis.
Gene
Accession.
number Sequence, 5¢-3¢. Forward (F), Reverse (R) Product (bp)
Annealing
temp (°C)
EF1a AF120093 (F)TCGCCTTGTGGAAGTTTGAGAC (R)AACATTGTCACCAGGGAGTGCC 665 56
acidic PR-3 M29868 (F)CAGGAGGGTATTGCTTTGTTAGGC (R)ATCTTCCACTGCGTCATTCCGTCC 356 58
basic PR-3 X16938 (F)GCCATAGGAGTGGACCTGCTAAAC (R)AAAAGACCTCTGGTTGCCGC 336 56
PAL X78269 (F)TTACGCCCTCAGAACATCACCC (R)GCTTGGATTCCTTCCTGCTGTC 301 56
CCoAOMT U62734 (F)ATTGGTGTTTTTACTGGTTACT (R)ATTGGTGTTTTTACTGGTTACT 410 56
OMT AF484252 (F)TTGATGTTGGAGGTGGTCTTGG (R)GTCTGGTTTCACTGGTAAAATGGC 302 50
HMGR AF004232 (F)GACACTTGCTGCTGTTTTCAACC (R)TTTCTTCACCACCTCTTCCGTG 310 56
STC AF272244 (F)TCAAGGTGGTGGAAAGATTTGG (R)GCTTAGGTATTCAGAAACAGGTGGC 447 56
T. Shinya et al. Novel b-glucan elicitor from A. alternata 102
FEBS Journal 273 (2006) 2421–2431 ª 2006 The Authors Journal compilation ª 2006 FEBS 2429

25–29 cycles. The annealing temperature and number of
cycles was adapted for each gene (see Table 2 for primer
sequence and annealing temperature). Control reactions to
normalize RT-PCR amplification were run with the EF1a
specific primers (Table 2) and five serial dilutions of each
first strand cDNA. PCRs were performed through 25
cycles and resulted in amplification linearly related to
RNA amounts. The PCR mixture contained 0.4 lm of
each specific primer, 0.5 unit of Taq polymerase (Eurobio,
Courtaboeuf, France). PCR products were separated on a
1% agarose gel and visualized after ethidium bromide
staining. Quantification of the PCR products was made in
gel using the Bio-Rad GelDoc apparatus (Bio-Rad,
Hercules, CA) together with the Bio-Rad quantity one
software. Using the cDNA from BY-2 cells, PCR was
performed with denaturing, annealing and extension
temperatures of 94 °C for 0.5 min, 50–58 °C for 0.5 min
and 72 °C for 0.5 min, respectively, for 24–34 cycles. The
annealing temperature and number of cycles was adapted
for each gene. Twenty-five serial dilutions of each first
strand cDNA. The PCR mixture contained 0.5 lm of each
specific primer, Taq polymerase (Ex-Taq, TAKARA BIO
INC., Shiga, Japan).
Acknowledgements
We thank Dr K. Takatori and Dr M. Aihara (National
Institute of Health Sciences, Tokyo, Japan) for their
advice and the kind gift of A. alternata 102. The
authors are thankful to Patrice de Ruffray for technical
help (IBMP, Strasbourg, France). We thank Mr Y.
Desaki of the Meiji University for help with the MS

analysis. We thank Dr M. Hisamatsu (Mie University,
Mie, Japan) for the kind gift of the cyclic b-1,2-glucan.
We are indebted to Yaizu Suisankagaku Industrial Co.
for the supply of chitosan oligosaccharides. This work
was partly supported by a Ministry of Education,
Culture, Sports, Science, and Technology Grant-in-Aid
for Scientific Research, Scientific Research of Priority
Areas: Single-Cell Molecular Technology.
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