including tobacco [13], potato [14], bean [15], and rice [16]. In our earlier study, two acidic b-1,3-glu-
canase isoforms and six basic b-1,3-glucanase iso- forms were detected in pepper stems infected with
Phytophthora capsici [17]. We further isolated a b-1,3-glucanase with antifungal activity from pep-
per stems [4]. The two acidic and six basic b-1,3- glucanases were also induced and accumulated in
pepper leaves infected with Xanthomonas campes- tris pv. 6esicatoria [18]. However, the role of b-1,3-
glucanase either in defense responses or in physiological and developmental processes has not
been well understood, since the enzyme activity exists in several isoforms that differ in amino acid
sequences, cellular localization and inducibility [19].
Based on amino acid sequence identities, b-1,3- glucanases from tobacco have been classified into
three structural classes [20]. The class I b-1,3- glucanase is approximately 33 kDa, basic proteins,
and accumulates intracellularly at a site presumed to be vacuoles [21]. This vacuolar localization
correlates with the presence of a distinctive C-ter- minal extension that contains sorting information
for proper targeting to the vacuole [19]. Class II b-1,3-glucanase includes the acidic isoforms such
as PR-2a, PR-2b, and PR-2c, formerly named PR-2, PR-N, and PR-O, respectively [13,20]. The
single representative of the class III isoforms is PR-Q’ found in TMV-infected tobacco leaves [20].
Both class II and III b-1,3-glucanases accumulate predominantly in the extracellular space, i.e. the
apoplast. In some cases, the acidic b-1,3-glu- canases were shown to be present in intercellular
washing fluid IWF from pepper tissues infected with pathogens [17,18].
To gain a better understanding of the role of PR-proteins including b-1,3-glucanase, we con-
structed a cDNA library from pepper leaves infected with avirulent strain Bv5-4a of X. campes-
tris pv. 6esicatoria [22]. More recently, some PR-gene clones encoding b-1,3-glucanase, chiti-
nase, thionin, and lipid transfer protein were isolated from this cDNA library using the differ-
ential hybridization technique [23]. In this paper, we report on the isolation and sequence analysis of
a full-length cDNA clone encoding a basic b-1, 3-glucanase from pepper leaves. We further show
that expression of the basic b-1,3-glucanase gene is induced in pepper leaves and stems by pathogen
infection and abiotic elicitor treatments.
2. Materials and methods
2
.
1
. Plant and pathogens Pepper Capsicum annuum L., cv. Hanbyul
plants were raised in a growth room to the four or six-leaf stages, as previously described [24].
The two strains Ds1 and Bv5-4a of X. campes- tris pv. 6esicatoria that were virulent and avirulent
to the pepper cultivar Hanbyul, respectively, were used in this study [25]. The bacteria strains were
grown overnight in YN medium 5 g yeast extract, 8 g nutrient broth and 1 l H
2
O at 28°C. The bacteria were harvested from broth cultures by
centrifugation, and suspended at 10
8
CFUml in sterile tap water prior to inoculation. Inoculation
with X. campestris pv. 6esicatoria was done by vacuum-infiltrating the bacterial suspension into
the abaxial side of fully expanded leaves of pepper plants at six-leaf stage with an automizer con-
nected to a compressor.
Two isolates, S197 and CBS 178.26, of Phytoph- thora capsici which are virulent and avirulent to
pepper cultivar Hanbyul, respectively, were used in this study [26]. The fungal pathogens were
grown on oatmeal plates for 10 days at 28°C in dark. Zoospores were harvested from sporulating
plates, and adjusted to 10
5
zoospores per ml with sterile tap water prior to inoculation. A small
quantity of cotton soaked in zoospore suspensions was placed on the bottom region of each pepper
stem. The inoculated sites were then covered with plastic tape to maintain moist conditions. Plant
samples were taken at various time intervals after inoculation. Harvested leaves and stems were im-
mediately frozen in liquid nitrogen, and stored at −
70°C until isolated for total RNA.
2
.
2
. Abiotic elicitor treatment Approximately 10 mM ethephon, 5 mM sali-
cylic acid SA, 100 mM methyl jasmonate MeJA, 2000 mgml DL-b-2-amino-n-butyric acid BABA,
and 20 mgml benzo-[1 – 3]-thiadiazole-7-carboth- ioic acid S-methyl ester BTH, CGA245704, a 98
active ingrediant formulation in sterile water were applied as a spray to pepper plants at six-leaf
stage. Phosphonic acid H
3
PO
3
or hydrochloric acid HCl, which are breakdown products gener-
ated during the conversion of 2-chloroethylphos- phonic acid to ethylene, was also applied at equal
molarity to ethephon to evaluate the effect of them. The pepper plants were incubated in a
growth-room at 28°C with 16-h day length. Plants applied with ethephon or MeJA were enclosed in a
vinyl bag. Pepper leaf samples were harvested at various time intervals after application, immedi-
ately frozen in liquid nitrogen, and stored at − 70°C until isolated for total RNA.
2
.
3
. Isolation of a basic b-
1
,
3
-glucanase cDNA clone
CABGLU To isolate pathogenesis related genes from pep-
per leaves, individual cDNA clones strongly or differentially expressed in the infected leaves were
selected using a differential hybridization tech- nique [23]. These cDNA genes were designated
CAIs Capsicum annuum induced genes. After the 5 partial sequencing of CAI genes using automatic
DNA sequencer ABI310, Applied Biosystem, the 5 end partial nucleotide and deduced peptide se-
quences obtained were analyzed using the PC Gene software system and BLAST network
services at the National Center for Biotechnology Information NCBI [27]. The partial nucleotide
sequence data of the CAI20 have been deposited in the EMBLGenBank database under accession
number AF082724 [23].
2
.
4
. DNA sequencing and analysis To determine a full-length sequence of CAI20,
the cDNA in the pBluescript SK − was se- quenced on ABI310 DNA sequencer Applied
Biosystem using Thermo-cycle sequenase kit Amersham with T3 or T7 primer according to
manufacture’s instruction. Progressive deletions were obtained by using Erase-a-Base system
Promega, Madison, WI, USA. The amino acid alignments were manually adjusted to compare
cDNA clones with those of other organisms.
2
.
5
. RNA gel blot analysis Total RNA was extracted from pepper leaves,
stems, root, flowers, and fruits by the guanidinium thiocyanate method [28]. The concentration and
integrity of total RNA in individual extracts were determined by UV absorbance and staining with
ethidium bromide, respectively. Total RNA 30 mg was denatured by heating at
65°C for 10 min in a formaldehyde gel-loading buffer, separated by electrophoresis on 7.4 form-
aldehyde gels, and transferred to nylon mem- branes Hybond N
+
, Amersham [29]. RNA was cross-linked on the blots by UV illumination. An
EcoRIXhoI restriction fragment in pBluescript SK − recombinant plasmid carrying the pepper
b-1,3-glucanase gene was
32
P-labeled with a ran- dom prime kit Boehringer Mannheim. Prehy-
bridization and hybridization was performed at 65°C in 5 wv dextran sulfate, 0.25 M disodium
phosphate pH 7.2, 7 wv sodium dodecyl sul- fate SDS, and 1 mM EDTA. The membranes
were washed twice with 2X SSC and 0.1 SDS for 10 min at room temperature and finally several
times with 0.1 × SSC and 0.1 SDS for 5 min at 65°C. To control equal transfer of RNA, the blots
were co-hybridized with a C. annuum 25S rRNA probe.
2
.
6
. DNA gel blot analysis C. annuum genomic DNA was prepared from
young leaves, as previously described by Hong et al. [30]. Each gram of tissue ground under liquid
nitrogen was suspended in 3 ml extraction buffer [8.0 M urea, 50 mM Tris – HCl pH 7.5, 20 mM
EATA, 350 mM NaCl, 2 wv SDS, 5 vv phenol and 20 mM EDTA 2-mercaptoethanol].
After successive extractions with phenolchloro- formisoamylalcohol25:24:1,
vvv, the
high molecular weight genomic DNA was recovered by
spooling. Twenty micrograms of genomic DNA were digested with appropriate restriction en-
zymes, according to the protocols described by Sambrook et al. [29]. After ethanol precipitation,
completely digested genomic DNA was resus- pended in 10 mM Tris and 1 mM EDTA prior to
gel electrophoresis. The DNA fragments were sep- arated on 0.8 agarose gel. The DNA was trans-
ferred to Hybond N + Amersham membrane, and hybridized to
32
P-labeled CABGLU gene probe at 65°C as described above.
3. Results
