Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Isolation and Characterization of Defense Genes Mediated by a Pathogen-Responsive MAPK Cascade in Tobacco

담배에서 병원균에 반응하는 MAPK 신호전달체계에 의해 매개되는 방어 유전자들의 분리 및 특성화

  • Jang, Eun-Kyoung (Department of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University) ;
  • Kang, Eun-Young (Department of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University) ;
  • Kim, Young-Cheol (Department of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University) ;
  • Cho, Baik-Ho (Department of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University) ;
  • Yang, Kwang-Yeol (Department of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University)
  • 장은경 (전남대학교 농업생명과학대학 식물생명공학부) ;
  • 강은영 (전남대학교 농업생명과학대학 식물생명공학부) ;
  • 김영철 (전남대학교 농업생명과학대학 식물생명공학부) ;
  • 조백호 (전남대학교 농업생명과학대학 식물생명공학부) ;
  • 양광열 (전남대학교 농업생명과학대학 식물생명공학부)
  • Published : 2008.08.30
Download PDF
⟨ Previous Next ⟩

Abstract

NtMEK2, which is the tobacco MAPK kinase that is upstream of SIPK and WIPK, was identified using the dexamethasone (DEX)-inducible gain-of-function transgenic system. Expression of $NtNEK2^{DD}$, a constitutively active mutant of NtNEK2, leads to HR-like cell death, which indicates that the NtMEK2-SIPK/WIPK cascade controls defense responses in tobacco. However, little is known about the downstream target substrates or defense-related genes that are regulated by the NtMEK2-SIPK/ WIPK cascade. In this study, ACP-based differential display RT-PCR was used to isolate the downstream effectors mediated by the NtMEK2-SIPK/WIPK cascade in $NtNEK2^{DD}$ transgenic plants. The results identified 6 novel differentially expressed genes (DEGs). These included pathogen induced protein 2-4 (pI2-4), monoterpene synthase 2 (MTS2), seven in absentia protein (SINA), cell death marker protein 1 (CDM1), hydroxyproline-rich glycoprotein (HRGP) and unknown genes (DEG45). The induction of these genes was confirmed by RT-PCR of samples obtained from $NtNEK2^{DD}$ plants. Additionally, when compared with other isolated DEGs, the pI2-4, CDM1 and HRGP genes were significantly up-regulated in response to treatment with salicylic acid and tobacco mosaic virus. Taken together, these results suggest that three novel DEGs were regulated by the NtMEK2-SIPK/WIPK cascade involved in disease resistance in tobacco.

SIPK와 WIPK의 상위 단계 인산화 효소로 알려진 NtMEK2가 DEX 유도성 시스템에 의해 밝혀졌다. 이 NtMEK2 유전자가 지속적으로 활성화된 돌연변이체인 $NtMEK2^{DD}$의 발현은 SIPK와 WIPK를 활성화 시켜 주므로 과민감 반응과 같은 세포 괴사를 야기하는 것으로 나타나 NtMEK2-SIPK/WIPK 체계가 담배에서 방어 반응을 조절하고 있음을 알 수 있었다. 그러나 NtMEK2-SIPK/WIPK 체계에 의해서 조절 되는 하위 기질이나 방어관련 유전자들에 대한 연구는 아직 미비한 상태이다. 그래서 본 연구는 NtMEK2-SIPK/WIPK 체계에 매개되는 하위 유전자들을 분리하기 위하여 $NtMEK2^{DD}$ 형질전환 식물체를 이용해 ACP에 기초한 DDRT-PCR을 수행하였다. 그 결과 본 연구를 통해 처음으로 pI2-4, MTS2, SINA, CDM1, HRGP 및 DEG45를 포함해 여섯 개의 DEG들을 선발하였다. 이 유전자들의 발현은 $NtMEK2^{DD}$ 형질전환에서 다시 확인하였으며 특히 pI2-4, CDM1, HRGP의 유전자 발현은 다른 유전자들과 비교해 볼 때 살리실산과 담배모자이크바이러스에 강하게 반응하여 증폭됨을 알 수 있었다. 이러한 결과를 볼 때 NtMEK2-SIPK/WIPK 체계에 의해 조절되는 세 개의 유전자는 병저항성에 관여하고 있음을 제시한다 하겠다.

Keywords

References

  1. Asai, T., G. Tena, J. Plotnikova, M. R. Willmann, W. L. Chi, L. Gomez-Gomez, T. Boller, F. M. Ausubel and J. Sheen. 2002. MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415, 977-983 https://doi.org/10.1038/415977a
  2. Corbin, D. R., N. Sauer and C. J. Lamb. 1987. Differential regulation of a hydroxyproline-rich glycoprotein gene family in wounded and infected plants. Mol. Cell Biol. 7, 4337-4344 https://doi.org/10.1128/MCB.7.12.4337
  3. Dean, J. D., P. H. Goodwin and T. Hsiang. 2002. Comparison of relative RT-PCR and northern blot analyses to measure expression of $\beta$-1,3-glucanase in Nicotiana benthamiana infected with Colletotrichum destructivum. Plant Mol. Biol. Rep. 20, 347-356 https://doi.org/10.1007/BF02772122
  4. Deepak, S., S. Shailasree, R. K. Kini, B. Hause, S. H. Shetty and A. Mithofer. 2007. Role of hydroxyproline-rich glycoproteins in resistance of pearl millet against downy mildew pathogen Sclerospora graminicola. Planta 226, 323-333 https://doi.org/10.1007/s00425-007-0484-4
  5. Durner, J., J. Shah and D. F. Klessing. 1997. Salicylic acid and disease resistance in plants. Trends Plant Sci. 2, 266-274 https://doi.org/10.1016/S1360-1385(97)86349-2
  6. Hwang, I. T., Y. J. Kim, S. H. Kim, C. I. Kwak, Y. Y. Gu and J. Y. Chun. 2003. Annealing control primer system for improving specificity of PCR amplification. Biotechniques 35, 180-1184
  7. Hwang, K. C., S. Y. Park, S. P. Park, J. H. Lim, X. S. Cui and N. H. Kim. 2005. Specific maternal transcripts in bovine oocytes and cleavaged embryos: identification with novel DDRT-PCR methods. Mol. Reprod. Dev. 71, 275-283 https://doi.org/10.1002/mrd.20282
  8. Jin, H., Y. Liu, K. Yang, C. Kim, B. Baker and S. Zhang. 2003. Function of a mitogen-activated protein kinase pathway in N gene-mediated resistance in tobacco. Plant J. 33, 719-731 https://doi.org/10.1046/j.1365-313X.2003.01664.x
  9. Kang, M. K., K. S. Park and D. Choi. 1998. Coordinated expression of defense-related genes by TMV infection or salicylic acid treatment in tobacco. Mol. Cells 8, 388-392
  10. Kim, C. Y. and S. Zhang. 2004. Activation of a mitogen- activated protein kinase cascade induces WRKY family of transcription factors and defense genes in tobacco. Plant J. 38, 142-151 https://doi.org/10.1111/j.1365-313X.2004.02033.x
  11. Kim, Y. J., C. I. Kwak, Y. Y. Gu, I. T. Hwang and J. Y. Chun. 2004. Annealing control primer system for identification of differentially expressed genes on agarose gels. Bio Techniques 36, 424-434
  12. Lee, S. C., S. H. Kim, S. H. An, S. Y. Yi and B. K. Hwang. 2006. Identification and functional expression of the pepper pathogen-induced gene, CAPIP2, involved in disease resistance and drought and salt stress tolerance. Plant Mol. Biol. 62, 151-164 https://doi.org/10.1007/s11103-006-9010-5
  13. Ligterink, W., T. Kroj. U. zur Nieden, H. Hirt and D. Scheel. 1997. Receptor-mediated activation of a MAP kinase in pathogen defense of plants. Science 276, 2054-2057 https://doi.org/10.1126/science.276.5321.2054
  14. Liu, Y., D. Ren, S. Pike, S. Pallardy, W. Gassmann and S. Zhang. 2007. Chloroplast-generated reactive oxygen species are involved in hypersensitive response-like cell death mediated by a mitogen-activated protein kinase cascade. Plant J. 51, 941-954 https://doi.org/10.1111/j.1365-313X.2007.03191.x
  15. Mellon, J. E and J. P. Helgeson. 1982. Interaction of a Hydroxyproline-rich glycoprotein from tobacco callus with potential pathogens. Plant Physiol. 70, 401-405 https://doi.org/10.1104/pp.70.2.401
  16. Park, J. S., I. S. Kim, M. S. Cho, S. Park and S. G. Park. 2006. Identification of differentially expressed genes involved in spine formation on seeds of Daucus carota L. (Carrot), using annealing control primer (ACP) system. J. Plant Biol. 49, 133-140 https://doi.org/10.1007/BF03031009
  17. Ren, D., H. Yang and S. Zhang. 2002. Cell death mediated by mitogen-activated protein kinase pathway is associated with the generation of hydrogen peroxide in Arabidopsis. J. Biol. Chem. 277, 559-565 https://doi.org/10.1074/jbc.M109495200
  18. Sauter, M., G. Rzewuski, T. Marwedel and R. Lorbiecke. 2002. The novel ethylene regulated gene OsUsp1 from rice encodes a member of a plant protein family related to prokaryotic universal stress proteins. J. Exp. Bot. 53, 2325-2331 https://doi.org/10.1093/jxb/erf096
  19. Sessa, G., X. Q. Yang, V. Raz, Y. Eyal and R. Fluhr. 1995. Dark induction and subcellular localization of the pathogenesis- related PRB-1b protein. Plant Mol. Biol. 28, 537-547 https://doi.org/10.1007/BF00020400
  20. Showalter, S. M., J. N. Bell, C. L. Cramer, J. A. Bailey, J. E. Varner and C. J. Lamb. 1985. Accumulation of hydroxyproline- rich glycoprotein mRNAs in response to fungal elicitor and infection. Proc. Natl. Acad. Sci. USA 82, 6551- 6555
  21. Steiner-Lange, S., A. Fischer, A. Boettcher, I. Rouhara, H. Liedgens, E. Schmelzer and W. Knogge. 2003. Differential defense reactions in leaf tissues of barley in response to infection by Rhynchosporium secalis and to treatment with a fungal avirulence gene product. Mol. Plant Microbe Interact 16, 893-902 https://doi.org/10.1094/MPMI.2003.16.10.893
  22. Suh, M., S. K. Oh, Y. C. Kim, H. S. Pai and D. Choi. 2003. Expression of a novel tobacco gene, NgCDM1, is preferentially associated with pathogen-induced cell death. Physiol. Mol. Plant Pathol. 62, 227-235 https://doi.org/10.1016/S0885-5765(03)00049-3
  23. van Schie, C. C. N. and M. A. Haring. 2007. Tomato linalool synthase is induced in trichomes by jasmonic acid. Plant Mol. Biol. 64, 251-263 https://doi.org/10.1007/s11103-007-9149-8
  24. Welsch, R., D. Maass, T. Voegel, D. DellaPenna and P. Beyer. 2007. Transcription factor RAP2.2 and its interacting partner SINAT2: stable elements in the carotenogenesis of Arabidopsis leaves. Plant Physiol. 145, 1073-1085 https://doi.org/10.1104/pp.107.104828
  25. Widmann, C., S. Gibson, M. B. Jarpe and G. L. Johnson. 2001. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol. Rev. 79, 143-180
  26. Yang, K. Y., Y. Liu and S. Zhang. 2001. Activation of a mitogen- activated protein kinase pathway is involved in disease resistance in tobacco. Proc. Natl. Acad. Sci. USA 98, 741-746
  27. Zhang, S. and D. F. Klessig. 1998. Resistance gene N-mediated de novo synthesis and activation of a tobacco mitogen- activated protein kinase by tobacco mosaic virus infection. Proc. Natl. Acad. Sci. USA 95, 7433-7438
  28. Zhang, S. and D. F. Klessig. 2001. MAPK cascades in plant defense signaling. Trends Plant Sci. 6, 520-527 https://doi.org/10.1016/S1360-1385(01)02103-3

Cited by

  1. Mitogen-Activated Protein Kinase Cascade in the Signaling for Polyamine Biosynthesis in Tobacco vol.50, pp.3, 2009, https://doi.org/10.1093/pcp/pcp009