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Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance

Nature Biotechnology volume 24, pages 1420–1428 (2006)Cite this article

A Corrigendum to this article was published on 01 February 2007

Abstract

Plant microRNAs (miRNAs) regulate the abundance of target mRNAs by guiding their cleavage at the sequence complementary region. We have modified an Arabidopsis thaliana miR159 precursor to express artificial miRNAs (amiRNAs) targeting viral mRNA sequences encoding two gene silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV). Production of these amiRNAs requires A. thaliana DICER-like protein 1. Transgenic A. thaliana plants expressing amiR-P69159 and amiR-HC-Pro159 are specifically resistant to TYMV and TuMV, respectively. Expression of amiR-TuCP159 targeting TuMV coat protein sequences also confers specific TuMV resistance. However, transgenic plants that express both amiR-P69159 and amiR-HC-Pro159 from a dimeric pre-amiR-P69159/amiR-HC-Pro159 transgene are resistant to both viruses. The virus resistance trait is displayed at the cell level and is hereditable. More important, the resistance trait is maintained at 15 °C, a temperature that compromises small interfering RNA–mediated gene silencing. The amiRNA-mediated approach should have broad applicability for engineering multiple virus resistance in crop plants.

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Figure 1: Design of amiRNA precursors and analysis of transgenic A. thaliana plants expressing amiRNAs.
Figure 2: Transgenic plants expressing amiR-P69159 and amiR-HC-Pro159 are resistant to TYMV and TuMV infection, respectively.
Figure 3: Effect of temperature on amiRNA production and virus resistance.

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Acknowledgements

S.-S.L. was supported by a fellowship from Ministry of Education, Taiwan. K.-C.C. and H.-W.W. are visiting students from the National Chung-Hsing University, Taiwan. We thank Jun Chen for TYMV, Chin-Chih Chen for TuMV and TuMV-GFP; Mengdai Xu for technical assistance, Chan-Sen Wang and Xuning Wang for assistance with microarray analysis and statistical treatment of the results; and Enno Krebbers, Richard Broglie, Karen Broglie and Barbara Mazur for helpful suggestions and stimulating discussions. This work was supported by a grant from DUPONT to N.-H.C.

Author information

Author notes

  1. Jose Luis Reyes

    Present address: Instituto de Biotecnología-UNAM, Ave. Universidad 2001, Cuernavaca, Morelos, 62215, Mexico

  2. Kuan-Chun Chen

    Present address: Department of Plant Pathology, National Chung Hsing University, 250 Kao Kung Road, Taichung, Taiwan, 402

  3. Qi-Wen Niu and Shih-Shun Lin: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Plant Molecular Biology, Rockefeller University, 1230 York Avenue, New York, 10021, New York, USA

    Qi-Wen Niu, Shih-Shun Lin, Jose Luis Reyes, Kuan-Chun Chen, Hui-Wen Wu & Nam-Hai Chua

  2. Department of Plant Pathology, National Chung Hsing University, 250 Kao Kung Road, Taichung, 402, Taiwan

    Shyi-Dong Yeh

  3. Instituto de Biotecnología-UNAM, Ave. Universidad 2001, Cuernavaca, Morelos, 62215, Mexico

    Kuan-Chun Chen

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Contributions

N.-H.C. and J.L.R. first conceived the idea of using amiRNAs to regulate gene expression. Q.-W.N., S.-S.L. and J.L.R. designed the amiRNAs. Q.-W.N. generated the transgenic plants. S.-S.L, Q.-W.N., K.-C.C. and H.-W.N. performed the virus challenge and related experiments. S.-D.Y. provided specific strains of TuMV and TuMV-GFP and advice on experimental design. All authors discussed the results and commented on the manuscript, which was written by N.-H.C. and S.-S.L.

Corresponding author

Correspondence to Nam-Hai Chua.

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Competing interests

Q.W.N., S.-S.L., J.L.R. & N.-H.C. are co-inventors on a patent application on the use of microRNAs to regulate plant gene expression and to confer virus resistance. The results described in this manuscript were used to support claims in the patent application.

Supplementary information

Supplementary Fig. 1

Transcript analysis of 6 predicted target genes of amiRNAs. (PDF 123 kb)

Supplementary Fig. 2

Transgenic plants expressing amiR-P69159 are resistant to TYMV infection. (PDF 64 kb)

Supplementary Fig. 3

Transgenic plants expressing amiR-HC-Pro159 are resistant to TuMV infection. (PDF 91 kb)

Supplementary Fig. 4

Expression of amiRNAs, endogenous miRNAs and ta-siRNA in WT and transgenic plants at 15 °C and 24 °C. (PDF 68 kb)

Supplementary Fig. 5

Alignment of amiR-P69159 and amiR-HC-Pro159 with P69 and HC-Pro sequences of different TYMV and TuMV strains. (PDF 274 kb)

Supplementary Table 1

Comparative microarray analysis of gene expression in WT and amiRNA transgenic plants. (PDF 65 kb)

Supplementary Table 2

Infectivity assay of amiR-TuCP159 transgenic plants challenged with TYMV or TuMV inocula. (PDF 17 kb)

Supplementary Table 3

Break-down of specific resistance amiR-P69159 and amiR-HC-Pro159 transgenic plants co-inoculated with TYMV and TuMV at 15 °C or 24 °C (PDF 17 kb)

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Niu, QW., Lin, SS., Reyes, J. et al. Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol 24, 1420–1428 (2006). https://doi.org/10.1038/nbt1255

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