Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Expression of $HpaG_{Xooc}$ Protein in Bacillus subtilis and its Biological Functions

  • Wu, Huijun (Key Laboratory of Monitoring and Management for Plant Diseases and Insects, Ministry of Agriculture, Department of Plant Pathology, Nanjing Agricultural University) ;
  • Wang, Shuai (Key Laboratory of Monitoring and Management for Plant Diseases and Insects, Ministry of Agriculture, Department of Plant Pathology, Nanjing Agricultural University) ;
  • Qiao, Junqing (Key Laboratory of Monitoring and Management for Plant Diseases and Insects, Ministry of Agriculture, Department of Plant Pathology, Nanjing Agricultural University) ;
  • Liu, Jun (Key Laboratory of Monitoring and Management for Plant Diseases and Insects, Ministry of Agriculture, Department of Plant Pathology, Nanjing Agricultural University) ;
  • Zhan, Jiang (Key Laboratory of Monitoring and Management for Plant Diseases and Insects, Ministry of Agriculture, Department of Plant Pathology, Nanjing Agricultural University) ;
  • Gao, Xuewen (Key Laboratory of Monitoring and Management for Plant Diseases and Insects, Ministry of Agriculture, Department of Plant Pathology, Nanjing Agricultural University)
  • Published : 2009.02.28
Download PDF
⟨ Previous Next ⟩

Abstract

$HpaG_{Xooc}$, from rice pathogenic bacterium Xanthomonas oryzae pv. oryzicola, is a member of the harpin group of proteins, eliciting hypersensitive cell death in non-host plants, inducing disease and insect resistance in plants, and enhancing plant growth. To express and secret the $HpaG_{Xooc}$ protein in Bacillus subtilis, we constructed a recombinant expression vector pM43HF with stronger promoter P43 and signal peptide element nprB. The SDS-PAGE and Western blot analysis demonstrated the expression of the protein $HpaG_{Xooc}$ in B. subtilis. The ELISA analysis determined the optimum condition for $HpaG_{Xooc}$ expression in B. subtilis WBHF. The biological function analysis indicated that the protein $HpaG_{Xooc}$ from B. subtilis WBHF elicits hypersensitive response(HR) and enhances the growth of tobacco. The results of RT-PCR analysis revealed that $HpaG_{Xooc}$ induces expression of the pathogenesis-related genes PR-1a and PR-1b in plant defense response.

Keywords

References

  1. Alfano, J. R. and A. Collmer. 2004. Type III secretion system effector proteins: Double agents in bacterial disease and plant defense. Annu. Rev. Phytopathol. 42: 385-414 https://doi.org/10.1146/annurev.phyto.42.040103.110731
  2. Asaka, O. and M. Shoda. 1996. Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14. Appl. Environ. Microbiol. 62: 4081-4085
  3. Brocmeier, U., M. Wendorff, and T. Eggert. 2006. Versatile expression and secretion vectors for Bacillus subtilis. Curr. Microbiol. 52: 143-148 https://doi.org/10.1007/s00284-005-0231-7
  4. Bauer, D. W., C. H. Zumoff, T. M. Theisen, A. J. Bogdanove, and S. V. Beer. 1997. Optimized production of Erwinia amylovora harpin and its use to control plant disease and enhance plant growth. Phytopathology 87: S7
  5. Bron, S. and E. Luxen. 1985. Segregational instability of pUB110- derived recombinant plasmids in Bacillus subtilis. Plasmid 14: 235-244 https://doi.org/10.1016/0147-619X(85)90007-1
  6. Chang, S. 1987. Engineering for protein secretion in Grampositive bacteria. Meth. Enzymol. 153: 507-516 https://doi.org/10.1016/0076-6879(87)53075-0
  7. Clarke, J. D., S. M. Volko, H. Ledford, F. M. Ausubel, and X. Dong. 2000. Roles of salicylic acid, jasmonic acid, and ethylene in cpr-induced resistance in Arabidopsis. Plant Cell 12: 2175- 2190 https://doi.org/10.1105/tpc.12.11.2175
  8. Dartois, V., J. Y. Coppée, C. Colson, and A. Baulard. 1994. Genetic analysis and overexpression of lipolytic activity in Bacillus subtilis. Appl. Environ. Microbiol. 60: 1670-1673
  9. Dixon, R. A. 2001. Natural products and plant disease resistance. Nature 411: 843-847 https://doi.org/10.1038/35081178
  10. Doi, R. H., S. L. Wong, and F. Kawamura. 1986. Potential use of Bacillus subtilis for secretion and production of foreign proteins. Trends Biotechnol. 4: 232-235 https://doi.org/10.1016/0167-7799(86)90116-2
  11. Dong, H. P., J. L. Peng, Z. L. Bao, X. D. Meng, J. M. Bonasera, G. Y. Chen, S. V. Beer, and H. S. Dong. 2004. Downstream divergence of the ethylene signaling pathway for harpin-stimulated Arabidopsis growth and insect defense. Plant Physiol. 136: 3628-3638 https://doi.org/10.1104/pp.104.048900
  12. Dong, X. 1998. SA, JA, ethylene, and disease resistance in plants. Curr. Opin. Plant Biol. 1: 316-323 https://doi.org/10.1016/1369-5266(88)80053-0
  13. Halling, S. M., F. J. Sanchez-Anzaldo, R. Fukuda, R. H. Doi, and C. F. Meares. 1977. Zinc is associated with the beta subunit of DNA-dependent RNA polymerase of Bacillus subtilis. Biochemistry 16: 2880-2884 https://doi.org/10.1021/bi00632a012
  14. Kameda, Y., S. Oira, K. Matsui, S. Kanatomo, and T. Hase. 1974. Antitumor activity of Bacillus natto. V. Isolation and characterization of surfactin in the culture medium of Bacillus natto KMD 2331. Chem. Pharm. Bull. (Tokyo) 22: 938-944 https://doi.org/10.1248/cpb.22.938
  15. Li, M., M. Shao, X. Z. Lu, and J. S. Wang. 2005. Biological activity of purified harpinXoo and harpinXoo detection in transgenic plants using its polyclonal antibody. Acta Biochim. Biophys. Sin. 37: 713-718 https://doi.org/10.1111/j.1745-7270.2005.00096.x
  16. Li, P., X. Z. Lu, M. Shao, J. Y. Long, and J. S. Wang. 2004. Genetic diversity of harpins from Xanthomonas oryzae and their activity to induce hypersensitive response and disease resistance in tobacco. Sci. China C Life Sci. 47: 461-469 https://doi.org/10.1360/03yc0152
  17. Liu, F. Q., H. X. Liu, Q. Jia, X. J. Wu, X. J. Guo, S. J. Zhang, F. Song, and H. S. Dong. 2006. The internal glycine-rich motif and cysteine suppress several effects of the HpaG(Xooc) protein in plants. Phytopathology 96: 1052-1059 https://doi.org/10.1094/PHYTO-96-1052
  18. Nakano, M. M., M. A. Marahiel, and P. Zuber. 1988. Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis. J. Bacteriol. 170: 5662-5668 https://doi.org/10.1128/jb.170.12.5662-5668.1988
  19. Nguyen, H. D., Q. A. Nguyen, R. C. Ferreira, L. C. S. Ferreira, L. T. Tran, and W. Schumann. 2005. Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability. Plasmid 54: 241-248 https://doi.org/10.1016/j.plasmid.2005.05.001
  20. Paulitz, T. C. and R. R. Bélanger. 2001. Biological control in greenhouse systems. Annu. Rev. Phytopathol. 39: 103-133 https://doi.org/10.1146/annurev.phyto.39.1.103
  21. Peng, J. L., Z. L. Bao, H. Y. Ren, J. S. Wang, and H. S. Dong. 2004. Expression of harpinXoo in transgenic tobacco induces pathogen defense in the absence of hypersensitive cell death. Phytopathology 94: 1048-1055 https://doi.org/10.1094/PHYTO.2004.94.10.1048
  22. Peypoux, F., J. M. Bonmatin, and J. Wallach. 1999. Recent trends in the biochemistry of surfactin. Appl. Microbiol. Biotechnol. 51: 553-563 https://doi.org/10.1007/s002530051432
  23. Ryals, J. A., U. H. Neuenschwander, M. G. Willits, A. Molina, H. Y. Steiner, and M. D. Hunt. 1996. Systemic acquired resistance. Plant Cell 8: 1809-1819 https://doi.org/10.1105/tpc.8.10.1809
  24. Ryu, C. M., M. A. Farag, C. H. Hu, M. S. Reddy, H. X. Wei, P. W. Pare, and J. W. Kloepper. 2003. Bacterial volatiles promote growth in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 100: 4927-4932 https://doi.org/10.1073/pnas.0730845100
  25. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, U.S.A
  26. Schisler, D. A., P. J. Slininger, R. W. Behle, and M. A. Jackson. 2004. Formulation of Bacillus spp. for biological control of plant diseases. Phytopathology 94: 1267-1271 https://doi.org/10.1094/PHYTO.2004.94.11.1267
  27. Spizizen, J. 1958. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleotide. Proc. Natl. Acad. Sci. U.S.A. 44: 1072-1078 https://doi.org/10.1073/pnas.44.10.1072
  28. Spoel, S. H., A. Koornneef, S. M. C. Claessens, J. P. Korzelius, J. A. V. Pelt, M. J. Mueller, et al. 2003. NPR1 modulates cross-talking between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol. Plant Cell 15: 760- 770 https://doi.org/10.1105/tpc.009159
  29. Stein, T., J. Vater, V. Kruft, A. Otto, B. Wittmann-Liebold, P. Franke, M. Panico, R. McDowell, and H. R. Morris. 1996. The multiple carrier model of nonribosomal peptide biosynthesis at modular multienzymatic templates. J. Biol. Chem. 271: 15428-15435 https://doi.org/10.1074/jbc.271.26.15428
  30. Tjalsma, H., H. Antelmann, J. D. H. Jongbloed, P. G. Braun, E. Darmon, R. Dorenbos, et al. 2004. Proteomics of Protein Secretion by Bacillus subtilis: separating the 'secrets' of the secretome. Microbiol. Mol. Biol. Rev. 68: 207-233 https://doi.org/10.1128/MMBR.68.2.207-233.2004
  31. Vollenbroich, D., G. Pauli, M. Özel, and J. Vater. 1997. Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis. Appl. Environ. Microbiol. 63: 44-49
  32. Wang, P. Z. and R. H. Doi. 1984. Overlapping promoters transcribed by Bacillus subtilis ${\sigma}^{55}$ and ${\sigma}^{37}$ RNA polymerase holoenzymes during growth and stationary phase. J. Biol. Chem. 259: 8619-8625
  33. Wei, Z. M., R.J. Laby, C. H. Zumoff, D. W. Bauer, S. Y. He, A. Collmer, and S. V. Beer. 1992. Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 257: 85-88 https://doi.org/10.1126/science.1621099
  34. Westers, L., D. S. Dijkstra, H. Westers, J. M. van Dijl, and W. J. Quax. 2006. Secretion of functional human interleukin-3 from Bacillus subtilis. J. Biotechnol. 123: 211-224 https://doi.org/10.1016/j.jbiotec.2005.11.007
  35. Wong, S. L. 1995. Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins. Curr. Opin. Biotechnol. 6: 517-522 https://doi.org/10.1016/0958-1669(95)80085-9
  36. Wu, S. C. and S. L. Wong. 2002. Engineering of a Bacillus subtilis strain with adjustable levels of intracellular biotin for secretory production of functional streptavidin. Appl. Environ. Microbiol. 68: 1102-1108 https://doi.org/10.1128/AEM.68.3.1102-1108.2002
  37. Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415: 389-395 https://doi.org/10.1038/415389a
  38. Zhang, X. Z., Z. L. Cui, Q. Hong, and S. P. Li. 2005. High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800. Appl. Environ. Microbiol. 71: 4101-4103 https://doi.org/10.1128/AEM.71.7.4101-4103.2005