Technical Report | Published:

The amplicon-plus system for high-level expression of transgenes in plants

Nature Biotechnology volume 20, pages 622625 (2002) | Download Citation

Subjects

Abstract

Many biotechnological applications require high-level expression of transgenes in plants. One strategy to achieve this goal was the production of potato virus X (PVX) “amplicon” lines: transgenic lines that encode a replicating RNA virus vector carrying a gene of interest1. The idea was that transcription of the amplicon transgene would initiate viral RNA replication and gene expression, resulting in very high levels of the gene product of interest. This approach failed, however, because every amplicon transgene, in both tobacco and Arabidopsis thaliana, was subject to post-transcriptional gene silencing (PTGS)1,2,3. In PTGS, the transgene is transcribed but the transcripts fail to accumulate as a result of sequence-specific targeting and destruction4,5. Even though the amplicon locus is silenced, the level of β-glucuronidase (GUS) activity in a PVX/GUS line is similar to that in some transgenic lines expressing GUS from a conventional (not silenced) GUS locus1. This result suggested that the very high levels of expression originally envisioned for amplicons could be achieved if PTGS could be overcome and if the resulting plants did not suffer from severe viral disease. Here we report that high-level transgene expression can be achieved by pairing the amplicon approach with the use of a viral suppressor of PTGS, tobacco etch virus (TEV) helper component–proteinase (HC-Pro). Leaves of mature tobacco plants co-expressing HC-Pro and a PVX/GUS amplicon accumulate GUS to 3% of total protein. Moreover, high-level expression occurs without viral symptoms and, when HC-Pro is expressed from a mutant transgene, without detrimental developmental phenotypes.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & Consistent gene silencing in transgenic plants expressing a replicating potato virus X RNA. EMBO J. 16, 3675–3684 (1997).

  2. 2.

    & Technical advance: potato virus X amplicon-mediated silencing of nuclear genes. Plant J. 20, 357–362 (1999).

  3. 3.

    , , & Potato virus X amplicons in Arabidopsis mediate genetic and epigenetic gene silencing. Plant Cell 12, 369–379 (2000).

  4. 4.

    , & Gene silencing as an adaptive defence against viruses. Nature 411, 834–842 (2001).

  5. 5.

    & RNA silencing in plants—defense and counterdefense. Science 292, 2277–2280 (2001).

  6. 6.

    et al. A viral suppressor of gene silencing in plants. Proc. Natl. Acad. Sci. USA 95, 13079–13084 (1998).

  7. 7.

    & A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95, 461–470 (1998).

  8. 8.

    et al. HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal. Plant Cell 13, 571–583 (2001).

  9. 9.

    et al. A calmodulin-related protein that suppresses posttranscriptional gene silencing in plants. Science 290, 142–144 (2000).

  10. 10.

    , & Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete processing. EMBO J. 9, 1347–1353 (1990).

  11. 11.

    , & The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants. Plant Mol. Biol. 15, 851–864 (1990).

  12. 12.

    & Expression of single copies of a strongly expressed 35S transgene can be silenced post-transcriptionally. Plant J. 9, 787–797 (1996).

  13. 13.

    , & A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103, 157–167 (2000).

  14. 14.

    , , , & Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses. Plant Cell 9, 859–868 (1997).

  15. 15.

    et al. Cell-to-cell movement of potexviruses: evidence for a ribonucleoprotein complex involving the coat protein and first triple gene block protein. Mol. Plant Microbe Interact. 13, 962–974 (2000).

  16. 16.

    , , & RNA-based silencing strategies in plants. Curr. Opin. Genet. Dev. 11, 221–227 (2001).

  17. 17.

    , & Mutational analysis of the tobacco etch potyviral 35-kDa proteinase: identification of essential residues and requirements for autoproteolysis. Virology 190, 298–306 (1992).

  18. 18.

    Engineering chloroplasts: an alternative site for foreign genes, proteins, reactions and products. Trends Biotechnol. 18, 257–263 (2000).

  19. 19.

    , & Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci. 7, 84–91 (2002).

  20. 20.

    & Evidence that the potyvirus P1 proteinase functions in trans as an accessory factor for genome amplification. J. Virol. 69, 3668–3674 (1995).

Download references

Acknowledgements

We thank Sue Angell for amplicon lines 155 and 163 and Art Hunt for the plant line expressing P1. This work was supported by grants to V.B.V. from the US Department of Agriculture, Competitive Grants Program, and by a grant to V.B.V. and L.H.B. from the National Institutes of Health. A.C.M. was supported in part by a National Science Foundation Industry/Graduate Research Traineeship. D.B. acknowledges the support of the Gatsby Charitable Foundation.

Author information

Affiliations

  1. University of South Carolina, Columbia, SC 29208.

    • Allison C. Mallory
    • , Graham Parks
    • , Matthew W. Endres
    • , Lewis H. Bowman
    • , Gail J. Pruss
    •  & Vicki B. Vance
  2. The Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK.

    • David Baulcombe

Authors

  1. Search for Allison C. Mallory in:

  2. Search for Graham Parks in:

  3. Search for Matthew W. Endres in:

  4. Search for David Baulcombe in:

  5. Search for Lewis H. Bowman in:

  6. Search for Gail J. Pruss in:

  7. Search for Vicki B. Vance in:

Competing interests

Two patents cover the use of HC-Pro to enhance gene expression in conjunction with conventional transgenes or amplicons, respectively: US patent 5,939,541 (V.B.V and G.J.P., coinventors), issued on August 17, 1999, and US patent application 09/338,397 (V.B.V., inventor), which is pending. Two additional pending patent applications cover amplicon vectors and their uses for gene silencing: international patent applications PCT/GB98/00442 and PCT/GB98/02862 (D.B., inventor on both).

Corresponding author

Correspondence to Vicki B. Vance.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nbt0602-622

Further reading