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.
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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.
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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).
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Mallory, A., Parks, G., Endres, M. et al. The amplicon-plus system for high-level expression of transgenes in plants. Nat Biotechnol 20, 622–625 (2002). https://doi.org/10.1038/nbt0602-622
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DOI: https://doi.org/10.1038/nbt0602-622
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