RNA Interference of Plant MAPK Cascades for Functional Studies

  • Juan XuEmail author
  • Shuqun ZhangEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1171)


Arabidopsis genome contains 20 genes encoding mitogen-activated protein kinases (MAPKs, or MPKs), and ten genes encoding MAPK kinases (MAPKKs, or MKKs), the upstream kinases that activate MAPKs in the signaling cascades. They play critical roles in many different biological processes ranging from growth/development to response to environmental stimuli and pathogen invasion. T-DNA knockout lines are not currently available for all these genes. There is also functional redundancy at both MAPK and MAPKK levels. In addition, embryo lethality is associated with some double mutant combinations, which makes it difficult to investigate their specific functions in plants. In such situation, the use of RNA interference technology by which mRNA of interested gene is targeted by double-stranded RNA (dsRNA) for degradation and gene silencing provides a powerful tool for loss-of-function analyses. In this chapter, we describe the hairpin-RNA interference (hpRNAi) method we employed to silence MPK3/MPK6 and their upstream MKK4/MKK5 in the model plant Arabidopsis, with particular emphasis on the generation of hpRNAi constructs for single gene RNAi, tandem RNAi of two MAPKK genes, and tissue-specific RNAi.

Key words

MAPK cascade RNA interference Hairpin RNAi construct Tandem RNAi Tissue/cell-specific RNAi 



This work was supported by the third stage of Zhejiang University 985 Project fund to Juan Xu and Shuqun Zhang.


  1. 1.
    Ichimura K, Shinozaki K, Tena G, Sheen J, Henry Y, Champion A, Kreis M, Zhang S, Hirt H, Wilson C, Heberle-Borse E, Ellisf BE, Morrisg PC, Innesh RW, Eckeri JR, Scheelj D, Klessigk DF, Machidal Y, Mundym J, Ohashin Y, Walker JC (2002) Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci 7:301–308CrossRefGoogle Scholar
  2. 2.
    Gudesblat GE, Iusem ND, Morris PC (2007) Guard cell-specific inhibition of Arabidopsis MPK3 expression causes abnormal stomatal responses to abscisic acid and hydrogen peroxide. New Phytol 173:713–721PubMedCrossRefGoogle Scholar
  3. 3.
    Wang H, Ngwenyama N, Liu Y, Walker JC, Zhang S (2007) Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis. Plant Cell 19:63–73PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Cho SK, Larue CT, Chevalier D, Wang H, Jinn T-L, Zhang S, Walker JC (2008) Regulation of floral organ abscission in Arabidopsis thaliana. Proc Natl Acad Sci U S A 105:15629–15634PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Meng X, Wang H, He Y, Liu Y, Walker JC, Torii KU, Zhang S (2012) A MAPK cascade downstream of erecta receptor-like protein kinase regulates Arabidopsis inflorescence architecture by promoting localized cell proliferation. Plant Cell 24:4948–4960PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Miles GP, Samuel MA, Zhang Y, Ellis BE (2005) RNA interference-based (RNAi) suppression of AtMPK6, an Arabidopsis mitogen-activated protein kinase, results in hypersensitivity to ozone and misregulation of AtMPK3. Environ Pollut 138:230–237PubMedCrossRefGoogle Scholar
  7. 7.
    Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811PubMedCrossRefGoogle Scholar
  8. 8.
    Perrimon N, Ni J-Q, Perkins L (2010) In vivo RNAi: today and tomorrow. Cold Spring Harb Perspect Biol 2:a003640PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Wesley SV, Helliwell CA, Smith NA, Wang M, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581–590PubMedCrossRefGoogle Scholar
  10. 10.
    Smith NA, Singh SP, Wang M-B, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Gene expression: total silencing by intron-spliced hairpin RNAs. Nature 407:319–320PubMedCrossRefGoogle Scholar
  11. 11.
    Rodriguez S, Petersen M, Mundy J (2010) Mitogen-activated protein kinase signaling in plants. Annu Rev Plant Biol 61:621–649PubMedCrossRefGoogle Scholar
  12. 12.
    Zhang S (2009) Mitogen-activated protein kinase cascades in plant signaling. In: Yang Z (ed) Annual plant reviews, vol 33: intracellular signaling in plants. Wiley-Blackwell, Oxford, UK, pp 100–136Google Scholar
  13. 13.
    Twell D, Yamaguchi J, McCormick S (1990) Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis. Development 109:705–713PubMedGoogle Scholar
  14. 14.
    Clough S, Bent A (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–778PubMedCrossRefGoogle Scholar
  15. 15.
    Aoyama T, Chua NH (1997) A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J 11:605–612PubMedCrossRefGoogle Scholar
  16. 16.
    Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.State Key Laboratory of Plant Physiology and Biochemistry, College of Life SciencesZhejiang UniversityHangzhou, ZhejiangChina
  2. 2.Division of Biochemistry, Interdisciplinary Plant Group, Christopher S. Bond Life Sciences CenterUniversity of MissouriColumbiaUSA

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