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Planta

, Volume 239, Issue 2, pp 455–468 | Cite as

Functional identification of multiple nucleocytoplasmic trafficking signals in the broad-spectrum resistance protein RPW8.2

  • Yan-Yan Huang
  • Yi Shi
  • Yang Lei
  • Yan Li
  • Jing Fan
  • Yong-Ju Xu
  • Xian-Feng Ma
  • Ji-Qun Zhao
  • Shunyuan Xiao
  • Wen-Ming WangEmail author
Original Article

Abstract

Nuclear localization signals (NLSs) and nuclear export signals (NESs) are important intramolecular regulatory elements for protein nucleocytoplasmic trafficking. This regulation confers spatial specificity to signal initiation and transduction in eukaryotic cells and thus is fundamental to the viability of all eukaryotic organisms. Here, we developed a simple and rapid method in which activity of putative NLSs or NESs was reported by subcellular localization of two tandem fluorescent proteins in fusion with the respective NLSs or NESs after agroinfiltration-mediated transient expression in leaves of Nicotiana benthamiana (Nb). We further demonstrated that the predicted NES from amino acid residue (aa) 9 to 22 and the NLS from aa91 to 101 in the broad-spectrum disease resistance protein RPW8.2 possess nuclear export and import activity, respectively. Additionally, by testing overlapping fragments covering the full length of RPW8.2, we identified another NLS from aa65 to 74 with strong nuclear import activity and two tandem non-canonical NESs in the C-terminus with strong nuclear export activity. Taken together, our results demonstrated the utility of a simple method to evaluate potential NLSs and NESs in plant cells and suggested that RPW8.2 may be subject to opposing nucleocytoplasmic trafficking forces for its subcellular localization and functional execution.

Keywords

Nuclear localization signal Nuclear export signal Powdery mildew RPW8.2 Cell death 

Notes

Acknowledgments

The project was partially supported by the National Natural Science Foundation of China (grant 31071670) and the Sichuan Research Program of International Cooperation and Exchanges (grant 01981) to W.-M.W., and by National Science Foundation (grant IOS-1146589) to S.X.

Supplementary material

425_2013_1994_MOESM1_ESM.jpg (616 kb)
Supplemental Fig. S1 Amino acid (aa) sequences of RPW8.2. The predicted NES from aa9 to 22 is highlighted green. The predicted bipartite and monopartite NLS are highlighted pink and red, respectively. (JPEG 616 kb)
425_2013_1994_MOESM2_ESM.jpg (1.3 mb)
Supplemental Fig. S2 Comparison of the nuclear-localized 2 × RFP-NLSSV40 with the indicated eYFP-based reporter constructs. Representative LSCM images show the eYFP signal from indicated eYFP-based reporter constructs at YFP channel and the RFP signal from P R82 ::2 × RFP-NLS SV40 in RFP channel that were transiently expressed in N. benthamiana leaves. Arrows indicated the nuclear location where there was no or background eYFP signal. Bars, 20 µm. (JPEG 1290 kb)
425_2013_1994_MOESM3_ESM.jpg (5.1 mb)
Supplemental Fig. S3 Comparison of the nuclear-localized 2 × RFP-NLSSV40 with the indicated eYFP-based reporter constructs. Representative LSCM images show the eYFP signal from indicated eYFP-based reporter constructs at YFP channel and the RFP signal from P R82 ::2 × RFP-NLS SV40 in RFP channel that were transiently expressed in N. benthamiana leaves. Arrows indicated the nuclear location where there was no or background eYFP signal. Arrowheads indicated yellowish shift of the color due to the co-localization of eYFP and RFP in nuclei. Bars, 20 µm. (JPEG 5226 kb)
425_2013_1994_MOESM4_ESM.docx (13 kb)
Supplemental Table S1 Primers used in the study. (DOCX 13 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Yan-Yan Huang
    • 1
  • Yi Shi
    • 1
  • Yang Lei
    • 1
  • Yan Li
    • 1
  • Jing Fan
    • 1
  • Yong-Ju Xu
    • 1
  • Xian-Feng Ma
    • 1
  • Ji-Qun Zhao
    • 1
  • Shunyuan Xiao
    • 2
  • Wen-Ming Wang
    • 1
    Email author
  1. 1.Rice Research InstituteSichuan Agricultural UniversityChengduChina
  2. 2.Institute for Bioscience and Biotechnology Research and Department of Plant Science and Landscape ArchitectureUniversity of MarylandRockvilleUSA

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