Plant Molecular Biology 34: 949–959, 1997.
c 1997 Kluwer Academic Publishers. Printed in Belgium.
949
Cloning and expression of a PR5-like protein from Arabidopsis: inhibition of
fungal growth by bacterially expressed protein
Xu Hu1 and A.S.N. Reddy
Department of Biology and Program in Cell and Molecular Biology, Colorado State University, Fort Collins, CO
80523, USA ( author for correspondence); 1 Present address: Plant Biotechnology Institute, National Research
Council Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9
Received 8 January 1997; accepted in revised form 7 May 1997
Key words: antifungal activity, Arabidopsis, osmotin, pathogenesis-related protein, plant defense, PR5 protein,
thaumatin-like protein
Abstract
Pathogenesis-related (PR)-5 proteins are a family of proteins that are induced by different phytopathogens in many
plants and share significant sequence similarity with thaumatin. We isolated a complementary DNA (ATLP-3)
encoding a PR5-like protein from Arabidopsis which is distinct from two other previously reported PR5 cDNAs
from the same plant species. The predicted ATLP-3 protein with its amino-terminal signal sequence is 245 amino
acids in length and is acidic with a pI of 4.8. The deduced amino acid sequence of ATLP-3 shows significant
sequence similarity with PR5 and thaumatin-like proteins from Arabidopsis and other plants and contains a putative
signal sequence at the amino-terminus. The expression of ATLP-3 and a related gene (ATLP-1) that we previously
isolated from Arabidopsis was induced by pathogen infection and salicylic acid, a known inducer of pathogenesisrelated genes. Southern blot analysis indicates that the ATLP-1 and ATLP-3 are coded by single-copy genes. To
study the effect of ATLP-1 and ATLP-3 proteins on fungal growth, the cDNA regions corresponding to putative
mature protein were expressed in Escherichia coli and the cDNA encoded proteins were purified. ATLP-1 and
ATLP-3 proteins cross-reacted with anti-osmotin and anti-zeamatin antibodies. ATLP-3 protein showed antifungal
activity against several fungal pathogens suggesting that ATLP-3 may be involved in plant defense against fungal
pathogens.
Introduction
Plants accumulate a large number of proteins called
pathogenesis-related (PR) proteins when confronted
with phytopathogens such as viruses, bacteria and
fungi. The accumulation of these proteins has been
shown to correlate with the development of systemic acquired resistance in plants [24, 40, 47]. The PR
proteins have been divided into five families (PR1 to
PR5) based on their mobility in native gels [43]. The
PR proteins are evolutionarily conserved in the plant
kingdom and are induced by various biotic and abiotic
stresses [3, 24, 40]. The PR1 family of proteins conThe nucleotide sequence data reported will appear in the EMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession number U83490.
*141196*
sists of mostly acidic proteins with a molecular mass of
15–17 kDa [24]. PR2 and PR3 proteins are identified
as -1,3-glucanases and chitinases, respectively, with
antifungal activity [19, 23, 27]. Members of the PR4
family are acidic proteins [24]. This group of proteins
have been shown to contain antifungal activity and act
synergistically with basic PR2 and PR3 proteins [7,
31]. PR5 proteins are also called thaumatin-like proteins because of their striking sequence similarity with
thaumatin, a sweet-tasting protein from Thaumatococcus daniellii [9, 11, 30, 41]. PR5 proteins have been
characterized from a wide range of plant species in both
dicotyledonous and monocotyledonous plants [5, 12,
15, 18, 21, 26, 32, 34, 44, 48]. Although the biological
function of thaumatin-like proteins has not yet been
established, members of this group have been shown
GR: 201001989, Pips nr. 141196 BIO2KAP
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to have antifungal activity against a broad spectrum
of fungal pathogens [1, 15, 18, 26, 44, 45, 48]. Furthermore, overexpression of osmotin, a member of the
PR5 protein family, in transgenic potato plants showed
enhanced resistance to Phytophthora infestans [25].
Although most thaumatin-like proteins are about 24–
25 kDa in size, there is a sub-group of PR5 proteins
from monocots that are substantially smaller in size
(about 17.5 kDa) due to an internal deletion of about
one-fourth of the amino acids [5, 12, 32, 34]. These
smaller thaumatin-like proteins which lack six out of
sixteen conserved cysteine residues are also induced
by pathogens and show antifungal activity [12, 32].
Arabidopsis plants infected with tobacco mosaic
virus (TMV), Pseudomonas syringae pv. tomato (Pst)
DC3000, or treated with 2,6-dichloroisonicotinic acid
(INA) or salicylic acid (SA) accumulate several PR
proteins resulting in induction of systemic acquired
resistance [40, 47]. PR5 mRNA and encoded protein (acidic PR5) are significantly induced when Arabidopsis plants are challenged by pathogens or systemic acquired resistance-inducing compounds [40]. A
cDNA encoding a basic PR5-like protein has also been
reported from Arabidopsis [17]. In addition, recently a
receptor protein kinase (PR5K) with an amino-terminal
domain that is closely related to PR5 family of antifungal proteins has also been isolated from Arabidopsis [46]. In this study, we isolated a cDNA (ATLP-3,
for Arabidopsis thaumatin-like protein 3) from Arabidopsis which shares significant sequence similarity
with PR5 proteins from Arabidopsis and other plants.
Expression studies and in vitro antifungal assays with
bacterially expressed protein suggest that the ATLP-3
may be involved in plant defense response to pathogen
attack.
Materials and methods
Materials
pET 28 expression vector and E. coli strains were
obtained from Novagen. Alkaline phosphatase conjugated antichicken antibodies, diaminobenzidine tetrahydrochloride and salicylic acid were purchased
from Sigma. Biotinylated anti-rabbit secondary antibodies and Vectastain ABC horseradish peroxidase
kit were from Vector Laboratories. Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate
were bought from Amresco. Pseudomonas syringae
pv. tomato (Pst) strain DC 3000 was kindly provided
by Dr Barbara Kunkel, University of Washington.
Trichoderma reesei, Fusarium oxysporum and Alternaria solani were obtained from Microbial Properties
Research, Peoria, IL. Isolation of Verticillium alboatrum and Verticillium dahliae has been described previously [16].
Isolation of cDNA clones
The ATLP cDNAs were isolated by screening a floral bud cDNA library of Arabidopsis thaliana (constructed in ZAP II vector) with a partial cDNA
for thaumatin-like protein obtained from Arabidopsis
Biological Resource Center. Screening of the cDNA
library was performed according to standard procedures [33, 36]. Probes were made by random-priming
method using an oligolabeling Kit (Pharmacia). The
cDNA inserts from phage recombinants were excised
in vivo in plasmid (pBluescript) form. The clones
were sequenced by the dideoxynucleotide chain termination method [37]. Nucleotide and amino acid
sequences were analyzed with MacVector software.
Sequence similarity searches were performed at the
National Center for Biotechnology Information using
the BLAST network service.
Growth and treatment of plants
Arabidopsis thaliana ecotype Columbia seeds were
sown in 7.6 cm 7.6 cm plastic pots in soil consisting of peat/perlite/vermiculite (1:1:1). Plants were
grown in a growth chamber under 12 h photoperiod at
22 . P. syringae pv. tomato (Pst) strain DC 3000 was
grown at 30 C on King’s Medium B [20] containing
rifampicin (100 g/l).
Four-week old plants were inoculated with Pst
DC3000 by briefly dipping the entire leaf rosette into
bacterial suspension (2 108 colony-forming units
(cfu)/ml) in 10 mM MgCl2 containing 0.02% Silwet
L-77 (Union Carbide). Control plants were dipped in
10 mM MgCl2 containing 0.02% Silwet L-77. Symptoms were scored 3 to 5 days after inoculation. Plants
were treated with salicylic acid by spraying about 10 ml
of salicylic acid (5 mM) solution onto leaves. Control
plants were treated the same way with sterile water.
Northern blotting
Plant material was ground in liquid nitrogen and total
RNA was prepared by the guanidine hydrochloride
method [36]. Total RNA (20 g) was separated in
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a 1.0% agarose gel containing formamide. Ethidium
bromide was included to verify equal loading of RNA.
After transfer onto Hybond N+ nylon membrane, filters were hybridized with 32P-labeled ATLP-1, ATLP-3
or PR2 cDNA probes [36]. A duplicate blot was hybridized with Arabidopsis ubiquitin cDNA probe [14].
Hybridization and washings were performed according to Church and Gilbert [8].
Southern blotting
Genomic DNA from 5-week old plants was isolated
and purified on a cesium chloride gradient [36]. A
10 g portion of DNA was digested with restriction
enzymes and fractionated on a 0.8% agarose gel. DNA
was transferred onto Hybond membrane and blots were
hybridized with 32 P-labeled ATLP-1 or ATLP-3 cDNA
probes. Filters were washed twice with 2 SSC, 0.1%
SDS at 65 C, once with 0:2 SSC, 0.1% SDS at 65 C
and exposed to X-ray film.
Expression of thaumatin-like proteins
cDNA regions of ATLP-1 and ATLP-3 corresponding to putative mature protein were amplified by polymerase chain reaction (PCR) to create a cloning site.
ATLP-1S (50 -CTCACGCTAGCACTGTAATCTTCTA-30 ) and ATLP-3S (50 -TGATGCTAGCACCGTATTCACTTTA-30 ) primers containing NheI site (in
underscored) were used with T7 primer to amplify ATLP-1 and ATLP-3 cDNAs, respectively. The
amplified PCR products were cloned into NheI/EcoRI
(for ATLP-1) or NheI/HindIII (for ATLP-3) sites of
pET28a vector. The constructs were introduced into
E. coli BL21 (DE3) strain. The cells were grown in
LB containing kanamycin and IPTG (final concentration 1 mM) was added to the cultures when the O.D.
600 reached 0.6. The cells were grown for 4 h in the
induction medium and collected by centrifugation at
5000 rpm for 5 min. Cells were resuspended in lysis
buffer (50 mM Tris pH 8.0, 8% sucrose, 5% Triton
X-100, 50 mM EDTA, lysozyme 0.1 mg/ml). Soluble and insoluble proteins from induced and uninduced cultures were prepared as described [33]. The
fusion proteins were found exclusively in insoluble
inclusion bodies. To purify the fusion protein, inclusion bodies were collected by spinning at 20 000 g
for 15 min. Pellets were resuspended and washed three
times in binding buffer (5 mM imidazole, 500 mM
NaCl, 20 mM Tris-HCl pH 7.9). Purified inclusion
bodies were dissolved in renaturation buffer consist-
ing of 6 M urea, 50 mM Tris pH 8.0, 19 mM CaCl2 ,
10 mM MgCl2 , 5 mM glutathione, and 0.5 mM oxidized glutathione. Renaturation of ATLP-3 fusion protein was conducted by gradually adding renaturation
buffer with decreasing concentrations of urea to the
above protein solution to 1 liter and concentrating the
protein down to 100 ml by ultra filtration (Amicon).
This process was repeated several times until urea concentration was less than 0.5 M. The amino-terminus
of the fusion protein was removed by digesting the
fusion protein with thrombin according to instructions
provided by Novagen. A 500 g portion of fusion protein was incubated with thrombin (5 units) for 16 h at
20 C in 1 cleavage buffer containing 20 mM TrisHCl pH 8.4, 150 mM NaCl and 2.5 mM CaCl2 . All
protein samples were dialyzed with 20 mM Tris-HCl
pH 8.0 overnight at 4 C and concentrated to about
1 mg/ml protein using Centricon-10 (Amicon). Protein
concentration was determined by the method of Bradford [4] using BSA as the standard. We were unable
to renature urea-solubilized ATLP-1 as it precipitated
upon removal of urea.
Protein gel blot analysis
Anti-osmotin antibodies (a gift from Dr R.A. Bressan)
[22] raised in chicken and anti-zeamatin antibodies (a
gift from Dr C.P. Selitrennikoff) raised in rabbits [44]
were used in western blot analyses.
Protein samples were electrophoresed on 12%
SDS-polyacrylamide gels and transferred onto nitrocellulose membrane using a BioRad transfer cell. The
membrane was blocked according to the instructions
provided with Vectastain ABC kit. The membranes
were incubated with either anti-osmotin (1:1000 dilution) or anti-zeamatin polyclonal antibodies (1:500
dilution) for 60 min. After washing the membranes,
the blot probed with osmotin antibodies was incubated
with alkaline phosphatase conjugated secondary antibodies (1:10 000 dilution) for 30 min. Immunoreactive bands were detected colorimetrically by immersing
the filter in substrate solution (0.3 mg/ml nitroblue tetrazolium and 0.15 mg/ml 5-bromo-4-chloro-3-indolyl
phosphate in 100 mM Tris-HCl pH 9.5, 100 mM NaCl,
5 mM MgCl2 ). The blot probed with zeamatin antibodies was incubated with biotinylated anti-rabbit IgG
(1:10 000 dilution) for 30 min. The blot was washed
and incubated with Vectastain ABC.HRP according
to the manufacturer’s instructions. Color development
was performed by placing the filters in a substrate solution containing 100 mM Tris-HCl pH 7.5, 0.8 mg/ml
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diaminobenzidine, 0.4 mg/ml nickel chloride, and
0.009% hydrogen peroxide.
Fungal growth inhibition assays
Pure bacterial expressed proteins were tested for fungal
growth inhibitory activity. Growth inhibition of Candida albicans was determined as described by Roberts
and Selitrennikoff [35]. Agar assay plates were made
by autoclaving 80 ml water, 20 ml 20% (w/v) carrot extract, and 1.5 g agar (Difco), cooling to 45 C,
and then adding C. albicans to a final concentration of
2 , 4 105 cells per ml. Ten ml aliquots of this warm
medium were poured into a Petri dish and allowed to
solidify before placing a paper disk on the surface of
the agar. 20 l of various concentrations of fusion protein were added to each disk, and plates were incubated
overnight at 37 C. Plates were examined for zones of
growth inhibition around each disk.
Fungal species (V. albo-atrum, V. dahliae,
F. oxysporum, A. solani, and T. reesei) were grown
on PDA plates at 24 C for 1–3 weeks. Spores were
washed off plates with sterile water and collected by
centrifugation at 5000 rpm for 10 min. Antifungal
activity was measured by microspectrophotometry as
described [6]. In a microplate well, 20 l of protein
solution was added to 100 l of spore suspension of
V. albo-atrum (5 104 cells/ml), V. dahliae (5 104
cells/ml), T. reesei (5 104 cells/ml), F. oxysporum
(2 104 cells/ml), or A. solani (2 104 cells/ml) in halfstrength PD Broth (Difco) or Nutrient Broth (Difco).
The control well contained 20 l of sterile water and
100 l of the fungal spore suspension. Percentage of
growth inhibition is defined as the 100 times the ratio
of the corrected absorbance at 495 nm of the control
microculture minus the corrected absorbance of the test
microculture over the corrected absorbance of the control microculture [6]. The corrected absorbance values
equal the absorbance at 495 nm of the culture measured
after 48 h minus the absorbance at 495 nm measured
after 30 min.
Results
Isolation of a thaumatin-like protein cDNA from
A. thaliana
Using a partial thaumatin-like cDNA as a probe, a fulllength cDNA encoding a PR5-like protein (ATLP-3)
was isolated from Arabidopsis. Restriction enzyme and
nucleotide sequence analysis of the ATLP-3 indicates
that it is distinct from previously reported thaumatinlike cDNAs from Arabidopsis [17, 40]. The nucleotide and predicted amino acid sequence of ATLP-3
is shown in Figure 1. The ATLP-3 protein with its
amino-terminal signal sequence is 245 amino acids in
length with a calculated molecular mass of 25.7 kDa
and a pI of 4.8. Database searches with predicted amino
acid sequence revealed significant sequence similarity
with PR5 and thaumatin-like proteins from Arabidopsis and other plants [17, 29, 38, 40]. Figure 2 shows the
alignment of ATLP-3 with similar proteins from Arabidopsis and other plants. ATLP-3 shows the highest
sequence similarity to acidic PR5 protein (74% amino
acid identity; 81% similarity) followed by ATLP-1
(51% amino acid identity; 63% similarity) from Arabidopsis [17, 40]. All 16 cysteine residues that are
present in PR5 and thaumatin-like proteins are conserved in ATLP-3 (Figure 2). Alignment with other
thaumatin-like proteins and a hydropathy plot show a
22 amino acid signal sequence at the amino terminus
(Figure 1). The lack of a C-terminal signal sequence
and the presence of an amino-terminal signal peptide
suggest that it is a secreted protein. However, both
ATLP-1 and ATLP-3 proteins have three additional
amino acids at the carboxy-terminus. Whether these
amino acids have any role in targeting these proteins
remains to be seen.
Accumulation of ATLP-1 and ATLP-3 transcripts in
response to pathogen infection and SA treatment
Although some thaumatin-like proteins are constitutively expressed, the expression of many of them
is induced by biotic and abiotic stresses [24, 26, 45,
46]. Salicylic acid (SA), a compound that plays a
central role in plant disease resistance and systemic
acquired resistance, has been shown to induce PR
proteins including PR5 protein in Arabidopsis [10,
13, 40]. Since ATLP-1 and ATLP-3 share significant
sequence similarity with PR5 genes, we tested whether the expression of ATLP-1 and ATLP-3 is inducible
by pathogens and compounds that are known to induce
systemic acquired resistance. Four-week old Arabidopsis plants were either inoculated with Pst DC3000 or
treated with 5 mM SA. Plants inoculated with Pst
DC3000 showed symptoms 3 days after inoculation.
Plant tissue was collected at 0, 24, 48 h after treatment and the RNA from these tissues was probed with
ATLP-1 and ATLP-3 cDNAs. Pst DC 3000 infection
and SA treatment increased the accumulation of tran-
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Figure 1. Nucleotide and deduced amino acid sequences of ATLP-3. The amino acid sequence is presented below the nucleotide sequence.
Translation initiation and termination codons are underlined. Putative amino-terminal signal sequence is underscored.
scripts hybridizing to ATLP-1 and ATLP-3 probes at
24 and 48 h after treatment, whereas no or low level
transcripts of ATLP-1 and ATLP-3 are detected in
untreated controls (Figure 3A, B). As a positive control
we used a PR-2 cDNA (Figure 3C) that is known to be
induced by Pst and salicylic acid treatment. These results demonstrate that both basic (ATLP-1) and acidic
(ATLP-3) thaumatin-like proteins are induced by Pst
DC 3000 and SA. In some systems, basic PR5 proteins are constitutively expressed in some tissues or
induced in response to developmental signals and are
retained intracellularly [28, 39]. Lack of a C-terminal
extension that targets the basic thaumatin-like proteins
to the vacuole and the presence of a signal sequence
at the amino-terminus in ATLP-3 suggests that it is
likely to be secreted to the extracellular space. Zlp,
a basic thaumatin-like protein in Zea mays, has been
shown to be targeted to the extracellular space [26]. A
blot probed with a constitutively expressed ubiquitin
cDNA (Figure 3D) showed that all lanes contained an
equal amount of RNA.
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Figure 2. Alignment of amino acid sequence of ATLP-3, with PR5 (Arab.PR-5 [40]) and thaumatin-like protein (ATLP-1) from Arabidopsis
[17], tobacco PR5 (Tob.PR-5 [29]) and osmotin (osmotin [38]). Dashes indicate aligned identical amino acids, upper case letters denote aligned
nonidentical amino acids, and dots denote gaps in alignment. Arrowheads show all 16 conserved cysteine residues among the aligned proteins.
ATLP-1 and ATLP-3 are single-copy genes
Southern blot of Arabidopsis genomic DNA probed
with full-length ATLP-1 and ATLP-3 cDNAs is shown
in Figure 4. The number of hybridizing bands and the
banding pattern suggest that both genes are present
in a single copy in the genome. ATLP-1 cDNA has
one EcoRI, two HindIII and no BamHI sites whereas
ATLP-3 cDNA has one BamHI and two EcoRI sites. As
expected, a single hybridizing band with BamHI and
two bands with EcoRI are detected with ATLP-1 cDNA
(Figure 4). However, in HindIII lane four hybridizing
bands were detected instead of three expected bands
based on HindII sites in cDNA. It is likely that ATLP-1
gene has an intron with an additional HindIII site which
could account for an additional band on Southern blots.
With ATLP-3 cDNA two and three hybridizing bands
were detected with BamHI and EcoRI, respectively,
which is consistent with the expected number of bands
based on the number of restriction sites in the cDNA.
Our Southern data shows that ATLP-1 and ATLP-3
cDNAs do not cross react as there were no common
bands between the two blots. This is consistent with
the finding that the nucleotide sequences of ATLP-1
and ATLP-3 do not have considerable sequence similarity at the nucleotide level. Such divergence in nucleotide sequence between different thaumatin-like proteins from the same systems is common [17, 38, 40,
42].
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Expression of thaumatin-like proteins in E. coli
Figure 3. Induction of expression of ATLP-1 and ATLP-3 genes by
Pst infection and SA treatment. Gel blot of RNA (20 g per lane)
isolated from leaves at 0 h (lane 1), 24 h (lane 2) and 48 h (lane
3) after subjecting plants to either Pst infection or salicylic acid
treatment. Mock treatment of control plants (lane 4) was carried out
as described in Materials and methods. Four identical gel blots were
prepared and hybridized with 32 P-labeled ATLP-3 (A), ATLP-1 (B),
and PR-2 (C) or ubiquitin (D) cDNA probes. Ubiquitin cDNA was
used to demonstrate equal amount of RNA in different lanes.
Since PR5 and thaumatin-like proteins are known to
have antifungal activity, we expressed and purified
ATLP-1 and ATLP-3 cDNAs to perform in vitro antifungal assays with the purified proteins. We cloned
cDNA regions corresponding to the putative mature
proteins into E. coli expression vector pET28. The proteins expressed as His.tag fusion were induced in the
presence of 1 mM IPTG and purified as described in
Materials and methods. Analysis of soluble and insoluble fractions from induced and uninduced cultures
has shown that the expressed ATLP-1 and ATLP-3
fusion proteins are present exclusively in the insoluble
fraction as inclusion bodies (data not shown). Both
cDNAs produced expected size fusion protein. Induction at room temperature in the presence of varying
concentrations of IPTG did not help in recovering the
fusion protein in soluble protein fraction. We used different detergents and denaturing agents to solubilize
the inclusion bodies to obtain soluble form of fusion
proteins. We were able to solubilize ATLP-3 fusion
protein in urea containing buffer and removed urea to
renature the protein using the procedure described in
Materials and methods. However, our attempts to completely remove urea and renature ATLP-1 fusion protein were unsuccessful. Since ATLP-1 required 4 M
urea to avoid precipitation, all studies with ATLP-1
were carried out in a buffer containing 4 M urea. Figure 5 shows a stained gel with crude, enriched and
cleaved ATLP-1 and ATLP-3 proteins.
To test if ATLP-1 and ATLP-3 proteins cross react
with antibodies to other thaumatin-like antifungal proteins, these proteins were blotted and probed with
anti-osmotin or anti-zeamatin antibodies. ATLP-1 and
ATLP-3 showed cross reactivity with anti-osmotin and
anti-zeamatin antibodies (data not shown). However,
ATLP-1 showed stronger cross reactivity with both
antibodies as compared to ATLP-3.
In vitro antifungal activity of purified proteins
Figure 4. Genomic DNA gel blot analysis. A 10 g portion of genomic DNA was digested with different restriction enzymes, viz.
BamHI (B), EcoRI (E), and HindIII (H), fractionated on 0.8%
agarose gel, and blotted onto nylon membrane (Hybond). Membranes were hybridized with ATLP-1 cDNA probe (left) and ATLP-3
cDNA probe (right).
To test antifungal activities of ATLP-1 and ATLP-3,
growth inhibition of C. albicans was determined by
adding various concentrations of cleaved protein to
blank paper disks on agar containing suspension of
C. albicans. After 12–14 h incubation at 37 C, clear
inhibition zones were seen around the discs containing
5, 10 or 20 g of ATLP-3 while no inhibition zone was
seen around control disc (Figure 6). ALTP-1 did not
show inhibitory activity (data not shown). However, it
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Figure 5. Expression of ATLP-1 (right) and ATLP-3 (left) cDNAs in E. coli. Fusion proteins were induced by adding IPTG and incubating the
culture at 37 C for 4 h. Total protein isolated from cell culture before (lane 1) and after (lane 2) induction, enriched fusion protein (lane 3) and
protein after removing N-terminal fusion part (lane 4) were separated on 12% SDS-PAGE gel and stained with Coomassie blue. Fusion protein
is shown with an arrow and the cleaved protein is shown with an arrowhead. M, molecular mass markers.
3 fusion protein showed strong antifungal activities
against five fungi. In contrast, no inhibition was seen
with ATLP-1 fusion protein even at five fold concentration. The concentration of each protein required to
obtain 50% growth inhibition after 48 h incubation is
presented in Table 1. ATLP-3 appears to inhibit the
growth by inhibiting the germination of spores (Figure 7). Boiling of ATLP-3 for 5 min. resulted in complete loss of antifungal activity in these assays. Similarily, induced extracts prepared from E. coli cells
containing pET28 without the ATLP-3 gene did not
show antifungal activity (data not shown). These results clearly show the ATLP-3, like zeamatin and osmotin, has antifungal activity. The concentration of ATLP3 required to obtain 50% growth inhibition is similar to osmotin and zeamatin. Furthermore, our results
demonstrate that bacterially expressed protein retained
its biological activity.
Figure 6. Inhibition of C. albicans growth by purified ATLP-3
protein. Paper disks containing varying amounts of ATLP-3 protein
were placed on agar containing C. albicans. After 12–14 h incubation
at 37 C, clear inhibition zones were seen around the paper disks.
1, 20 g; 2, 10 g; 3, 5 g; 4, 2 g of protein; 5, paper disks with
buffer only.
should be mentioned that ATLP-1 protein was in 4 M
urea-containing buffer.
A microspectrophotometric assay was used to test
the effect of cleaved ATLP-1 and ATLP-3 on growth
of five different fungal spores. Osmotin and zeamatin, two known antifungal proteins, were also used in
parallel experiments. Osmotin, zeamatin and ATLP-
Discussion
We have isolated and characterized a new thaumatinlike cDNA (ATLP-3) from A. thaliana that is distinct
from previously reported thaumatin-like cDNAs from
the same plant species [17, 40, 46]. The data presented
here show that the ATLP-3 protein has structural and
functional properties characteristic of thaumatin-like
proteins. The amino acid sequence of ALTP-3 has a
very high sequence identity with thaumatin-like proteins and contained all 16 conserved cysteine residues
(Figure 2). Like some other thaumatin-like proteins,
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Table 1. Effect of osmotin, zeamatin and purified ATLP-1 and ATLP-3
on the growth of V. albo-atrum (Va), V. dahliae (Vd), T. reesei (Tr),
F. oxysporum (Fo), and A. solani (As).
Protein
Concentration of protein (g/ml) required for IC50 Va
Vd
Tr
Fo
As
Osmotin
Zeamatin
ATLP-3
ATLP-1
25
30
35
>200
25
25
30
160
20
15
25
100
30
35
25
>200
30
20
40
>200
Serial dilutions of osmotin, zeamatin, purified ATLP-3 and ATLP-1
fusion proteins were applied to fungi grown in a half strength PDA and
the percent growth inhibition measured by microspectrophotometry. The
concentration required for 50% growth inhibition after 48 h of incubation
was taken as the IC50 value, which was calculated from the dose-response
curves. IC50 values are averaged numbers from three independent experiments.
ATLP-1 fusion protein was in renaturation buffer containing 4 M urea.
Control assays were performed in 4 M urea containing buffer.
Figure 7. Inhibition of germination and growth of A. solani (top)
and T. reesei (bottom) by ATLP-3. Fungal spores were allowed to
germinate and grow in 120 l half-strength PD broth alone (left) or
half-strength PD Broth containing 20 g of purified ATLP-3 (right).
Pictures were taken after 18 h of incubation at 24 C.
ATLP-3 is acidic and contained a signal sequence at
the amino-terminus (Figure 1) suggesting that it is
a secreted protein. The expression of ATLP-3 was
induced by pathogen infection and by compounds that
are known to elicit systemic acquired resistance (Figure 3). Antibodies to two extensively characterized
thaumatin-like proteins, zeamatin (also called permatin) from Zea mays [44, 45] and osmotin [1, 38, 39]
from tobacco, showed cross reactivity with ATLP-3
protein. In addition, in vitro antifungal experiments
with bacterially expressed ATLP-3 have shown strong
antifungal activity against fungal pathogens V. alboatrum, V. dahliae, F. oxysporum and A. solani, as well
as other fungi, T. reesei and C. albicans (Figures 6 and
7, Table 1). These data strongly suggest that the ATLP3 is a bonafide member of thaumatin-like proteins with
antifungal activity and may have a biological role as a
pathogen-induced antifungal factor.
Previously, two thaumatin-like proteins, an acidic
(pI 4.5) protein with a molecular mass of 22.7 kDa [40]
and a basic (pI 9.6) protein of 25.9 kDa [17], have been
isolated from Arabidopsis. One of these was shown
to be induced by pathogens and systemic acquired
resistance-inducing compounds [40]. However, it is
not known if these proteins have antifungal activity. So
far, antifungal activity has been demonstrated for only
ATLP-3 protein (this report). Difficulty in renaturation
of ATLP-1 fusion protein from E. coli inclusion bodies prevented the use of this protein in native soluble
condition in in vitro antifungal assays. Under denatured conditions, ATLP-1 showed little or no effect on
fungal growth. Whether ATLP-1 has antifungal activ-
pla423us.tex; 10/07/1997; 14:44; v.7; p.9
958
ity or not can only be answered if antifungal assays are
performed with native protein.
Nucleotide sequence and Southern data suggest that
there are at least three thaumatin-like proteins in Arabidopsis. Of these, two are acidic [40, this report] and
one is basic [17]. In addition, another acidic thaumatinlike protein that is an integral part of a receptor protein kinase has been reported recently [46] suggesting involvement of thaumatin-like proteins in other
roles, possibly in recognizing the signals (e.g. pathogen) at the plasma membrane. The results presented
here together with published reports suggest that there
are at least four (possibly more) related thaumatin-like
proteins in Arabidopsis.
Thaumatin-like proteins have no known enzymatic
activity [26, 35]. The antifungal effect of thaumatinlike proteins is due to inhibition of hyphal growth,
spore lysis and/or reduction in spore germination or
viability of germinated spores [1, 35, 45, 48]. Zeamatin
has been shown to cause fungal hyphae to leak and rupture just below their apex [45]. Osmotin causes membrane leakage and dissipation of pH gradient across
the plasma membrane [1]. However, the mechanism by
which thaumatin-like proteins bring about these effects
are not completely understood. It was proposed that
zeamatin-induced lysis may be due to its insertion into
plasma membrane to form pores [35]. Recently solved
crystal structure of zeamatin suggests that it is unlikely
to be involved directly in forming pores in the plasma
membrane [2]. Based on crystal structure of zeamatin,
it was suggested that all thaumatin-like proteins have
an electrostatically polarized surface that may be critical for antifungal activity of this group of proteins [2].
These structural and other studies indicate the existence of membrane receptors on sensitive pathogens [1,
2]. Future studies should help identify such protein(s)
that interact with this group of proteins.
also thank Dr Irene Day and Dr Soma Narasimhulu
for their comments on the manuscript. Comparison of
the nucleotide and protein sequence with sequences
in the data bases was performed at NCBI using the
BLAST network service. This work was supported by
an Interdisciplinary Research Grant, a seed grant from
Colorado Institute for Research in Biotechnology, and
Colorado Potato Administrative Committee (Area II)
to A.S.N.R.
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We thank Elliot Meyerowitz, California Institute of
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Pst DC 3000 strain, Kerry O’Donnell of Microbial
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