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.
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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.
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425_2013_1994_MOESM1_ESM.jpg
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
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
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)
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Huang, YY., Shi, Y., Lei, Y. et al. Functional identification of multiple nucleocytoplasmic trafficking signals in the broad-spectrum resistance protein RPW8.2. Planta 239, 455–468 (2014). https://doi.org/10.1007/s00425-013-1994-x
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DOI: https://doi.org/10.1007/s00425-013-1994-x