---

Research Paper	Pdf file on Synergy omments
Acta Biochim Biophys Sin 2005,37:713-718
doi:10.1111/j.1745-7270.2005.00096.x

Biological Activities of Purified Harpin_Xoo and Harpin_Xoo 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, harpin_Xoo, in Escherichia coli BL21/pGEX-hpa1. Harpin_Xoo 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 harpin_Xoo 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 harpin_Ea of Erwinia amylovora. The purified harpin_Xoo showed a similar ability to induce tobacco mosaic virus resistance in tobacco as harpin_Ea. Its antibody worked well in detecting the purified harpin_Xoo, harpin_Xoo in the total protein of E. coli BL21/pGEX-hpa1 and an hpa1 transgenic rice.

Key words harpin_Xoo; 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 harpin_Ea [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 harpin_Xoo and the corresponding gene designated hpa1 (GenBank accession No. AB045311) [19]. hpa1 was previously expressed in Escherichia coli in our laboratory, but harpin_Xoo was not purified [11,20]. The purification of harpin_Xoo is indispensable to the study of its 3-D structure and the preparation of anti-harpin_Xoo 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 harpin_Xoo 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 锟斤拷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 锟斤拷l culture was inoculated into 10 ml of 2锟斤拷YT medium and incubated overnight at 37 ºC under the same conditions. Thereafter, the culture was diluted at 1:100 in fresh 2锟斤拷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. Harpin_Xoo was cut from the GST-harpin_Xoo fusion protein and eluted according to the instructions of the Thrombin cleavage capture kit (Novagen, San Diego, USA). Harpin_Xoo 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 harpin_Xoo. The GST was eluted according to the instructions of the manufacturer. The concentration of purified harpin_Xoo 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 harpin_Xoo 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-harpin_Xoo 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-harpin_Xoo serum using 40% saturated ammonium sulfate according to an ammonium sulfate precipitation protocol [22]. Anti-harpin_Xoo antibodies precipitated from 1 ml of anti-harpin_Xoo 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 harpin_Xoo 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 harpin_Xoo were measured according to Wen and Wang [11], and the commercial product Messenger containing 3% harpin_Ea, 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 锟斤拷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, harpin_Xoo and harpin_Ea, 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锟斤拷an, China) [1]. HR was examined after 24 h.

We also investigated the induction of resistance for tobacco mosaic virus. Harpin_Ea and purified harpin_Xoo 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锟斤拷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-harpin_Xoo fusion protein was expressed in the presence of IPTG and more soluble GST-harpin_Xoo 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-harpin_Xoo protein was purified by the Bulk GST purification module, and the GST tag was removed by thrombin (Fig. 2). The normally purified harpin_Xoo 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 harpin_Xoo per liter of culture.

The polyclonal antibody against harpin_Xoo was produced. The titer of the antibody against harpin_Xoo reached 1:3000. Western blot analysis showed the antibody could be used to detect the purified harpin_Xoo, harpin_Xoo in the total protein of E. coli BL21/pGEX-hpa1, or harpin_Xoo 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 harpin_Xoo. 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-harpin_Xoo 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 harpin_Xoo. 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 harpin_Xoo.

The purified harpin_Xoo 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 harpin_Xoo is sensitive to protease K but stable to heat treatment (Fig. 4). The capacity of harpin_Xoo to induce HR was restrained after treatment by various plant metabolic inhibitors (Fig. 4). These results indicate that the HR induced by harpin_Xoo requires active plant metabolism and is not a result of direct toxicity.

Both harpin_Ea and harpin_Xoo could induce tobacco mosaic virus (TMV) resistance in tobacco, but harpin_Xoo was more effective than harpin_Ea (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 harpin_Xoo was expressed in E. coli, but not purified, and antibody against harpin_Xoo was not produced [11,20]. In this research, the GST gene fusion system was used and a large amount of purified harpin_Xoo protein (up to 6 mg per liter of culture) was obtained. The inhibition of harpin_Xoo-induced HR by some plant metabolic inhibitors and the induction of TMV resistance, together with the physical characteristics of harpin_Xoo, suggest that the protein we purified is a typical harpin, similar to harpin_Pss [9] and harpin_Ea [1]. The purification of harpin_Xoo 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 harpin_Xoo from Xoo [34,35]. Thus, the purification of harpin_Xoo from E. coli is easier than from Xoo.

Our result showed harpin_Xoo was more effective than harpin_Ea in inducing TMV resistance in tobacco. It has also been reported that harpin_Xoo shows better results than harpin_Ea in controlling Fusarium wilt and Verticillium wilt on cotton in the field [36]. The probable reason was that the molecular weight of harpin_Xoo (14 kDa) is smaller than that of harpin_Ea (44 kDa), and the molarity of harpin_Xoo would be higher than that of harpin_Ea at the same concentration (W/V) of protein solution.

We found that GST-harpin_Xoo fusion proteins expressed in culture conditions at 28 锟斤拷C were more soluble than those at 37 锟斤拷C. The possible reason is that the lower temperature is favorable to the accurate folding of GST-harpin_Xoo fusion proteins [37].

The hypothesized dimer of harpin_Xoo was found in Western blot analysis of this study. A similar phenomenon was reported in harpin_Pss when it was dissolved in a neutral buffer system [38]. The possibility of dimer formation of harpin_Xoo 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 锟斤拷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 harpin_Xoo [20] may form disulfide bond, leading to the formation of harpin_Xoo 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 harpin_Xoo [11] and harpin_Ea [1], indicating that the protein we purified is harpin_Xoo. The polyclonal antibody has been successfully used in detection of harpin_Xoo 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.

References

1 Wei ZM, Laby RJ, Zumoff CH, Bauer DW, He SY, Collmer A, Beer SV. Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 1992, 257: 85-88

2 Wei ZM, Beer SV. Harpin from Erwinia amylovora induces plant resistance. Acta Hortic 1996, 411: 223-225

3 Dong H, Delaney TP, Bauer DW, Beer SV. Harpin induces disease resistance in Arabidopsis through the systemic acquired resistance pathway mediated by salicylic acid and the NIM1 gene. Plant J 1999, 20: 207-215

4 Bauer DW, Zumoff CH, Theisen TM, Bogdanove AJ, Beer SV. Optimized production of Erwinia amylovora harpin and its use to control plant disease and enhance plant growth. Phytopathology 1997, 87: S7

5 Qiu D, Wei ZM, Bauer DW, Beer SV. Treatment of tomato seed with harpin enhances germination and growth and induces resistance to Ralstonia solanacearum. Phytopathol 1997, 87: S80

6 Zitter TA, Beer SV. Harpin for insect control. Phytopathology 1998, 88: S104-S105

7 Collmer A, Lindeberg M, Petnicki-Ocwieja T, Schneider DJ, Alfano JR. Genomic mining type III secretion system effectors in Pseudomonas syringae yields new picks for all TTSS prospectors. Trends Microbiol 2002, 10: 462-469

8 Gal锟斤拷n JE, Collmer A. Type III secretion machines: Bacterial devices for protein delivery into host cells. Science 1999, 284: 1322-1328

9 He SY, Huang HC, Collmer A. Pseudomonas syringae pv. syringae harpin_Pss: A protein that is secreted via the Hrp pathway and elicits the hypersensitive response in plants. Cell 1993, 73: 1255-1266

10 Wen WG, Shao M, Chen GY, Wang JS. Defense response in plants induced by harpin_Xoo, an elicitor of hypersensitive response from Xanthomonas oryzae pv. oryzae. Journal of Agricultural Biotechnology 2003, 11: 192-197

11 Wen WG, Wang JS. Cloning and expressing a harpin gene from Xanthomonas oryzae pv. oryzae. Acta Phytopathologica Sinica 2001, 31: 295-300

12 Zhu WG, Magbanua MM, White FF. Identification of two novel hrp-associated genes in the hrp gene cluster of Xanthomonas oryzae pv. oryzae. J Bacteriol 2000, 182: 1844-1853

13 Arlat M, Van Gijsegem F, Huet JC, Pernollet JC, Boucher CA. PopA1, a protein which induces a hypersensitive-like response on specific Petunia genotypes, is secreted via the Hrp pathway of Pseudomonas solanacearum. EMBO J 1994, 13: 543-553

14 Charkowski AO, Alfano JR, Preston G, Yuan J, He SY, Collmer A. The Pseudomonas syringae pv. tomato HrpW protein has domains similar to harpins and pectate lyases and can elicit the plant hypersensitive response and bind to pectate. J Bacteriol 1998, 180: 5211-5217

15 de Gara L, de Pinto MC, Tommasi F. The antioxidant systems vis-锟斤拷-vis reactive oxygen species during plant-pathogen interaction. Plant Physiol Biochem 2003, 41: 863-870

16 Willis DK, Rich JJ, Hrabak EM. hrp genes of phytopathogenic bacteria. Mol Plant Microbe Interact 1991, 4: 132-138

17 Kariola T, Palomäki TA, Brader G, Palva ET. Erwinia carotovora subsp. Carotovora and Erwinia-derived elicitors HrpN and PehA trigger distinct but interacting defense responses and cell death in Arabidopsis. Mol Plant Microbe Interact 2003, 16: 179-187

18 Bonas U. hrp genes of phytopathogenic bacteria. Curr Top Microbiol Immunol 1994, 192: 79-98

19 Ochiai H, Inoue Y, Hasebe A, Kaku H. Construction and characterization of a Xanthomonas oryzae pv. oryzae bacterial artificial chromosome library. FEMS Microbiol Lett 2001, 200: 59-65

20 Li P, Lu XZ, Shao M, Long JY, Wang JS. Genetic diversity of harpins from Xanthomonas oryzae and their activity to induce hypersensitive response and disease resistance in tobacco. Sci China C Life Sci 2004, 47: 461-469

21 Fang J, Ewald D. Expression cloned cDNA for 10-deacetylbaccatin III-10-O-acetyltransferase in Escherichia coli: A comparative study of three fusion systems. Protein Expr Purif 2004, 35: 17-24

22 Harlow E, Lane D. Antibodies: A Laboratory Manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press 1988

23 Sambrook J, Russell DW. Molecular cloning: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press 2001

24 He SY, Bauer DW, Collmer A, Beer SV. The hypersensitive response elicited by Erwinia amylovora harpin requires active plant metabolism. Mol Plant Microbe Interact 1994, 7: 289-292

25 Fang ZD. The Methods of Plant Pathology Research. 3rd ed. Beijing: China Agricultural Science and Technology Press 1998

26 Shao M, Wang JS Transformation of rice with hrfA_Xoo gene and resistance of transgenic plants to bacterial leaf blight. Journal of Nanjing Agricultural University 2004, 27: 36-40

27 Alfano JR, Collmer A. The type III (Hrp) secretion pathway of plant pathogenic bacteria: Trafficking harpins, Avr proteins and death. J Bacteriol 1997, 179: 5655-5662

28 Rahme LG, Mindrinos MN, Panopoulos NJ. Plant and environmental sensory signals control the expression of hrp genes in Pseudomonas syringae pv. phaseolicola. J Bacteriol 1992, 174: 3499-3507

29 Xiao Y, Lu Y, Heu S, Hutcheson SW. Organization and environmental regulation of the Pseudomonas syringae pv. syringae 61 hrp cluster. J Bacteriol 1992, 174: 1734-1741

30 Snoeijers SS, P锟斤拷rez-Garc锟斤拷a A, Joosten MHAJ, de Wit PJGM. The effect of nitrogen on disease development and gene expression in bacterial and fungal plant pathogens. Euro J Plant Pathol 2000, 106: 493-506

31 Arlat M, Gough CL, Zischek C, Barberis PA, Trigalet A, Boucher CA. Transcriptional organization and expression of the large hrp gene cluster of Pseudomonas solanacearum. Mol Plant Microbe Interact 1992, 5: 187-193

32 Schulte R, Bonas U. Expression of the Xanthomonas campestris pv. vesicatoria hrp gene cluster, which determines pathogenicity and hypersensitivity on pepper and tomato, is plant inducible. J Bacteriol 1992, 174: 815-823

33 Lu XZ, Shao M, Wei WG, Wang JS. Purification and characteristics of a harpin-like protein from Xanthomonas oryzae pv. oryzicola. Acta Phytopathologica Sinica 2004, 34: 43-48

34 Goel AK, Rajagopal L, Nagesh N, Sonti RV. Genetic locus encoding functions involved in biosynthesis and outer membrane localization of xanthomonadin in Xanthomonas oryzae pv. oryzae. J Bacteriol 2002, 184: 3539-3548

35 Kumar A, Sunish Kumar R, Sakthivel N. Compositional difference of the exopolysaccharides produced by the virulent and virulence-deficient strains of Xanthomonas oryzae pv. oryzae. Curr Microbiol 2003, 46: 251-255

36 Zhang S, Miao WG, Nurziya, Li JH, Jin W, Tan ZH. The study of a new biotic product to cotton Fusarium wilt and Verticillium wilt in field control. J Shihezi Univ (Natural Science) 2004, 22:71-73

37 Yang Q, Li M, Xu JQ, Bao YM, Lei XY, An LJ. Expression of gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, in Esherichia coli. Biotechnol Lett 2003, 25: 101-104

38 Chen CH, Lin HJ, Feng TY. An amphipathic protein from sweet pepper can dissociate harpin_Pss multimeric forms and intensify the harpin_Pss-mediated hypersensitive response. Physiol Mol Plant Pathol 1998, 52: 139-149

39 Wu DH, Yang HT, Xue XY, Liang WX, Miao XY, Chen SJ, Pang H. Oligomerization study of NHR3 and NHR4 domains from ETO protein involved in t (8;21)-associated acute myeloid leukemia. Chinese Sci Bull 2005, 50: 875-879

40 Bhakdi S, Bayley H, Valeva A, Walev I, Walker B, Weller U, Kehoe M et al. Staphylococcal alpha-toxin, streptolysin-o, and Escherichia coli hemolysin: Prototypes of pore-forming bacterial cytolysins. Arch Microbiol 1996, 165: 73-79