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Nuclear import of Maize chlorotic mottle virus capsid protein is mediated by importin-α

Abstract

Maize chlorotic mottle virus (MCMV), one of the important pathogens causing corn lethal necrosis disease, possesses a positive-sense single-stranded RNA genome. Subcellular localization of MCMV coat protein (CP) was investigated by tagging with fluorescent proteins to explore its role in virus infection. Our results showed that the CP fusion proteins can be imported into the nucleus, and the N-terminal region rich in basic amino acids is essential for its nuclear localization. Bimolecular fluorescence complementation assays in maize protoplasts demonstrated that the MCMV CP interacted with the nuclear import factor importin-α1a (ZmIMPα1a) and importin-α1b (ZmIMPα1b), which suggests that the nuclear import of MCMV CP is mediated by the classical importin-α/β pathway in plants. We also found that both ZmIMPα1a and ZmIMPα1b expression were up-regulated in maize tissues at 7 d and 10 d post MCMV inoculation, respectively. Silencing ZmIMPα1a and ZmIMPα1b expression simultaneously using the Brome mosaic virus-based silencing vector decreased MCMV accumulation significantly. Our results demonstrated the nuclear import of MCMV CP and imply a role of ZmIMPα1a and ZmIMPα1b during MCMV infection.

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Acknowledgments

We thank Dr. Kay Scheets from Oklahoma State University for the gift of MCMV clone, Dr. Jörg Kudla from Universität Münster for providing vectors used in the BiFC assay, Dr. Zejian Guo from China Agricultural University for providing the plasmid pCAM35S-GFP, Dr. Andrew Jackson from University of California, Berkeley for providing vectors pGDG and pGDR, Dr. David Ehrhardt from Carnegie Institute of Washington for providing the plasmid pEZS-NL and Dr. Richard S. Nelson from Samuel Roberts Noble Foundation for providing BMV vectors. This study was supported by the National Basic Research Program of China (#2012CB114004), a grant from the Ministry of Agriculture of China (2014ZX08003-001), and the 111 Program (B13006) from the Ministry of Education of China.

Author contributions

B. Z. conducted most of the experiments, analyzed data and wrote the manuscript. F. L. performed partial experiments and analyzed data. T. Z. revised the manuscript. Z. F. designed the experiment, supervised the study and revised the manuscript.

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Correspondence to Zaifeng Fan.

Electronic supplementary material

Fig. S1
figure7

Phylodendrogram of the 7 maize importin-α paralogs Alignments were performed using ClustalW and the tree was prepared in MEGA6. The sequences of proteins used in the analysis can be found at MaizeGDB with the accession numbers. The importin-α paralogs investigated in this study were denoted. (GIF 972 kb)

Fig. S2
figure8

The relative expression level of ZmIMPα2 during MCMV infection Relative expression levels of ZmIMPα2 were determined at 4, 7 and 10 dpi in the first systemically infected maize leaves by qRT-PCR. Three independent experiments were conducted with four biological replicates each. Bars represent the grand means ± SD. Data were analyzed using a two-sample t-test. The P values are shown. (GIF 328 kb)

Table S1

Primers used for PCR amplification (PDF 278 kb)

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High resolution image (TIFF 1.11 mb)

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Zhan, B., Lang, F., Zhou, T. et al. Nuclear import of Maize chlorotic mottle virus capsid protein is mediated by importin-α. Eur J Plant Pathol 146, 881–892 (2016). https://doi.org/10.1007/s10658-016-0966-y

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Keywords

  • Capsid protein
  • Subcellular localization
  • Nuclear import
  • Importin-α
  • BMV-VIGS