Research Paper |
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Acta Biochim Biophys Sin |
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doi:10.1111/j.1745-7270.2005.00096.x |
Biological Activities of
Purified HarpinXoo and HarpinXoo Detection in Transgenic
Plants Using Its Polyclonal Antibody
Ming LI, Min SHAO, Xu-Zhong
LU, and Jin-Sheng WANG*
Key
Laboratory of Monitoring and Management of Plant Diseases and Insects, Ministry
of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
Received: April 8,
2005
Accepted: August 5,
2005
This work was supported
by the grants from the Major State Basic Research Development Program of China
(No. 2003CB114204) and the National Natural Science Foundation of China (No.
30230240)
*Corresponding
author: Tel/Fax, 86-25-84399072; E-mail, [email protected]
Abstract Many harpins have been found
in plant pathogen bacteria that can elicit disease and insect resistance in
plants, and promote plant growth. In this work, we overexpressed and purified Xanthomonas
oryzae pv. oryzae harpin, harpinXoo, in Escherichia coli
BL21/pGEX-hpa1. HarpinXoo was fused to the C-terminus of
glutathione S-transferase (GST) and purified using the Bulk GST purification
module and thrombin cleavage capture kit. Purified harpinXoo protein was sensitive to
protease K and stable to heat treatment, and could not induce a hypersensitive
response after treatment with various plant metabolic inhibitors; these
characteristics were similar to harpinEa of Erwinia amylovora.
The purified harpinXoo showed a similar ability to induce
tobacco mosaic virus resistance in tobacco as harpinEa. Its antibody worked well in
detecting the purified
harpinXoo, harpinXoo in the total protein of E.
coli BL21/pGEX-hpa1 and an hpa1 transgenic rice.
Key words harpinXoo; glutathione S-transferase (GST);
biological activity; polyclonal antibody
Harpin is a group of glycine-rich, heat-stable and protease
K-sensitive proteins that are able to elicit disease and insect resistance in
plants, induce many plant-reaction phenotypes, and promote plant growth, yield
and quality [1–6]. The first harpin, encoded by the gene hrpN, was isolated
from Erwinia amylovora, and named harpinEa [1]. To date,
harpins from four genera of bacterial plant pathogens, Erwinia, Pseudomonas,
Xanthomonas and Ralstonia, have been reported [7–14].
Plant-pathogen interactions can be classified into two types:
compatible and incompatible. In a compatible interaction, the host plant does
not mount an effective defense response, and the pathogen causes disease; in an
incompatible interaction, the resistant variety or non-host plant effectively
prevents the invasion and spread of the pathogen by initiating defense
responses or its pre-formed barriers and compounds. Hypersensitive response
(HR) is an important characteristic in the defense mechanisms of some
incompatible interactions. HR is a localized plant cell death at the site of
pathogen infection. The dead cell surrounding the pathogen forms a physical
barrier to the invasion of the pathogen. In addition, compounds released from
the dead cells may be toxic to the invading pathogen [15]. Earlier research
indicates that phytopathogenic bacteria are likely to have the same factors
that are responsible for triggering HR in non-host plants and are required for
pathogenicity in host plants [16]. Harpin can induce HR in non-host plants, but
the role of harpin in sensitive hosts is not clear [17]. Harpins were
identified and isolated to study how plant pathogenic bacteria interact with
host and non-host plants. Harpins are encoded by hypersensitive response and
pathogenicity gene (hrp) clusters of plant bacteria [18].
In Xanthomonas oryzae pv. oryzae (Xoo),
a Gram-negative bacterium causing bacterial leaf blight on rice, the hrp gene
cluster was dissected. The protein was given the name harpinXoo
and the corresponding gene designated hpa1 (GenBank accession No.
AB045311) [19]. hpa1 was previously expressed in Escherichia coli in
our laboratory, but harpinXoo was not purified [11,20]. The
purification of harpinXoo is indispensable to the study of its
3-D structure and the preparation of anti-harpinXoo polyclonal
antibody. The glutathione S-transferase (GST) gene fusion system is a simple
and fast technique to express and purify proteins [21]. This technique needs
few apparatus and obtains abundant purified protein in two days.
In this work, we purified harpinXoo using the
GST gene fusion system, studied its characteristics, and reported its detection
in transgenic rice plants by polyclonal antibody.
Experimental Procedures
The gene hpa1 was amplified from X. oryzae pv.
oryzae strain JxoIII genomic DNA by a standard polymerase chain reaction
(PCR). The primers were 5‘-TTCGGATCCATGAATTCTTTGAACACACAATT-3‘
(forward, BamHI site is in italic) and 5‘-GGTGAATTCTTACTGCATCGATGCGCT-3‘
(reverse, EcoRI site is in italic). PCR amplifications were performed
for one cycle of 5 min at 95 °C; 35 cycles of 45 s at 95 ºC, 45 s at 56 ºC, 45
s at 72 ºC; and a final extension step of 7 min at 72 ºC. The PCR product was
cloned into pGEM-T easy vector (Promega, Madison, USA), a high copy T vector.
The positive insertion was confirmed by the automated DNA sequencing in TaKaRa
(Dalian, China). The recombinant vector containing hpa1 (pGEM-hpa1) was
digested with BamHI and EcoRI, then ligated into pGEX-2T vector
(Pharmacia, Uppsala, Sweden) digested with the same enzymes. The recombinant
plasmid pGEX-hpa1 was transformed into E. coli BL21(DE3) (Pharmacia),
and the resulting strain was named E. coli BL21/pGEX-hpa1.
Ten microliters of glycerol stock of E. coli BL21/pGEX-hpa1
was inoculated into 1 ml of 2´YT medium supplemented with 100 mg/ml ampicillin. After 6 h incubation
at 37 ºC, 100 μl culture was inoculated into 10 ml of 2×YT medium and incubated
overnight at 37 ºC under the same conditions. Thereafter, the culture was
diluted at 1:100 in fresh 2×YT medium with 100 mg/ml ampicillin and
incubated at 37 ºC. When the A600 reached 0.6–0.8, 0.1 mM
isopropyl-b–D-1-thiogalactopyranoside (IPTG) was added, and the mixture was
incubated for additional 4 h at 28 ºC or 37 ºC to induce the protein
expression.
The bacterial cells were harvested and sonicated, and the
glutathione Sepharose 4B was prepared according to the instructions of the Bulk
GST purification module (Pharmacia). Glutathione Sepharose 4B was added to the
cell supernatant, then mixed gently for 30 min at room temperature. The mixture was loaded
onto a column provided in the Bulk GST purification module. HarpinXoo
was cut from the GST-harpinXoo fusion protein and eluted according to
the instructions of the Thrombin cleavage capture kit (Novagen, San Diego,
USA). HarpinXoo was then subjected to a boiling water bath for 10 min and centrifuged
at 10,000 g for 5 min to remove insoluble materials. The supernatant contained
purified harpinXoo. The GST was eluted according to the instructions of the
manufacturer. The concentration of purified harpinXoo was measured
using the BAC-100 protein quantitative analysis kit (Biocolor Biotech,
Shanghai, China).
New Zealand white rabbits (College of Veterinary Medicine, Nanjing
Agricultural University, Nanjing, China) were immunized subcutaneously at
multiple sites with 200 mg of purified harpinXoo in 0.5 ml of isotonic saline
emulsified with an equal volume of Freund complete adjuvant. Animals were
boosted at weekly intervals with 30 mg of antigen emulsified with Freund incomplete
adjuvant. Six weeks later, the rabbits were bled and the sera were tested for
antibody by immunoblotting. Anti-harpinXoo serum titer
was determined by enzyme-linked immunosorbent
assay (ELISA) method. Pre-immune sera were also obtained and analyzed in
parallel. IgG was isolated from anti-harpinXoo serum using
40% saturated ammonium sulfate according to an ammonium sulfate precipitation
protocol [22]. Anti-harpinXoo antibodies precipitated from 1 ml of
anti-harpinXoo serum were redissolved in the same volume of phosphate-buffered
saline (PBS) and dialyzed overnight against the same buffer.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
was carried out on a 13% slab gel according to the method of Sambrook et al.
[23] and samples were boiled for 5 min before loading. Proteins on the
polyacrylamide gel were stained with 0.25% Coomassie brilliant blue R-250
(Amresco, Ohio, USA). In immunoblotting, the heat-treated proteins separated by
SDS-PAGE were electrotransferred to a nitrocellulose membrane. The membrane was
reacted with polyclonal antibody against harpinXoo and
horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Sino-American
Biotech, Luoyang, China). Finally the membrane was detected with 3,3‘-diaminobenzidine
tetrahydrochloride.
The sensitivity to protease K and heat-stability of purified harpinXoo
were measured according to Wen and Wang [11],
and the commercial product Messenger containing 3% harpinEa,
kindly provided by Dr. Zhong-Min WEI (Eden Bioscience, Annapolis, USA), was
used as a control. In brief, protease K or protease K+phenyl methyl sulphonyl
fluoride (PMSF, a protease inhibitor, final concentration 0.5 mM) was added to
harpin (10 mg/ml) in PBS with final concentration of protease K being 0.2 mg/ml and maintained
at 37 °C for 15 min; heat-stability assay was performed with a boiling water
bath for 10 min. Harpins were treated with eucaryotic metabolic inhibitors,
cycloheximide, actinomycin D and lanthanum chloride, according to He et al.
[24].
To induce HR, harpinXoo and harpinEa,
which were treated with boiling water, cycloheximide, actinomycin, lanthanum
chloride, protease K, or protease K+PMSF, were infiltrated into Nicotiana
tabacum L. cv. Xanthi tobacco leaves, the seeds of which were from the
Tobacco Laboratory of Shandong Agricultural University (Tai’an, China) [1]. HR
was examined after 24 h.
We also investigated the induction of resistance for tobacco mosaic
virus. HarpinEa and purified harpinXoo were dissolved in distilled water to
give a concentration of 30 mg/ml, and sprayed on nine leaves
of three N. tabacum L. cv. Xanthi tobacco plants. Water was used as a
control. Tobacco plants were grown in a greenhouse for 6–7 weeks before
use. After harpin’s being sprayed for 16 h, the tobacco mosaic viruses, kindly
provided by Dr. Yi-Jun ZHOU (Jiangsu Academy of Agricultural Science, Nanjing,
China), were inoculated according to the method described by Fang [25]. Three
to four days later, the plants were assayed for typical necrotic lesion
production.
Results
GST-harpinXoo fusion protein was expressed in the presence
of IPTG and more soluble GST-harpinXoo protein was
obtained in culture conditions at 28 ºC
than that at 37 ºC for an additional 4 h after 0.1 mM IPTG was added (Fig.
1). The crude GST-harpinXoo protein was purified by the Bulk GST
purification module, and the GST tag was removed by thrombin (Fig. 2). The
normally purified harpinXoo was visualized as multiple bands by
SDS-PAGE, but visualized as a single band after a boiling water bath (Fig. 2,
lanes 2 and 3). The yield was about 6 mg harpinXoo per liter of
culture.
The polyclonal antibody against harpinXoo was
produced. The titer of the antibody against harpinXoo reached
1:3000. Western blot analysis showed the antibody could be used to detect the purified harpinXoo, harpinXoo
in the total protein of E. coli BL21/pGEX-hpa1, or harpinXoo
from the hpa1 transgenic rice line TR19 as reported in [26] (Fig. 3).
A band with a molecular weight of 14 kDa was detected in all materials presumed
to contain harpinXoo. The molecular weight of 14 kDa was equal to the expected size
based on sequence data of gene hpa1. A band of about 40 kDa was found in
the total protein of E. coli BL21/pGEX-hpa1, which should be the
GST-harpinXoo fusion protein (the molecular weight of GST is about 26 kDa). An
additional band was found with a molecular weight of 29 kDa, more than double
that of the 14 kDa band. This band might be a dimer of harpinXoo.
No bands appeared in negative controls, including the purified GST, the total
protein of E. coli BL21/pGEX-2T and rice variety R109. These results indicated
that the polyclonal antibody developed in this experiment was specific against
harpinXoo.
The purified harpinXoo lost the ability to elicit HR after
treatment with protease K but could still elicit HR after heat treatment in a
boiling water bath and treatment with protease K+PMSF, suggesting that harpinXoo
is sensitive to protease K but stable to heat treatment (Fig. 4). The
capacity of harpinXoo to induce HR was restrained after treatment by various plant
metabolic inhibitors (Fig. 4). These results indicate that the HR
induced by harpinXoo requires active plant metabolism and is not a result of direct
toxicity.
Both harpinEa and harpinXoo could induce
tobacco mosaic virus (TMV) resistance in tobacco, but harpinXoo
was more effective than harpinEa (Table 1).
Discussion
Harpins are a special family of proteins produced by plant bacteria.
They are able to elicit HR on some non-host plants and, at the same time,
induce a series of defense reactions. Recently, evidence has shown that crude
harpins from two pathovars of X. oryzae pv.
oryzae and X. oryzae pv. oryzicola (Xooc) have these basic
properties [20]. HR, in general, is different from damage caused by toxins. HR
is a rapid cell apoptosis induced by avirulent pathogens or elicitors produced
by avirulent pathogens [27].
In our previous study, the hpa1 gene was inserted into
pET-30a(+), and harpinXoo was expressed in E. coli, but
not purified, and antibody against harpinXoo was not produced
[11,20]. In this research, the GST gene fusion system was used and a large
amount of purified harpinXoo protein (up to 6 mg per liter of
culture) was obtained. The inhibition of harpinXoo-induced HR
by some plant metabolic inhibitors and the induction of TMV resistance,
together with the physical characteristics of harpinXoo, suggest
that the protein we purified is a typical harpin, similar to harpinPss
[9] and harpinEa [1]. The purification of harpinXoo and production
of its antibody make it possible to further study on its structure and
function, as well as monitor the protein produced in recombinant
microorganisms or transgenic plants.
Previous reports indicate that the plant-pathogenic bacteria do not
produce harpins in culture because they are induced in nutrient-poor conditions
that mimic the plant apoplast [28–32]. Recently, two harpin-like proteins have
been isolated in a small amount from bacterial cells of Xoo and Xooc cultured
in NB medium [33]. In addition, abundant pigment and lipopolysaccharide
produced by Xoo make it difficult to purify the harpinXoo
from Xoo [34,35]. Thus, the purification of harpinXoo
from E. coli is easier than from Xoo.
Our result showed harpinXoo was more
effective than harpinEa in inducing TMV resistance in tobacco.
It has also been reported that harpinXoo shows better
results than harpinEa in controlling Fusarium wilt and Verticillium wilt on
cotton in the field [36]. The probable reason was that the molecular weight of
harpinXoo (14 kDa) is smaller than that of harpinEa
(44 kDa), and the molarity of harpinXoo would be
higher than that of harpinEa at the same concentration (W/V)
of protein solution.
We found that GST-harpinXoo fusion
proteins expressed in culture conditions at 28 °C were more soluble than those
at 37 °C. The possible reason is that the lower temperature is favorable to the
accurate folding of GST-harpinXoo fusion proteins [37].
The hypothesized dimer of harpinXoo was found in
Western blot analysis of this study. A similar phenomenon was reported in
harpinPss when it was dissolved in a neutral buffer system [38]. The possibility
of dimer formation of harpinXoo may be due to incomplete protein
denaturation. Wu et al. [39] reported that the NHR3 domain of the ETO
protein was a tight tetramer, but four bands, representing tetramer, trimer,
dimer and monomer, could be observed by SDS-PAGE, although the loaded sample
was boiled for several minutes at 90 °C. They proposed that the oligomerization
of the NHR3 domain might be due to the combining strength of elements such as
salt bridge, hydrophobic strength and hydrogen bond, because the NHR3 domain
contains no cysteines and does not involve formation of a disulphide bond [39].
However, a cysteine present at position 45
at the N terminus of harpinXoo [20] may form disulfide bond, leading
to the formation of harpinXoo dimer. The formation of disulfide bond
would be the main reason of dimer in transgenic rice. The oligomerization of
harpin may affect the interaction between harpin and the plant cell membrane
[38]. The similar function of the oligomerization of animal bacterial protein
toxins such as a-toxin, streptolysin-O and hemolysin, has been reported [40].
In summary, we have expressed and purified a harpin protein in E.
coli and produced polyclonal antibody against it. The purified protein has
similar biological characteristics as the previously reported crude harpinXoo
[11] and harpinEa [1], indicating that the protein we purified is harpinXoo.
The polyclonal antibody has been successfully used in detection of harpinXoo
expression in transgenic rice.
Acknowledgements
We thank Prof. Gen-Xing XU and Dr. Geng-Feng FU of the Institute of
Molecular Medicine (Nanjing Military Medical College, Nanjing, China) for
useful suggestions in partial experimental design. We are also grateful to Dr.
Zhong-Li CUI of Nanjing Agricultural University (Nanjing, China) for helpful
comments on manuscript preparation.
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