Biotechnol Lett (2010) 32:539–546
DOI 10.1007/s10529-009-0192-1
ORIGINAL RESEARCH PAPER
Chitinolytic and antifungal activity of a Bacillus pumilus
chitinase expressed in Arabidopsis
Ali Dehestani • Kamal Kazemitabar • Gholamreza Ahmadian
Nadali Babaeian Jelodar • Ali Hatef Salmanian •
Mehdi Seyedi • Heshmat Rahimian • Seyedhadi Ghasemi
Received: 19 November 2009 / Revised: 8 December 2009 / Accepted: 9 December 2009 / Published online: 25 December 2009
Ó Springer Science+Business Media B.V. 2009
Abstract The Bacillus pumilus SG2 chitinase gene
(ChiS) and its truncated form lacking chitin binding
(ChBD) and fibronectin type III (FnIII) domains were
transformed to Arabidopsis plants and the expression,
functionality and antifungal activity of the recombi-
nant proteins were investigated. Results showed that
while the two enzyme forms showed almost equal
hydrolytic activity toward colloidal chitin, they exhib-
ited a significant difference in antifungal activity.
Electronic supplementary material The online version of
this article (doi:10.1007/s10529-009-0192-1) contains
supplementary material, which is available to authorized users.
A. Dehestani
K. Kazemitabar
N. B. Jelodar

S. Ghasemi
Department of Plant Breeding, Faculty of Agronomic
Sciences, Sari Agricultural Sciences and Natural
Resources University, P. O. Box 578, Sari, Iran
A. Dehestani
G. Ahmadian (&)
S. Ghasemi
Department of Molecular Genetics, National Institute
for Genetic Engineering and Biotechnology (NIGEB),
P. O. Box 14155-6343, Tehran, Iran
e-mail: gholamrezaahmadian@yahoo.ca
A. Dehestani
A. H. Salmanian
M. Seyedi
Department of Plant Biotechnology, National Institute
for Genetic Engineering and Biotechnology (NIGEB),
P. O. Box 14155-6343, Tehran, Iran
H. Rahimian
Department of Plant Protection, Faculty of Agronomic
Sciences, Sari Agricultural Sciences and Natural
Resources University, P. O. Box 578, Sari, Iran
•
Recombinant ChiS in plant protein extracts displayed
a high inhibitory effect on spore germination and
radial growth of hyphae in Alternaria brassicicola,
Fusarium graminearum and Botrytis cinerea, while
the activity of the truncated enzyme was strongly
abolished. These findings demonstrate that ChBD and
FnIII domains are not necessary for hydrolysis of
colloidal chitin but play an important role in hydrolysis
of chitin–glucan complex of fungal cell walls. Twenty
microgram aliquots of protein extracts from ChiS
transgenic lines displayed strong antifungal activity
causing up to 80% decrease in fungal spore germina-
tion. This is the first report of a Bacillus pumilus
chitinase expressed in plant system.
Keywords Antifungal activity

Arabidopsis thaliana
Bacillus pumilus

Chitin binding domain
Chitinase activity
Introduction
Chitinases play an important role in plant defense
against attack by a wide range of phytopathogenic
organisms. Inhibitory action of the plant chitinases is
achieved either by direct hydrolysis of the chitin in
the fungal cell walls or by releasing polysaccharide
oligomers stimulating plant defense reactions (Grover
and Gowthaman 2003; Kasprzewska 2003). Based on
amino acid sequence similarity, chitinases are clas-
sified into two glycosyl hydrolase families, 18 and 19.
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Family 19 chitinases are generally found in plants
and some bacteria, while family 18 chitinases are
found in a broad range of organisms including
viruses, bacteria, fungi and animals as well as plants
(Bishop et al. 2000). Most of the chitinase genes
transferred to plants are from plant or fungal sources
(Grover and Gowthaman 2003; Punja 2006) and there
have been only a few reports of bacterial chitinase
expression in plants (Suslow et al. 1988; Lund et al.
1989; Itoh et al. 2003; Hassan et al. 2009).
The ChiS gene (GenBank accession No. DQ355512)
was isolated from Bacillus pumilus SG2 and consists of
an N-terminal glycosyl hydrolase family 18 catalytic
domain (GH18) which is connected to a C-terminal
chitin binding domain (ChBD) by an intervening
fibronectin type-III-like domain (FnIII) (Ahmadian
et al. 2007). It was previously demonstrated that
purified recombinant ChiS expressed in E. coli inhib-
ited the in vitro growth of some fungal pathogens at
low concentrations (unpublished results). Hence, it was
hypothesized that it can be used to produce transgenic
plants with enhanced resistance against phytopatho-
genic fungi.
This study was conducted to determine whether
the ChiS protein can be expressed in Arabidopsis
plants, and to evaluate if these recombinant proteins
retain their biological activity.
Furthermore, to elucidate the role of ChBD and
FnIII on chitinolytic and antifungal activities, a
truncated form of the enzyme lacking the ChBD
and FnIII regions was also studied.
In this study we showed that Arabidopsis-
expressed ChiS exhibited antifungal activity against
Alternaria brassicicola, Botrytis cinerea, and Fusar-
ium graminearum in in vitro assays.
Furthermore, it was demonstrated that the ChBD
and FnIII domains of the ChiS enzyme play an
important role in antifungal activity, but not in
hydrolytic activity toward colloidal chitin.
Materials and methods
Vector construction and Arabidopsis
transformation
The ChiS and GH18 coding sequences were amplified
by PCR from pQE30-ChiS plasmid as template using
primers Chi-F1: 50 -GGGGGTCTAGAATGAGTTCT
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Biotechnol Lett (2010) 32:539–546
GACAAAAGTTA-30 (Forward) and Chi-R3: 50 -GGGG
GGAGCTCGAGCCCACTCTCTCTTTA-30 (Reverse)
for ChiS and Chi-F1 and Chi-R4: 50 -GGGGGGAG
CTCTTATGATTGAATAAAATC-30 for GH18. The
underlined sequences in forward and reverse primers
correspond to XbaI and SacI sites, respectively.
Amplified fragments were cloned into the XbaI and
SacI sites between the cauliflower mosaic virus
(CaMV) 35S promoter and the nopaline synthase gene
(nos) polyadenylation site of pBI121binary vector
(Clontech laboratories, California, USA) to yield
pBI121-ChiS and pBI121-GH18 recombinant vectors.
The vectors were transformed into Agrobacterium
tumefaciens strain GV3850 by the heat shock method
(Ausubel et al. 1995).
Arabidopsis thaliana ecotype Col-0 plants were
grown in a growth chamber at 22°C with a 16 h
photoperiod at 200 lE m-2 s-1and transformed by
recombinant Agrobacterium cells using vacuum
infiltration method (Bechtold et al. 1993). Putative
transgenic plants were selected on MS agar plates
containing 75 mg kanamycin l-1, transferred to soil
and grown to set seeds (T2). The T2 homozygous
progenies were selected for further analysis.
DNA extraction and PCR
Total DNA was isolated from leaf tissues of the
transgenic lines using a CTAB method and the
presence of the transgenes was detected by PCR using
the gene specific primers used for vector construction.
RNA isolation and RT-PCR
Total RNA was isolated using Trizol reagent (Invitrogen,
USA) according to the manufacturer’s instructions. The
first strand cDNA synthesis was performed with Revert-
Aid kit (Fermentas) using oligo (dT)18 primers and 2 lg
of RNA as template. Synthesized cDNA was subjected to
PCR using the gene specific primers used for vector
construction. The Actin gene of Arabidopsis was ampli-
fied with the primers Act-F: 50 -GGCGATGAAGCT-
CAATCCAAACG-30 and Act-R: 50 -GGTCACGACC
AGCAAGATCAAGACG-30 to serve as control.
Protein extraction and immunoblot analysis
Total soluble proteins were isolated from plant leaf
tissues with 100 mM sodium acetate buffer (pH 6.5)
Biotechnol Lett (2010) 32:539–546
containing 1 mM PMSF and 8 mM 2-mercap-
toethanol. The protein content of the extracts was
determined using the Bradford method with BSA as
the standard. Samples, 20 lg, were separated on 10%
SDS-PAGE and transferred onto PVDF membranes.
ChiS or GH18, 0.1 lg, expressed in E. coli, were
included to serve as positive controls. The blots were
probed with rabbit anti-ChiS (1:2000) or anti-GH18
(1:2000) polyclonal antibodies. Horseradish peroxi-
dase-conjugated goat anti-rabbit IgG (1:5000) was
used as secondary antibody and the blots were
developed with 4-chloro-1-naphthol.
ELISA analysis
The expression levels of ChiS and GH18 proteins
were analyzed by ELISA. Microtiter plates were
coated with 20 lg protein extracts and detection of
recombinant proteins was performed using the same
antibodies described for immunoblot analysis.
Colorimetric measurement of the reactions was car-
ried out with ABTS at 405 nm using a microplate
reader. Serial dilutions of purified ChiS or GH18
(10–200 ng) were included to plot a graph of absor-
bance against concentration. The quantities of recom-
binant ChiS and GH18 in protein extracts of different
transgenic lines were expressed as percentage of total
soluble protein (TSP). Five transgenic lines with
different expression levels were selected for chitinase
activity and spore germination inhibitory assays.
Chitinase activity assay
The enzyme activity was estimated using the method
described by Boller et al. (1983) with some modifi-
cations. The reaction mixture consisted of 400 ll
100 mM sodium acetate buffer (pH 6.5) containing
60 lg of protein from transgenic lines and 100 ll
0.5% colloidal chitin. After 1 h at 37°C, the reaction
was stopped by adding 200 ll 1 M NaOH solution.
After 5 min centrifugation at 60009g, liberated
N-acetylglucosamine (GlcNAc) content was esti-
mated. Briefly, 500 ll supernatant was transferred
to a glass tube and 100 ll potassium tetraborate
buffer was added. The solution was boiled for 3 min
and 3 ml DMAB reagent (10% (w/v) 4-(dimethyl
amino) benzaldehyde in glacial acetic acid/11.5 M
HCl (7:1 v/v)) was added. After 20 min at 37°C,
the absorbance at 544 nm was measured using a
541
microplate reader. The enzyme activity was calcu-
lated from a standard curve developed using known
concentrations of GlcNAc. One unit of chitinase
activity was defined as the amount of enzyme which
liberates 1 lmol GlcNAc per min at pH 6.5 and 37°C.
Specific activity was expressed as units per mg total
protein.
In vitro antifungal activity assay
Protein extracts from different transgenic lines were
equalized for specific chitinase activity (1 mU ll-1)
and 25 ll protein extracts from different transgenic
lines were dispensed into wells of a microtiter plate.
Subsequently, 75 ll Alternaria brassicicola or Botry-
tis cinerea spore suspensions (2 9 104 spores ml-1)
were added to each well. The plates were incubated at
24°C for 36 h and spore germination was evaluated
by measuring the OD595 value. The turbidimetric
values were normalized to the mean turbidity of the
control (only buffer) and the effects of different
extracts on spore germination were estimated as
described by Moravcikova et al. (2007).
Antifungal activity of the protein extracts was also
evaluated by the hyphal growth inhibition assay
(Schlumbaum et al. 1986) with some modifications.
Mycelial plugs from actively growing fungi including
A. brassicicola, B. cinerea and Fusarium graminea-
rum were placed in the center of PDA Petri plates
(100 mm). After 24–48 h, when the colony diameters
were about 3–4 cm, sterile filter paper disks (5 mm)
were laid on the agar surface 20 mm from the edge of
the fungal colonies. Approximately 40 ll 50 mM
sodium acetate buffer (pH 6.5) containing 80 lg
protein extracts from transgenic and non-transgenic
plants were applied to each disk. Paper disks loaded
with only buffer served as negative controls. After
18 h, an additional 20 ll protein solution (40 lg) was
added to each disk. The plates were incubated at
24°C until the inhibition zones around the disks were
developed.
Experiment design and data analysis
Each of the ELISA, chitinase activity and spore
germination assays were performed as three inde-
pendent experiments with five replicates within each
experiment. Data were analyzed using the general
linear model (GLM) procedure of the SAS (Statistical
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542 Biotechnol Lett (2010) 32:539–546

Analysis System Inc., NC, USA). When treatment
effects were significant (P \ 0.05), means compari-
son was performed using the least significant differ-
ence (LSD) (P = 0.05).
Results
Molecular analysis of transgenic plants
Ninety-seven kanamycin-resistant transgenic plants
were tested by PCR. Out of these, 91 plants showed
the ChiS or GH18 expected fragments (1729 and
1155 bp, respectively), while DNA from control
plants showed no amplification (Supplementary
Fig. 1).
RT-PCR analysis revealed that all the analyzed
PCR-positive plants had the ChiS and GH18 tran-
scripts which were not present in non-transformed
control plants (Supplementary Fig. 2). No amplifica-
tion was observed when PCR reaction was performed
on the same set of RNA samples without reverse
transcription.
Expression of the ChiS and GH18 proteins was
confirmed by Immunoblot analysis. The ChiS trans-
genic lines showed positive immunological reaction
and a distinct 63 kDa band corresponding to ChiS was
detected. Line AChiS24, a ChiS-containing line as
confirmed by PCR and RT-PCR, failed to express the
corresponding protein (Supplementary Fig. 3A). All
of the tested GH18 transgenic lines had the corre-
sponding 45 kDa band indicating high fidelity trans-
lation of the transcripts (Supplementary Fig. 3B). The
recombinant ChiS and GH18 co-migrated with the
control proteins (recombinant ChiS and GH18 pro-
duced in E. coli).
Comparison of the expression levels in different
transgenic lines revealed that there was a significant
difference among the transgenic lines (Table 1). The
highest level of ChiS expression of 0.19% of total
soluble protein (TSP) was achieved in line AChiS11,
while the maximum GH18 expression (0.17% of
TSP) was observed in transgenic line AG31.

Table 1 Expression levels, chitinase activities and spore germination inhibitory effects of five ChiS and five GH18 transgenic lines
Line Total Specific Relative increased Recombinant Spore germination
activity (U)a activity (U mg-1)b activity (%)c protein (% TSP)d inhibitory activity (%)e

Non-transgenic – 0.1 0 – 13
AChiS5 0.7 1.8 14 0.12 51
AChiS11 1.1 2.8 23 0.19 80
AChiS12 0.6 1.6 13 0.11 45
AChiS18 1 2.4 20 0.16 69
AChiS32 0.6 1.5 12 0.1 45
Mean (ChiS) 2
AG4 0.6 1.6 13 0.1 17
AG12 0.8 2.1 18 0.15 21
AG15 0.8 1.9 16 0.12 19
AG20 0.8 2.2 18 0.15 22
AG31 0.9 2.4 20 0.17 23
Mean (GH18) 2
The data presented are means of three independent experiments each with five replications (standard errors of the means are not
shown)
a
One unit of chitinase activity was defined as the amount of enzyme, which liberates 1 lmol of GlcNAc per min at pH 6.5 and 37°C.
Total activity was calculated using 0.4 mg protein extract
b
Specific activity was expressed as units per milligram of total extracted proteins
c
Increased chitinase activity over the non-transgenic control
d
Percentage of total soluble protein
e
Inhibitory activity of protein extracts on spore germination was estimated using turbidimetric value (595 nm) of each sample
according to the method described by Moravcikova et al. (2007)

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Chitinase activity of the recombinant proteins
Chitinase activity assays revealed that, compared to
untransformed control plants, both ChiS and GH18
transgenic lines experienced up to 23-fold increases
in chitinolytic activity (Table 1).
Low chitinase activities in the protein extracts
from non-transformed control plants (Table 1) might
be attributed to endogenous chitinases. The chitinase
activities of ChiS and GH18 transgenic lines corre-
lated with their expression levels (Fig. 1a). The
results revealed that the mean chitinase activity of
the GH18 transgenic lines did not show any signif-
icant difference with the mean chitinase activity of
the ChiS lines indicating chitinolytic activity toward
colloidal chitin was not affected by elimination of
ChBD and FnIII domains (Table 1).
Fig. 1 a Correlation of chitinase activities of protein extracts
from various ChiS and GH18 transgenic lines with their
expression levels as percentage of total soluble protein
(% TSP). b Correlation of spore germination inhibitory
activities of protein extracts from various ChiS and GH18
transgenic lines with their expression levels as percentage of
total soluble protein (% TSP)
543
Antifungal activity of recombinant proteins
Spore germination assay of Alt. brassicicola and Bot.
cinerea revealed significant differences between
antifungal activities of ChiS and GH18 transgenic
lines. The recombinant ChiS and GH18 proteins
(from AChiS11 and AG31lines, respectively), which
were equalized for chitinases activity, showed differ-
ent inhibitory activities against fungal spore germi-
nation (Table 2).
Protein extracts from GH18 transgenic lines had a
low inhibition of spore germination (17–23% inhibi-
tion), while the extracts from ChiS transgenic lines
decreased spore germination by 45–80% (Table 1).
The expression levels of the ChiS transgenic lines
correlated with their spore germination inhibitory
activities, while the inhibitory activities of the GH18
transformed lines were not significantly correlated to
their expression levels (Fig. 1b).
The highest inhibitory activity of 80% was for
AChiS11 which showed the highest level of ChiS
expression and chitinase activity. In contrast, AG31
which had a high chitinase activity, exhibited a very
low inhibitory effect on fungal spore germination
(Table 1).
Hyphal growth inhibition assays of Fusarium
graminearum, Alt. brassicicola and Bot. cinerea were
performed using protein extracts from AChiS11 or
AG31 lines which had the highest chitinase activities
within each of the ChiS or GH18 transgenic lines.
Results showed that protein extract from line AG31
restrained Alt. brassicicola hyphal growth to some
extent (Fig. 2c), but did not show any inhibitory
activity against Bot. cinerea and F. graminearum. In
Table 2 Antifungal activities of the protein extracts from non-
transgenic Arabidopsis (wild type) or transgenic lines AChiS11
and AG31 on spore germination of Botrytis cinerea and
Alternaria brassicicola was defined by turbidity measurement
of the fungal spore suspensions at 595 nm
Line Spore germination (OD595)
Bot. cinerea Alt. brassicicola
Non-transgenic 0.56 0.53
AChiS11 0.12 0.14
AG31 0.43 0.45
The data presented are means of three independent experiments
each with five replications (standard error bars of the means are
not shown)
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Fig. 2 The antifungal activity of Bacillus pumilus SG2
chitinase (ChiS) and its catalytic domain (GH18) against a
Bot. cinerea, b F. graminearum, and c Alt. brassicicola. In
vitro hyphal growth-inhibition assay was performed in 100 mm
PDA plates as described in materials and methods. Protein
extracts from AChiS11 and AG31transgenic lines (expressing
contrast, the protein extract from line AChiS11
inhibited the hyphal growth of all fungi tested
(Fig. 2), suggesting a higher antifungal activity for
ChiS in comparison with GH18.
Discussion
Chitinases protect plants from invading fungal patho-
gens by degrading the chitin of fungal cell walls
(Kasprzewska 2003; Bishop et al. 2000). Several
chitinases have been isolated from different organ-
isms, but not all show antifungal activity in in vitro
assays (Patil et al. 2000). Chitinase, ChiS, from
Bacillus pumilus SG2 exhibits in vitro antifungal
activity against fungal plant pathogens (unpublished
results) and the present study the ChiS gene and its
truncated form lacking ChBD and FnIII domains
were transformed to Arabidopsis plants and expres-
sion, functionality and antifungal activity of the
recombinant proteins were investigated.
The ChiS and GH18 proteins were efficiently
expressed by Arabidopsis plants without any post-
translational modifications (Supplementary Fig. 3).
Two previous studies indicated that the plant-
expressed bacterial chitinases migrated as a series of
discrete bands with slower or similar mobility to the
native proteins (Lund et al. 1989; Itoh et al. 2003).
The expression level of transgenic lines (up to
0.19% of TSP) is in accordance with other reported
values of recombinant proteins expressed in trans-
genic plants under the control of the CaMV 35S
promoter (Suslow et al. 1988; Gidoni et al. 1988).
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Biotechnol Lett (2010) 32:539–546
ChiS and GH18, respectively) were used for hyphal growth-
inhibition assay. Paper disks were loaded with approximately
120 lg protein aliquots from lines AChiS11 (disk 2), AG31
(disk 3) and non-transgenic control plant (disk 4) in 50 mM
acetate buffer (pH 6.5). Acetate extraction buffer alone (disk 1)
served as buffer blank
The chitinase activities of extracts from ChiS or
GH18 transgenic lines were positively correlated with
their expression levels (Fig. 1a). Mean chitinase
activities of the extracts from ChiS transgenic lines
showed no significant differences with the extracts of
GH18 transformed lines (Table 1), indicating that the
chitinase activity of the enzyme is not influenced by
ChBD and FnIII domains. The effect of ChBDs on
chitinolytic activity of chitinases is still contradictory
in the literature. While some previous reports indi-
cated ChBDs do not significantly contribute to the
chitinolytic activity of chitinase enzymes (Kuranda
and Robbins 1991; Fung et al. 2002), there is some
evidence suggesting that presence of a ChBD endows
the enzyme with a higher chitinolytic activity
(Yamagami and Funatsu 1996; Itoh et al. 2002).
Although careful examinations are necessary to
investigate the role of FnIII in the chitinase enzymes,
the reports to date suggest that FnIII domains of the
chitinases do not directly influence the chitinolytic or
antifungal activities, but they can improve the
function of ChBD by stabilizing the enzyme bound
to the substrate (Watanabe et al. 1994).
Recombinant ChiS effectively inhibited the spore
germination and hyphal growth of F. graminearum,
Alt. brassicicola and Bot. cinerea, while the anti-
fungal activity of the GH18 was strongly abolished.
The significant decrease in GH18 antifungal activity
implies the important role of ChBD and FnIII in the
antifungal activity of the enzyme. It was previously
shown that truncated chitinase proteins without a
ChBD, exhibited decreased antifungal activities (Iseli
et al. 1993; Suarez et al. 2001). Watanabe et al.
Biotechnol Lett (2010) 32:539–546
(1994) reported that hydrolytic activity of the chiti-
nase A1 from Bacillus circulans was not affected by
removal of the ChBD and FnIII domains.
This study indicated that ChBD and FnIII are
essential for efficient binding of the enzyme to the
chitin in the fungal cell wall complex, while they are
not necessary for the enzyme binding to the colloidal
chitin. It was previously proposed that ChBDs in
various chitinases convert tightly packed chitin into a
more amorphous structure, which allows or a more
efficient hydrolysis by chitinolytic enzymes (Morim-
oto et al. 1997).
Strong antifungal activity of the ChiS proteins
resulted in spore germination inhibition rates of
45–80% in different ChiS transformed lines. Further-
more, fungi sensitivity to ChiS appeared to be
concentration dependent, as shown in Fig. 1b and
Table 1. It was demonstrated that the protein extracts
from transgenic plants expressing the ChiS protein,
inhibited spore germination and hyphal growth of
important phytopathogens, F. graminearum, Alt.
brassicicola and Bot. cinerea. This is the first report
describing a Bacillus pumilus chitinase gene expres-
sion in Arabidopsis plants with a high in vitro activity
against plant fungal pathogens.
Acknowledgments This research was supported by Inter-
national Center for Genetic Engineering and Biotechnology
(ICGEB, Italy) and National Institute for Genetic Engineering
and Biotechnology (NIGEB, Iran).
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