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Systemic Agrobacterium tumefaciens–mediated transfection of viral replicons for efficient transient expression in plants

Nature Biotechnology volume 23, pages 718–723 (2005)Cite this article

Abstract

Plant biotechnology relies on two approaches for delivery and expression of heterologous genes in plants: stable genetic transformation and transient expression using viral vectors. Although much faster, the transient route is limited by low infectivity of viral vectors carrying average-sized or large genes. We have developed constructs for the efficient delivery of RNA viral vectors as DNA precursors and show here that Agrobacterium–mediated delivery of these constructs results in gene amplification in all mature leaves of a plant simultaneously (systemic transfection). This process, called 'magnifection', can be performed on a large scale and with different plant species. This technology combines advantages of three biological systems (the transfection efficiency of A. tumefaciens, the high expression yield obtained with viral vectors, and the post-translational capabilities of a plant), does not require genetic modification of plants and is faster than other existing methods.

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Figure 1: Constructs maps and quantification of the efficiency of initiation of replication.
Figure 2: Performance of different synthetic vectors.
Figure 3: Efficiency of 'agrodelivery' and of gene expression of the final, fully optimized construct.
Figure 4: Expression of GFP in N. benthamiana plants and Beta vulgaris.
Figure 5: Time course of GFP expression in N. benthamiana and tobacco.

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Acknowledgements

We thank Robert Erwin and Yuri Dorokhov for useful discussions.

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Author notes

  1. Sylvestre Marillonnet and Carola Thoeringer: These authors contributed equally to this work.

Authors and Affiliations

  1. Icon Genetics, Biozentrum Halle, Weinbergweg 22, Halle (Saale), D-06120, Germany

    Sylvestre Marillonnet, Carola Thoeringer, Romy Kandzia, Victor Klimyuk & Yuri Gleba

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  1. Sylvestre Marillonnet
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  2. Carola Thoeringer
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  3. Romy Kandzia
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  4. Victor Klimyuk
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Correspondence to Yuri Gleba.

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The authors are employees of a private company.

Supplementary information

Supplementary Fig. 1

Comparison of the patterns of GFP expression obtained with viral and non-viral vectors. (PDF 39 kb)

Supplementary Fig. 2

Computer prediction of coding and non-coding sequence features in original and synthetic viral vectors. (PDF 515 kb)

Supplementary Table 1

Quantification of the efficiency of initiation of viral replication of the original and modified GFP-expressing viral vectors. (PDF 55 kb)

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Marillonnet, S., Thoeringer, C., Kandzia, R. et al. Systemic Agrobacterium tumefaciens–mediated transfection of viral replicons for efficient transient expression in plants. Nat Biotechnol 23, 718–723 (2005). https://doi.org/10.1038/nbt1094

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