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GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing

Nature Plants volume 3, pages 875–884 (2017)Cite this article

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Abstract

The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.

Transgene silencing is a significant challenge for plant genetic engineering1 as it leads to phenotypic variability and eventual loss of transgene expression2,3,4. Precocious silencing is especially detrimental to biotechnological applications of agriculturally important crops, such as maize and canola. Many of these species are laborious to transform and have long breeding cycles; thus, cost and risks of failure for product development increase when silencing occurs. Improved transgene design could help achieve the desired levels of transgene expression and ensure that expression is stable over multiple generations. Insights into mechanisms of silencing have been gained through studies of transposable element (TE) inactivation and silencing. These pathways are largely conserved between eudicots such as Arabidopsis and monocots such as maize5. Two major routes leading to heritable TE silencing have been described: identity-based and expression-dependent silencing6. Identity-based silencing relies on a small RNA (sRNA)-mediated transfer of silencing from an endogenous, inactivated TE to a newly introduced, active TE. Expression-dependent silencing afflicts heterologous TEs with no sequence similarity to the host genome and depends on the de novo recruitment of silencing machinery to an actively transcribed TE6,7.

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Fig. 1: Establishment of transgene silencing is alleviated in Arabidopsis mutants defective in TGS or PTGS gene silencing.
Fig. 2: Transgenes with distinct construct designs encountered different degrees of transgene silencing.
Fig. 3: DNA cytosine methylation correlates with the loss of LC-PUFA accumulation in plants containing pDAB454, pDAB588 and pDAB525 transgenes.
Fig. 4: Cry protein accumulation in transgenic maize plants.
Fig. 5: Molecular analyses of transgenic plants with different cry CDS biases.

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Acknowledgements

At Dow AgroSciences, we thank M. German for his review and comments on the manuscript; S. Dhavala for initial consultations regarding statistical data analysis; W. Case for Arabidopsis mutant validation, development of high throughput qPCR assays and transgene copy number and zygosity analyses in Arabidopsis and maize; R. Hampton, C. Larsen and M. Foster for Arabidopsis protein analyses; and C. Ransom, V. Stoltz and D. Gachotte for LC-PUFA analyses. We thank S. Jamidar (University of Delaware) and R. McEwan (Dow AgroSciences) for assistance on NGS analyses of maize transgene-derived sRNAs.

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Author notes

  1. Tzuu-fen Lee

    Present address: Dupont Pioneer, Johnston, IA, 50131, USA

  2. Xiaozeng Yang

    Present address: Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, 100097, Beijing, China

  3. Lyudmila V. Sidorenko and Tzuu-fen Lee contributed equally to this work.

Authors and Affiliations

  1. Dow AgroSciences LLC., 9330 Zionsville Road, Indianapolis, IN, 46268, USA

    Lyudmila V. Sidorenko, Aaron Woosley, William A. Moskal, Scott A. Bevan, P. Ann Owens Merlo, Terence A. Walsh, Xiujuan Wang, Staci Weaver, Todd P. Glancy, PoHao Wang, Xiaozeng Yang & Shreedharan Sriram

  2. Delaware Biotechnology Institute, Department of Plant and Soil Sciences, University of Delaware, Newark, DE, 19716, USA

    Tzuu-fen Lee & Blake C. Meyers

  3. Donald Danforth Plant Science Center, St. Louis, MO, 63132, USA

    Blake C. Meyers

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Contributions

L.V.S., T.L., A.W. and B.C.M. designed studies, executed experiments and interpreted data. L.V.S., T.L., A.W., T.A.W. and B.C.M. cowrote the paper with participation from all authors. Specifically, L.V.S. designed and led execution of Arabidopsis and maize experiments and worked with W.A.M. and S.W. on experiment planning, sample collection, data generation and analyses. T.L. conducted Arabidopsis NGS library preparation and related data analysis. A.W. developed maize transgenes, including CDSs biasing, and led analyses of protein accumulation in maize transgenic plants. W.A.M. conducted Arabidopsis molecular analysis to identify and select plants for studies, and coordinated transfer of materials between labs. S.A.B. developed canola-biased CDS optimizations and designed and participated in building of all of the constructs used in the Arabidopsis study. T.P.G. provided protein data analyses and contributed to writing. P.H.W. designed and executed a subset of maize experiments and contributed to writing. X.Y. and S.S. executed maize sRNA data analyses, interpreted results and prepared figures. X.W. carried out statistical analysis and contributed to writing. P.A.O.M., T.A.W. and B.C.M. conceived the project and provided overall guidance for teams at Dow AgroSciences (P.A.O.M., T.A.W.) and the University of Delaware (B.C.M.).

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Correspondence to Blake C. Meyers.

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Competing interests

This work was part of the research and development programs of Dow AgroSciences LLC, a for-profit agricultural technology company. Part of the work was performed within a research collaboration with BCM (affiliated with University of Delaware, DE and Donald Danforth Plant Science Center, MO). L.V.S., A.W., W.A.M., S.A.B., P.A.O.M., T.A.W., X.W., S.W., T.P.G., P.H.W., X.Y. and S.S. were employees of Dow AgroSciences LLC. T.L. and B.C.M. were employees of the University of Delaware, DE and Donald Danforth Plant Science Center, MO. Some of the data was used in patent applications US2013 0150599 A1, US2014 0359900, WO2015 081270 A1.

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Sidorenko, L.V., Lee, Tf., Woosley, A. et al. GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing. Nature Plants 3, 875–884 (2017). https://doi.org/10.1038/s41477-017-0040-6

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