Rho GTPases pp 393-399 | Cite as

Reconstruction of ROP GTPase Domains on the Plasma Membrane in Tobacco Leaves

  • Yoshihisa OdaEmail author
  • Yoshinobu Nagashima
  • Hiroo Fukuda
Part of the Methods in Molecular Biology book series (MIMB, volume 1821)


Rho-type small GTPases (Rho GTPases) play central roles in various cellular events. Rho GTPases are often activated locally on the plasma membrane, forming plasma membrane domains, which induce downstream signaling. We describe an experimental procedure designed for inducing the production of de novo plasma membrane domains using Arabidopsis ROP11 GTPase. Introduction of ROP11 and its activator and inactivator into the tobacco leaf epidermis leads to formation of ROP11-activated plasma membrane domains on the plasma membrane. Effectors and marker genes can also be introduced alongside ROP11. This reconstruction system allows identifying molecules regulating Rho GTPase polarization.

Key words

ROP GTPase ROPGAP ROPGEF Nicotiana benthamiana Microtubule Plant Xylem Cell wall 



This work was supported by grants from MEXT KAKENHI (grant no. 16H01247 to YO and 15H05958 to HF), the JSPS KAKENHI (grant no. 16H06172 to YO and 16H06377 to HF), and the Naito Foundation to HF. YN is a special joint researcher of the National Institute of Genetics.


  1. 1.
    Yang Z, Lavagi I (2012) Spatial control of plasma membrane domains: ROP GTPase-based symmetry breaking. Curr Opin Plant Biol 15:601–607CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Schaefer A, Reinhard NR, Hordijk PL (2014) Toward understanding RhoGTPase specificity: structure, function and local activation. Small GTPases 5:6CrossRefGoogle Scholar
  3. 3.
    Wu CF, Lew DJ (2013) Beyond symmetry-breaking: competition and negative feedback in GTPase regulation. Trends Cell Biol 23:476–483CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Oda Y, Fukuda H (2012) Initiation of cell wall pattern by a Rho- and microtubule-driven symmetry breaking. Science 337:1333–1336CrossRefPubMedGoogle Scholar
  5. 5.
    Betsuyaku S, Takahashi F, Kinoshita A, Miwa H, Shinozaki K, Fukuda H, Sawa S (2011) Mitogen-activated protein kinase regulated by the CLAVATA receptors contributes to shoot apical meristem homeostasis. Plant Cell Physiol 52:14–29CrossRefGoogle Scholar
  6. 6.
    Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33:949–956CrossRefPubMedGoogle Scholar
  7. 7.
    Zuo J, Niu QW, Chua NH (2000) Technical advance: an estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J 24:265–273CrossRefPubMedGoogle Scholar
  8. 8.
    Sugiyama Y, Wakazaki M, Toyooka K, Fukuda H, Oda Y (2017) A novel plasma membrane-anchored protein regulates xylem cell-wall deposition through microtubule-dependent lateral inhibition of Rho GTPase domains. Curr Biol 27:2522–2528CrossRefPubMedGoogle Scholar
  9. 9.
    Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Oda Y, Iida Y, Kondo Y, Fukuda H (2010) Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein. Curr Biol 20:1197–1202CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Yoshihisa Oda
    • 1
    • 2
    Email author
  • Yoshinobu Nagashima
    • 1
    • 3
  • Hiroo Fukuda
    • 3
  1. 1.Center for Frontier ResearchNational Institute of GeneticsShizuokaJapan
  2. 2.Department of GeneticsSOKENDAI (Graduate University for Advanced Studies)ShizuokaJapan
  3. 3.Department of Biological Sciences, Graduate School of ScienceThe University of TokyoTokyoJapan

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