Expression of the Capsicuum annum (Chili) Defensin Gene in Transgenic Tomatoes Confers Enhanced Resistance to Fungal Pathogens
Plant defensins are a group of pathogenesis-related
defense mechanism proteins. Transgenic tomato plants expressing the chili
defensin gene under the control of a Cauliflower Mosaic Virus 35S (CaMV
35S) promoter were generated. The 5 kDa peptide, corresponding to the
chili defensin protein was detected in the total protein fraction extracted
from the transgenic plants. When compared to partially-purified peptide
extracts from untransformed tomato plants, those from transgenic plant
possessed the ability to reduce the growth of several fungi in vitro.
T2 transgenic plants were selected and tested for resistance
against Fusarium sp. and Phytophthora infestans. The transgenic
lines were more resistant to infection by these pathogens than the control
Plants have evolved a variety of different mechanisms to cope with the constant
threat posed by phytopathogenic microorganisms. For example, they have developed
physical barriers and antimicrobial compounds that form in advance of a pathogenic
attack (Van Loon et al., 2006). After infection
by pathogens, these constitutive defenses are then supported by other induced
mechanisms. For example, the cell wall can be reinforced by oxidative cross-linking.
Additionally, hypersensitive cell death is triggered to isolate the pathogens
from the healthy part of the plant causing the production of antimicrobial compounds
such as phytoalexins and Pathogenesis-Related (PR) proteins (Epple
et al., 1997; Gara et al., 2003). During
recent years, it has become evident that, in addition to the above-mentioned
defense mechanisms, several families of small, cysteine-rich, basic polypeptides
play a role in the defense of plants against microorganisms (Stec,
2006). These are similar to the variety of antimicrobial polypeptides in
epithelial tissues and macrophages that support the vertebrate immunoglobulin
system and antimicrobial polypeptides located in the fat body that serve as
part of the insect immune response (Aerts et al.,
2006). In plants, this group of polypeptides has been classified into eight
families that encompass thionins, defensins, lipid transfer proteins, hevein-type
peptides, knottin-like peptides and snakins (Lay et al.,
Plant defensins were originally termed γ-thionins because they have a
similar size (5 kDa) and the same number of disulfide bridges (four) as α-
and β-thionins. However, further studies revealed that γ-thionins
were structurally different from those of α- and β-thionins and rather
similar to insect and mammalian defensins in terms of its structure and function.
Thus, this class of plant peptides was termed plant defensins (Aerts
et al., 2007). The first members of this protein family have been
isolated from wheat and barley and named γ-hordothionin and γ-purothionin.
Since then, a variety of plant defensins have been isolated and characterized
from many plant species including monocotyledons and dicotyledons. Plant defensins
have 45-54 amino acid residues containing eight conserved cysteine residues
linked to disulfide bridges. The global structure of plant defensins is composed
of a Cysteine-Stabilized αβ motif (CSαβ motif) consisting
of an α-helix and a triple-stranded β-sheet, organized in a βαββ
architecture and stabilized by four disulfide bridges (Thevissen
et al., 2003; Liu et al., 2006).
Plant defensins have been detected in different organs of plants such as leaves,
flowers, pods, seeds and tubers, although, most plant defensins isolated to
date are seed-derived (Wong and Ng, 2005; Song
et al., 2005; Abre and Melane, 2008). Furthermore,
the expression of some defensin genes is developmentally regulated, while others
are greatly elevated in response to biotic and abiotic external stimuli. Defensin
genes induced upon pathogen infection have been identified in peas, pepper and
Arabidopsis (Mirouze et al., 2006. In
Arabidopsis, at least five different defensin genes (pdf) have
been identified; for example pdf 2.3, expressed constitutively in seedling,
rosettes and flowers and pdf 1.2, induced by methyl jasmonate as well
as by a fungal pathogen (Mirouze et al., 2006;
Oh et al., 1999; Hanks et
al., 2005; Manners et al., 1998). In
addition, transgenic tobacco plants expressing the radish seed antifungal protein
1 (Rs-AFP1) exhibited enhanced resistance to fungal pathogens. Thus, plant defensins
are considered to play an important role in plant defense mechanisms due to
their antimicrobial activity in vitro and enhanced resistance of transgenic
plants against phytopathogens. However, it has not been clearly demonstrated
whether the variety of plant defensin isoforms differ in biological significance
(Park et al., 2002). In this study, we report
the generation of transgenic tomato plants (Lycopersicon esculentum cv.
Alisa craig) constitutively expressing the chili defensin (cdef1) gene,
which exhibit enhanced resistance against fungi.
MATERIALS AND METHODS
The chili defensin coding sequence was amplified by PCR from the corresponding
cDNA with following primers:
cd.1: 5´ GGG GGG GGA TCC ATG GCT GGC TTT TCC
AAA GTA 3´
cd.2: 5´ GCC GCC GAG CTC TTA AGC ACA GGG CTT CGT GCA 3´
Primer cd.1 introduced a BamHI site at the start codon without altering
the nucleotide sequence of the coding region and primer cd.2 introduced an SstI
site just behind the stop codon. The PCR product was digested with BamHI
and SstI (Promega), gel-purified, then cloned into the vector pBI121
(Jefferson, 1987). The correct sequence was verified
by DNA sequencing and the recombinant vector was designated pBI-CD.
Generation of Transgenic Tomato Plants
The recombinant plasmid was transferred to Agrobacterium tumefaciens
strain LBA 4404 using the freeze-thaw method and used for tomato transformation.
Transformation of tomatoes (Lycopersicon esculentum cv. Alisa craig)
was carried out according to the methods of Ling et al.
(1998). Cotyledons and hypocotyls (10 days after germination) were used
as the source of explant material for co-cultivation with A. tumefaciens.
Tissue was selected on 2ZI (Murashige-Skoog (MS) salts, R3 vitamins, 2 mg L-1
zeatin, 0.1 mg L-1 indole-3-acetic acid (IAA) medium containing 150
mg L-1 gentamycin and 150 mg L-1 (timentin) and transformed
calluses were excised and transferred to regeneration medium. Upon regeneration,
plantlets were rooted with antibiotic selection and transferred to soil.
Genomic DNA from transformed and non-transformed plants was isolated following
the method described by Tabaeizadeh et al. (1999).
DNA (30 μg) was digested overnight with HindIII, electrophoresed
on a 0.7% agarose gel and transferred to nylon membranes (Hybond N+, Amersham
Biosciences). Membranes were hybridized with a 613 bp 32P-labeled
probe derived from the purified DNA fragment encompassing the CaMV 35S promoter
and the chili defensin sequence. Hybridization and autoradiography procedures
were carried out according to Sambrook et al. (1989).
Total RNA was prepared from leaf tissues as described by Chomczynski
and Sacchi (1987). To start, 20 μg of total RNA was separated on a
denaturing 1.2% agarose gel (Ausubel et al., 1994).
Ethidium bromide was included in the gel to verify equal loading of RNA. After
transfer to membranes (Hybond N+, Amersham Biosciences), filters were hybridized
with a 228 bp 32P-labeled probe, prepared from the corresponding
cDNA. Filters were washed with 2xSSC, 0.1% SDS at room temperature for 10 min
each, then once with 1xSSC, 0.1% SDS at room temperature for 15 min. Finally,
the membranes were washed at high stringency with 0.1xSSC, 0.1% SDS at 65°C
for 15 min. Filters were exposed to film (Kodak) at -70°C for 2 to 6 days.
Total protein from transgenic and control plants were extracted according
to Andresen et al. (1992). Protein extracts were
subjected to a polypeptide SDS-PAGE (Schagger and Von Jagow,
1987) and transferred to a nitrocellulose membrane (Sigma, 0.2 μm pore
size) by electroblotting using a Hofer miniVE Blotter (Amersham Biosciences).
Membranes were incubated with an antibody raised in rabbits against chili defensin
and the bands were visualized with a goat anti-rabbit IgG conjugated with alkaline
phosphatase (Promega). Western blot and immunodetection conditions were set
according to the methods described by Sambrook et al.
Production of Antibody
Polyclonal antibodies were raised in rabbits using a purified CD obtained
from the expression of the chili defensin gene cloned in plasmid pMALc2x
(New England Biolabs) and expressed in E. coli strain TB1. The
recombinant fusion protein was purified by amylose-affinity chromatography.
All procedures were performed according to manufacturers protocols.
Disease Resistance Analysis
Seeds from the control (non-transgenic plant) and transgenic plants (T2
generation) were surface-sterilized and grown on ½ MS, 2% w/v sucrose
and 75 μg mL-1 gentamycin and grown at 25°C in a 16 h light/8
h dark regime. After 1 month, surviving plants were transferred to soil and
grown for 1 month at 25°C. Disease tests were performed by applying three
needle-prick wounds to the leaves and covering the fresh wounds with 5 μL
drops of a mixture of Phytophthora infestans and Fusarium sp.
spores (conidial concentration was adjusted to 5x105 spores per mL
using a hemocytometer and Tween 20 was added to a concentration 0.05% just before
inoculation). Plants were covered with a plastic bag and kept at high humidity
for 2 days to stimulate infection. Disease symptoms were checked 7 days after
infection (Thomma and Broekaert, 1998).
For the in vitro assay, crude protein was extracted according to Francois
et al. (2004). Thirty grams of tomato leaves (2 month-old plants,
T2 generation plants and control plants) were frozen in liquid nitrogen,
homogenized in extraction buffer (10 mM KCl, 9.6 mM NaH2PO4.2H2O,
15.2 mM Na2HPO4.2H2O, 150 mM NaCl, pH 7.0,
50 mL) and total protein extracts were boiled at 100°C for 10 min. After
centrifugation at 13,000 rpm for 30 min, the supernatant was precipitated with
ammonium sulfate at 70% relative saturation. The precipitated protein fraction
was collected by centrifugation at 13,000 rpm for 30 min at 4°C. The pellet
dissolved in 2 mL of dH2O and dialyzed overnight at 4°C against
distilled water using 2000 kDa cut-off dialysis tubing (Sigma) and filter sterilized
(0.2 μm). Protein concentration was quantified using the Bradford assay
with bovine serum (BSA) as standard.
Antifungal activity of crude protein isolated from leaves was determined according
to Lai et al. (2002). Plant extracts (serial
two fold dilution, 2, 4, 8, 32, 64 and 128 mg L-1) were pipetted
into the wells of a 96 well microtiter plate (TPP, Immonumaxi) containing 50
μL of one quarter strength Potato Dextrose Yeast extract (PD medium). After
mixing, 50 μL of fungal spores (Phytophthora infestans, Fusarium
sp., Colletotrichum gloeosporioides and Curvularia sp.; 40,000
spores mL-1 of water) were added to each well and the plates incubated
in the dark at room temperature. As a control, protein extracted from a control
plant (non-transgenic) was applied to all wells. Fungal spore germination and
growth were measured with a microplate reader at 595 nm from 3 days to 1 week
RESULTS AND DISCUSSION
The pBI-CD plasmid, harboring 228 bp of the chili defensin cDNA under
the control of a CaMV 35S promoter, was used for Agrobacterium tumefaciens
mediated transformation and selected for gentamycin resistance (Fig. 1).
For further characterization, 49 plants (T0 generation) originating
from independent transformation events were grown.
In order to confirm the integration of cdef1 in the putative transgenic
lines, Southern blots of genomic DNA digested with HindIII were
performed. The DNA blot is shown in Fig. 2 indicates that selected lines
were derived from independent transformation events. In line T5
and T13, two signals were prominent, reflecting the integration
of two copies of the transgene in the genome of the transgenic plants.
In lines T2, T9 and T12, only one signal
was observed, which shows the presence of one transgene in the genomes
of transgenic plants. No hybridization signal was detected in control
Transcription of the integrated cdef1 was analyzed by Northern
blot by hybridization with a chili defensin-specific probe. This indicated
that the selected lines analyzed (T2, T5, T9,
T12 and T13) stably expressed the cdef1 transgene
as shown in Fig. 3, which confirms that cdef1 has been integrated
into the tomato genome and expresses mRNA in transgenic plants. Expression
of cdef1 was also analyzed by immuno-blot analysis of leaf crude
protein fractions using the raised antibody. Protein extracts of leaves
from transformants and non-transgenic control plants were electrophoresed
on a SDS-PAGE gel, transferred to a nitrocellulose membrane and incubated
with antibodies raised against chili defensin. The raised antibody detected
a protein band with an estimated molecular mass 5 kDa as compared to a
pre-stained molecular mass standard (Fig. 4). No correlation could be
found between the number of inserts and transcript or protein levels.
||Schematic diagram of the pBI-CD vector constructed for
transformation of cdef1 in transgenic tomato plants. LB: Left
border, RB: Right border, nptII: Neomycin phosphotransferase gene
conferring kanamycin resistance, hptII: Conferring hygromycin resistance,
Nos pro: Nophaline synthase promoter, Nos ter: Terminator, LacZ: β-D-galactosidase
||Autoradiogram of the Southern hybridization. The different
signal patterns suggest the multiple integration of cdef1 in plant
genome. Lane M: 1 kb marker; Lane-1-5: Transgenic plants; Lane C-:
||Autoradiogram of the Northern hybridization. Total RNA
isolated from leaves oftransgenic tomato was separated on a 1.2% denaturing
agarose gel, blotted onto a nylon membrane and hybridized with radio-labeled
probes as indicated. Lane T2-T13: Transgenic plants; Lane C-: Non-transformed
||Western blot analysis of plant defensin in the leaves
of transgenic tomato. Lane M: Protein marker; Lane C-: Non-transformed
plant; Lane T2-T13: Transgenic tomato plants
||Progeny segregation of survived lines
|The inheritance of the transgene was tested by estimating
the percentage of the seedlings that grew on gentamycin
||Antifungal activity of leaf extracts from transgenic
tomato plants (T) expressing chili defensin
|Result are summarized from four replicated experiments
and expressed as the percentage of fungal growth over extracts from
untransformed (Ctrl) tomato plants. *Indicate significant differences
between leaf extracts from transgenic plants and control extracts
Primary transformants (line T2, T5, T9, T12
and T13) were allowed to self-fertilize and seeds of the subsequent
(segregation) T1 generation were selected with gentamycin(75
mg L-1). Among the transgenic plants tested, the progeny population
from 4 lines (T2, T5, T9, T12)
showed conventional Mendelian inheritance (3:1), while line T13
showed a 5:1 ratio (Table 1). Gentamycin and resistant
T1 seedlings were grown in a greenhouse to produce T2
To test the antifungal activity of cdef1, partially purified extracts
from five independent transgenic plants (T2 generation of lines
T2, T5, T9, T12 and T13,
three plants for each line) were prepared. Crude protein extract fractions
were tested for in vitro antifungal activity against a pathogenic
fungus using a quantitative microplate assay. Crude extracts were added
to cultures of Phytophthora infestans, Fusarium sp.,
Colletotrichum gloeosporioides and Curvularia sp. and their
effects on fungal growth were assessed. When compared to similar extracts
from untransformed tomato leaves (control), the transformed leaves expressing
the chili cdef1 resulted in significant growth inhibition of the
four fungi tested. In some lines (line T2-13, T5-12,
T9-13, T12-11 and T13-12), up to 60%
fungal growth inhibition was observed (Table 2), while in other samples
(lines T2-12, T5-11, T5-13, T12-12,
T12-13 and T13-13), growth decreased up to 50%.
This result indicates that the chili defensin (cdef1) possesses
potent antifungal activity.
To assess whether transgenic tomato plants could acquire enhanced resistance
to fungal pathogens, 30 transgenic tomato plants (T2 generation,
6 plants from each line) were selected using gentamycin. Phytophthora
infestans and Fusarium sp. fungal pathogens were used to infect
transgenic and control plants and ten days after infection, disease symptoms
started to appear on the control plants, but not the transgenic plants.
Fourteen days after infection, the control plants displayed disease symptoms
such as brown lesions, necrotic spots and leaf wilting. However, transgenic
lines remained relatively healthy without appreciable disease symptoms,
with the exception of a slight yellowing of the bottom leaves that had
contacted the infecting fungus (Fig. 5a-c).
Plants have developed various defense mechanisms against environmental
stresses such as infection by foreign pathogens. Upon contact with pathogens,
a signal is transmitted and a number of antimicrobial proteins are expressed
to fight off pathogens.
||Disease symptoms on leaves of control and transgenic
plants. Picture were taken 14 days after infection. (a and b) The
control plants showed disease symptoms such as brown lesions, necrotic
spots and leaf wilting. (c) The transgenic lines remained relatively
healthy without appreciable disease symptoms
Plant defensins were suggested to be involved in plant defense mechanisms because
these proteins have either antifungal or antibacterial properties (Moreno
et al., 1994; Terras et al., 1995).
In addition, the steady-state levels of several plant defensin transcripts increase
upon infection (Chiang and Hadwiger, 1991; Terras
et al., 1995).
To validate the presumed role of chili defensin in host defense, we generated
transgenic tomato plants containing cdef1 by Agrobacterium mediated
transformation. The CaMV promoter was used in this study as it is extremely
strong and expected to drive high levels of chili defensin expression in transgenic
tomato plants. Southern blots were generated out to further confirm T-DNA integration
and results clearly show variation in pattern of the integration (one to two
copies) among transgenic plants, indicating independent transformation events.
The 5 kDa peptide, which corresponds to the mature form of the chili defensin
peptide was detected in the total protein fraction extracted from transgenic
tomato leaves by western blot analysis. These chili-defensin extracts significantly
reduced the growth of phytopathogenic fungi. However, due to the use of partially-purified
plant extracts rather than purified peptides, it remains possible that some
of the enhanced antifungal activity associated with the transgenic extracts
was due to synergistic interactions between the chili defensin and endogenous
tomato compounds. In this context, the plant extracts tested in this study had
been partially purified to remove enzymes through protein denaturation and size
fractionation (> 2000 Da). Therefore, it is unlikely that these extracts
contained hydrolytic enzymes such as chitinases and glucanases, which have been
speculated to interact synergistically with defensins (Lai
et al., 2002). We cannot rule out the possibility of interactions
between the chili defensin and other small, heat-stable compounds from tomato
that may have been in the extracts. Because of the difficulties associated with
obtaining proper disulfide linkages, chemical synthesis of defensin peptides
is not a viable option (Lai et al., 2002).
Present analysis of the antifungal activities of the chili defensin gene is
consistent with previous suggestions that plant defensins participate in host
defense responses against pathogens. For example, extracts from tobacco plants
expressing pea defensin genes significantly reduced the growth of several phytopathogenic
fungi including Ascochyta pinodes, A. pisi, Fusarium oxysporum,
F. solani, Alternaria alternate, Ascochyta lentis,
Leptosphaeria maculans and Trichoderma reesei (Lai
et al., 2002). Meyer et al. (1996)
also showed that purified chili fruit-specific defensin was effective in suppressing
the growth of the fungi Fusarium oxysporum and Botrytis cinerea.
Florack et al. (1994) demonstrated that the hordothionin
(HTHs) from transgenic tobacco plants were biologically active and inhibited
the growth of Clavibacter michiganesis subsp. michiganesis. However,
considerable variation was observed in the effectiveness of chili defensin against
different fungal strains. Chili defensin had a higher antifungal activity against
Phytophthora infestans and Fusarium sp. compared to Colletotrichum
gloeosporioides and Curvularia sp. This shows that the antifungal
activity of plant defensins is strongly dependent on the target fungus. Park
et al. (2002) showed that antifungal activities of the BSD1 (Brassica
Stamen-specific plant Defensin 1) protein by radial growth-inhibition assays
were most effective against Neurospora crassa, Fusarium oxysporium,
Phytophthora parasitica and Alternaria solani and did not show
growth inhibition against Botrytis cinrea, Pythium apharudermatum
and Rhizoctonia solani.
The transgenic T2 lines also exhibited enhanced resistance to the
fungal pathogen. Different levels of fungi resistance in T2 transformants
may be caused by two factors. First, transcriptional or post-transcriptional
silencing of the transgenes could have affected the level of disease resistance.
The silencing of transgenes has been reported for many plant species in transgenic
rice plants harboring a chitinase gene, silencing of the transgene was reported
in T2 and T3 progeny (Chareonpornwattana
et al., 1999; Hart et al., 1992).
Second, positional effects within the genome may cause differences in transgene
expression (Meyer, 1995).
Additional evidence for a role of plant defensins in plant defense came from
the analysis of the disease tolerance of transgenic tobacco plants constitutively
expressing Rs-AFP2. The leaves of such Rs-AFP2-expressing plants displayed a
significantly decreased susceptibility to Alternaria longipes infection
relative to untransformed control plants (Terras et al.,
1995). It is already known that transgenic rice plants overproducing wasabi
defensin are expected to possess a durable and wide-spectrum resistance (i.e.,
field resistance) against various rice blast races (Kanzaki
et al., 2002). A possible role for plant defensins in fungal pathogen
defense is also suggested by the finding that the over expression of a pea defensin
gene (DRR230-a) in canola reduced disease symptoms following infection with
Leptosphaeria maculans (Wang et al., 1999).
However, additional research will be required to determine whether expression
of chili defensin is an effective means of increasing disease resistance.
Finally, conclusive evidence for the involvement of chili defensins in
the plants defense response could be obtained by inactivating these genes
or expressing different transgenes under the control of a single promoter
sequence. With the development of effective genetic transformation methods,
this is now feasible in plants through antisense or gene tagging strategies.
This study was partially supported by a grant from the IRPA (Grant No. IRPA:
09-02-02-0081-EA209) and UKM-OUP-KPB-33-171/2008 awarded to Dr. Ismanizan Ismail
and Dr. Zamri Zainal. Authors thank to Tee Choon Yang for critical Aertsing
of this manuscript.
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