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
3
.
1
. Sequence analysis of CABGLU cDNA By using a differential hybridization technique,
Jung and Hwang [23] identified a cDNA clone
encoding a putative basic b-1,3-glucanase that was expressed in pepper leaves undergoing HR. To
determine a full-length nucleotide sequence of the cDNA clone, one set of nested deletions was
constructed. The full-length cDNA sequence was designated
as CABGLU
C. annuum
basic b-1,3-glucanase. The CABGLU gene contains
1332 bp with an apparent single open reading frame initiated by ATG codon 25 base pairs far
from the 5 end data not shown. The open reading frame ends with two successive stop codons at
position 1104 TGA and TAA. There is a potential polyadenylation signal AATAAA starting 42 bases
upstream from this polyadenylation tail. The predicted
CABGLU protein
has a
typical hydrophobic signal peptide cleavage site located
between residues 20 glycine and 21 glutamine. The pepper CABGLU cDNA encodes a putative
polypeptide of 359 amino acids with a predicted molecular mass of 39 226 Da and a predicted
isoelectric point pI of 9.46. Moreover, the CABGLU gene has a C-terminal extension signal
sequence, which is necessary for targeting to the vacuole [9,31]. An interesting feature in the amino
acid sequence of the CABGLU gene is the existence of a consensus sequence for N-glycosylation
Asn-Ala-Thr at amino acid positions 349 – 351.
The predicted polypeptide sequence of the CABGLU cDNA encoding a putative pepper basic
b-1,3-glucanase was compared with the previously sequenced
b-1,3-glucanases from
other plant
species Fig.
1. The
amino acid
sequence homology search revealed that the protein encoded
by CABGLU shares a significant identity with other known plant basic b-1,3-glucanases. The
basic b-1,3-glucanase of pepper has 77.4 identity with a tobacco Nicotiana plumbaginifolia basic
b-1,3-glucanase [32,33], 64.6 identity with a potato Solanum tuberosom basic b-1,3-glucanase
[34],
and 58.7
identity with
a tomato
Lycopersicon esculentum basic b-1,3-glucanase [35]. The amino acid sequence of CABGLU has
only 42.6 identity with tomato tomQ’a gene encoding
acidic b-1,3-glucanase
[19]. The
alignment shows that the basic b-1,3-glucanase has a C-terminal extension which does not contain
acidic b-1,3-glucanase.
3
.
2
. DNA gel blot analysis of CABGLU cDNA DNA gel blot analysis was performed on C.
annuum genomic DNA digested with EcoRI, EcoRV and XbaI which do not possess specific
recognition sites in the CABGLU cDNA sequence. To avoid cross-hybridization of several isoforms
of pepper b-1,3-glucanase gene, hybridization and blot washing conditions were performed at high
stringency. Two major bands were detected in EcoRI digests, while one major band was detected
in both EcoRV and XbaI digests Fig. 2. These results indicated that at high stringency, the
CABGLU gene is present as one or two copies per C. annuum genome.
3
.
3
. Organ-specific expression of CABGLU gene To examine organ-specific expression of the
CABGLU gene, RNA gel blot analysis was per- formed using transcripts of various tissues of pep-
per plants Fig. 3. The CABGLU mRNAs were not detectable in healthy leaves and fruits. In
contrast, the expression of the CABGLU gene was strong in roots, but remained at low levels in stems
and flowers.
3
.
4
. Expression of CABGLU gene by pathogen infection
To study CABGLU gene expression after bacte- rial infection, pepper leaves were inoculated with
the strains Ds1 or Bv5-4a of X. campestris pv. 6
esicatoria which were virulent or avirulent to pepper cultivar Hanbyul, respectively. Fig. 4
shows the CABGLU mRNA expression pattern in the compatible and incompatible interactions be-
tween pepper and X. campestris pv. 6esicatoria. No transcripts homologous to CABGLU gene were
detected in the healthy pepper leaves. In the com- patible interaction, accumulation of CABGLU
mRNA was slightly detected at 18 h after inocula- tion, continued to rise thereafter, and remained at
a high level to 30 h after inoculation. In the incompatible
interaction, accumulation
of CABGLU mRNA was more inducible than in the
compatible interaction. In particular, the accumu- lation of CABGLU mRNA drastically increased in
the infected leaves at 24 h after inoculation of the avirulent strain.
To examine CABGLU mRNA expression pat- tern in the compatible and incompatible interac-
tions between pepper and P. capsici Fig. 5, pepper stems were also inoculated with the two
isolates of P. capsici S197 or CBS178.26 that were
virulent or avirulent to pepper cultivar Hanbyul, respectively. CABGLU mRNA was slightly de-
tected in healthy or wounded pepper stems. In the compatible and incompatible interactions, some
induction of CABGLU mRNA was found at 24 h after inoculation. The highest level of the tran-
scripts was detected in the compatible response at 48 h after inoculation, but the mRNA levels grad-
ually declined from 48 to 96 h after inoculation, when the infected stems completely withered. In
contrast, the mRNA levels of CABGLU gene in the incompatible response gradually increased
from 24 to 72 h after inoculation.
3
.
5
. Induction of CABGLU mRNA by ethephon and methyl jasmonate
The levels of expression of the CABGLU mRNA in the pepper leaves by abiotic elicitor
treatment were examined by RNA gel blot analy- sis Fig. 6. Following treatment with ethephon
and methyl jasmonate MeJA, the CABGLU
Fig. 1. Comparison of the deduced amino acid sequences of CABGLU cDNA with basic b-1,3-glucanases from tobacco Nicotiana plumbaginifolia, accession no. M63634, [32,33], potato Solanum tuberosom, accession no. U01901, [34], tomato Lycopersicon
esculentum, accession no. M80608, [35], and with acidic b-1,3-glucanase from tomato tomQ’a accession no. X74905, [19]. Arrow indicates the position of a putative leader cleavage site in pepper CABGLU. Asterisk and bar indicate the complete match
sequence of phenylalanine-glycine and N-glycosylation site, respectively. Dashes mark the spacing in amino acid sequences that was done for optimal alignment.
Fig. 2. Genomic DNA gel blot analysis of CABGLU gene encoding a basic b-1,3-glucanase in pepper plants. The en-
zymes that were used for digestion of genomic DNA are indicated above each lane. The numbers on the left indicate
the length of the fragments in kilobases. Twenty micrograms of digested genomic DNA were separated in each lane.
remained at a high level by 24 h after treatment Fig. 7A. In pepper leaves treated with MeJA,
CABGLU transcripts were detected at 12 h after treatment and increased drastically thereafter Fig.
7B.
4. Discussion
