Skip to main content
SpringerLink
Log in

Eplt4 Proteinaceous Elicitor Produced in Pichia pastoris Has a Protective Effect Against Cercosporidium sofinum Infections of Soybean Leaves

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

A complementary DNA library was constructed from the mycelium of Trichoderma asperellum T4, and a highly expressed gene fragment named EplT4 was found. In order to find a more efficient and cost-effective way of obtaining EplT4, this study attempted to produce EplT4 using a Pichia pastoris expression system. The gene encoding EplT4, with an additional 6-His tag at the C-terminus, was cloned into the yeast vector pPIC9K and expressed in the P. pastoris strain GS115 to obtaining more protein for the further research. Transformants of P. pastoris were selected by PCR analysis, and the ability to secrete high levels of the EplT4 protein was determined. The optimal conditions for induction were assayed using the shake flask method and an enzyme-linked immunosorbent assay. The yield of purified EplT4 was approximately 20 mg/L by nickel affinity chromatography and gel-filtration chromatography. Western blot and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis revealed that the recombinant EplT4 was expressed in both its monomers and dimers. Soybean leaves treated with the EplT4 monomer demonstrated the induction of glucanase, chitinase III-A, cysteine proteinase inhibitor, and peroxidase genes. Early cellular events in plant defense response were also observed after incubation with EplT4. Soybean leaves protected by EplT4 against the pathogen Cercosporidium sofinum (Hara) indicated that EplT4 produced in P. pastoris was biologically active and would be potentially useful for improving food security.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Verma, M., Brar, S. K., Tyagi, R. D., Surampalli, R. Y., & Valéro, J. R. (2007). Biochemical Engineering Journal, 37, 1–20.

    Article  Google Scholar 

  2. Harman, G. E. (2006). Phytopathology, 96, 190–194.

    Article  CAS  Google Scholar 

  3. Harman, G. E., Herrera-Estrella, A. H., Horwitz, B. A., & Lorito, M. (2012). Microbiology, 158, 1–2.

    Article  CAS  Google Scholar 

  4. Shoresh, M., Yedidia, I., & Chet, I. (2005). Phytopathology, 95, 76–84.

    Article  CAS  Google Scholar 

  5. Yedidia, I., Benhamou, N., & Chet, I. I. (1999). Applied and Environmental Microbiology, 65, 1061–1070.

    CAS  Google Scholar 

  6. Yedidia, I., Shoresh, M., Kerem, Z., Benhamou, N., Kapulnik, Y., & Chet, I. (2003). Applied and Environmental Microbiology, 69, 7343–7353.

    Article  CAS  Google Scholar 

  7. Kloepper, J., Schroth, M., & Miller, T. (1980). Phytopathology, 70, 1078–1082.

    Article  Google Scholar 

  8. De Meyer, G., Bigirimana, J., Elad, Y., & Höfte, M. (1998). European Journal of Plant Pathology, 104, 279–286.

    Article  Google Scholar 

  9. Hanson, L. E. (2000). Journal of Cotton Science, 4, 224–231.

    Google Scholar 

  10. Djonovic, S., Vargas, W. A., Kolomiets, M. V., Horndeski, M., Wiest, A., & Kenerley, C. M. (2007). Plant Physiology, 145, 875–889.

    Article  CAS  Google Scholar 

  11. Rotblat, B., Enshell-Seijffers, D., Gershoni, J. M., Schuster, S., & Avni, A. (2002). Plant Journal, 32, 1049–1055.

    Article  CAS  Google Scholar 

  12. Viterbo, A., Wiest, A., Brotman, Y., Chet, I., & Kenerley, C. (2007). Molecular Plant Pathology, 8, 737–746.

    Article  CAS  Google Scholar 

  13. Seidl, V., Marchetti, M., Schandl, R., Allmaier, G., & Kubicek, C. P. (2006). FEBS Journal, 273, 4346–4359.

    Article  CAS  Google Scholar 

  14. Vargas, W. A., Djonovic, S., Sukno, S. A., & Kenerley, C. M. (2008). Journal of Biological Chemistry, 283, 19804–19815.

    Article  CAS  Google Scholar 

  15. Djonovic, S., Pozo, M. J., Dangott, L. J., Howell, C. R., & Kenerley, C. M. (2006). Molecular Plant–Microbe Interactions, 19, 838–853.

    Article  CAS  Google Scholar 

  16. Liu, Z., Yang, X., Sun, D., Song, J., Chen, G., Juba, O., & Yang, Q. (2010). Molecular Biology Reports, 37, 3673–3681.

    Article  CAS  Google Scholar 

  17. Carresi, L., Pantera, B., Zoppi, C., Cappugi, G., Oliveira, A. L., Pertinhez, T. A., Spisni, A., Scala, A., & Pazzagli, L. (2006). Protein Expression and Purification, 49, 159–167.

    Article  CAS  Google Scholar 

  18. Terpe, K. (2006). Applied Microbiology and Biotechnology, 72, 211–222.

    Article  CAS  Google Scholar 

  19. Wang, Y., Song, J. Z., Yang, Q., Liu, Z. H., Huang, X. M., & Chen, Y. (2010). Applied Biochemistry and Biotechnology, 162, 843–854.

    Article  CAS  Google Scholar 

  20. Daly, R., & Hearn, M. T. (2005). Journal of Molecular Recognition, 18, 119–138.

    Article  CAS  Google Scholar 

  21. Cregg, J. M., Vedvick, T. S., & Raschke, W. C. (1993). Nature Biotechnology, 11, 905–910.

    Article  CAS  Google Scholar 

  22. Macauley-Patrick, S., Fazenda, M. L., McNeil, B., & Harvey, L. M. (2005). Yeast, 22, 249–270.

    Article  CAS  Google Scholar 

  23. Sambrook, J., & Russell, D. (2001). Molecular cloning: A laboratory manual (3rd ed.). New York: Cold Spring Harbor Laboratory.

    Google Scholar 

  24. Adamska, M., Leoñska-Duniec, A., Maciejewska, A., Sawczuk, M., & Skotarczak, B. (2011). Folia Biologica-Krakow, 59, 115–120.

    Article  CAS  Google Scholar 

  25. Plantz, B. A., Sinha, J., Villarete, L., Nickerson, K. W., & Schlegel, V. L. (2006). Applied Microbiology and Biotechnology, 72, 297–305.

    Article  CAS  Google Scholar 

  26. Shevchenko, A., Henrik Tomas, J. H., Olsen, J. V., & Mann, M. (2007). Nature Protocols, 1, 2856–2860.

    Article  Google Scholar 

  27. Fitzgerald, H. A., Chern, M. S., Navarre, R., & Ronald, P. C. (2004). Molecular Plant–Microbe Interactions, 17, 140–151.

    Article  CAS  Google Scholar 

  28. Livak, K. J., & Schmittgen, T. D. (2001). Methods, 25, 402–408.

    Article  CAS  Google Scholar 

  29. Bradford, M. M. (1976). Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  30. Dixon, R. A., Harrison, M. J., & Lamb, C. J. (1994). Annual Review of Phytopathology, 32, 479–501.

    Article  CAS  Google Scholar 

  31. Hutcheson, S. W. (1998). Annual Review of Phytopathology, 36, 59–90.

    Article  CAS  Google Scholar 

  32. McHunu, N. P., Singh, S., & Permaul, K. (2009). Journal of Biotechnology, 141, 26–30.

    Article  CAS  Google Scholar 

  33. Buensanteai, N., Mukherjee, P. K., Horwitz, B. A., Cheng, C., Dangott, L. J., & Kenerley, C. M. (2010). Protein Expression and Purification, 72, 131–138.

    Article  CAS  Google Scholar 

  34. De Schutter, K., Lin, Y. C., Tiels, P., Van Hecke, A., Glinka, S., Weber-Lehmann, J., Rouze, P., Van de Peer, Y., & Callewaert, N. (2009). Nature Biotechnology, 27, 561–566.

    Article  Google Scholar 

  35. Bayry, J., Aimanianda, V., Guijarro, J. I., Sunde, M., & Latge, J. P. (2012). PLoS Pathogens, 8, e1002700.

    Article  CAS  Google Scholar 

  36. Baker, C. J., & Orlandi, E. W. (1995). Annual Review of Phytopathology, 33, 299–321.

    Article  CAS  Google Scholar 

  37. Glazener, J. A., Orlandi, E. W., & Baker, C. J. (1996). Plant Physiology, 110, 759–763.

    CAS  Google Scholar 

  38. Yang, Y., Zhang, H., Li, G., Li, W., Wang, X., & Song, F. (2009). Plant Biotechnology Journal, 7, 763–777.

    Article  CAS  Google Scholar 

  39. Frias, M., Gonzalez, C., & Brito, N. (2011). New Phytologist, 192, 483–495.

    Article  CAS  Google Scholar 

  40. Heath, M. C. (2000). Plant Molecular Biology, 44, 321–334.

    Article  CAS  Google Scholar 

  41. Watanabe, A., Nong, V. H., Zhang, D., Arahira, M., Yeboah, N. A., Udaka, K., & Fukazawa, C. (1999). Bioscience Biotechnology and Biochemistry, 63, 251–256.

    Article  CAS  Google Scholar 

  42. Gijzen, M., Kuflu, K., Qutob, D., & Chernys, J. T. (2001). Journal of Experimental Botany, 52, 2283–2289.

    Article  CAS  Google Scholar 

  43. Onishi, M., Tachi, H., Kojima, T., & Shiraiwa, M. (2006). Plant Physiology and Biochemistry, 44, 574–580.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National High Technology Research and Development Program of China (2011AA10A205), the Significant Scientific and Technical Project of Heilongjiang Province, China (GA08B101), and the Natural Science Foundation of Heilongjiang (C201118). Thanks also go to KI Tull and C Luprasong from the Faculty of Pharmacy, Rangsit University for editorial assistance with this paper.

Author information

Authors and Affiliations

  1. Department of Life Science and Engineering, Harbin Institute of Technology, Harbin, 150001, China

    Yun Wang, Jinzhu Song, Yingjie Wu, Margaret Odeph, Zhihua Liu, Ping Yang, Lin Yao, Lei Zhao & Qian Yang

  2. School of Botany, The University of Melbourne, 3010, Melbourne, Victoria, Australia

    Barbara J. Howlett

  3. Institute of Soil Fertilizer and Environment Resources, Heilongjiang Academy of Agricultural Sciences, Harbin, China

    Shuang Wang

Authors
  1. Yun Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jinzhu Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yingjie Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Margaret Odeph
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhihua Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Barbara J. Howlett
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shuang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ping Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Lin Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Lei Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Qian Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Qian Yang.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplemental Fig. 1

Alignment and phylogenetic tree analysis of EplT4. a Alignment of EplT4 with other known fungal proteins. Fungal proteins used for alignment were from Trichoderma asperellum T4 (ADP09352), Trichoderma virens (Q1KHY4), and Trichoderma atroviridis (DQ464903). Signal peptides at N-terminal are indicated by a line below the sequences. Four conserved cysteines are indicated by filled triangles. b Phylogenetic tree analysis of EplT4 with other proteins from various fungal species. Phylogenetic tree was constructed using CLASTALW program in MEGA software. The fungal proteins used were from Trichoderma asperellum T4 (ADP09352), Trichoderma virens (Q1KHY4), Trichoderma atroviridis (DQ464903), Ceratocystis platani(ABM63513), Auricularia delicate (EJD54220), Fomitiporia mediterranea (EJD01882), Ceratocystis fimbriata (ABM63507), Colletotrichum higginsianum (CCF39177), Aspergillus oryzae (Q2UF42), Emericella nidulans (Q5AZK7), Gibberella zeae (Q5PSV7), Gibberella pulicaris (Q5PSV6), and Leptosphaeria maculans(Q8J0U4) (DOC 745 kb)

Supplemental Fig. 2

Schematic description of the construct pPIC9K-EplT4. a Sequence of EplT4 gene cloned into pPIC9K. b Amplified EplT4 cDNA. Lane M molecular marker (DL2000); lane 1 EplT4 cDNA (DOC 344 kb)

Supplemental Fig. 3

Western blot of recombinant EplT4 from high-cell-density fermentation. Lanes 1–5 supernatant from medium at 0, 24, 48, 72, and 96 h, respectively (DOC 201 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, Y., Song, J., Wu, Y. et al. Eplt4 Proteinaceous Elicitor Produced in Pichia pastoris Has a Protective Effect Against Cercosporidium sofinum Infections of Soybean Leaves. Appl Biochem Biotechnol 169, 722–737 (2013). https://doi.org/10.1007/s12010-012-0015-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-012-0015-z

Keywords

Search

Navigation