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Design of orthogonal regulatory systems for modulating gene expression in plants

Abstract

Agricultural biotechnology strategies often require the precise regulation of multiple genes to effectively modify complex plant traits. However, most efforts are hindered by a lack of characterized tools that allow for reliable and targeted expression of transgenes. We have successfully engineered a library of synthetic transcriptional regulators that modulate expression strength in planta. By leveraging orthogonal regulatory systems from Saccharomyces spp., we have developed a strategy for the design of synthetic activators, synthetic repressors, and synthetic promoters and have validated their use in Nicotiana benthamiana and Arabidopsis thaliana. This characterization of contributing genetic elements that dictate gene expression represents a foundation for the rational design of refined synthetic regulators. Our findings demonstrate that these tools provide variation in transcriptional output while enabling the concerted expression of multiple genes in a tissue-specific and environmentally responsive manner, providing a basis for generating complex genetic circuits that process endogenous and environmental stimuli.

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Fig. 1: Design and characterization of a library of synthetic promoters.
Fig. 2: Teasing apart the contribution of DNA elements reveals generalizable trends in promoter expression strength.
Fig. 3: Using synthetic promoters for the coordinated expression of multiple stacked transgenes.
Fig. 4: Fusions of TADs to both full-length and minimal DNA-binding domains provides a level of modularity to trans-element design.
Fig. 5: Using synthetic repressors enables synthetic promoter compatibility with repressor logic.
Fig. 6: Hybrid promoters incorporate cis-elements with binding sites for multiple TF families.

Data availability

The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information Files are available from the corresponding author upon request. All vectors and resources described are publicly available and can be found through the Inventory of Composable Elements at https://acs-registry.jbei.org/. Once logged in, the plasmids and strains are listed under the JBEI Public Registry tab.

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Acknowledgements

This work was part of the Department of Energy Early Career Award and the Department of Energy JBEI (http://www.jbei.org) supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research through contract DE-AC02-05CH11231 (P.M.S., D.L. and H.V.S.) between the Lawrence Berkeley National Laboratory and the US Department of Energy. This work was also funded by BASF, Rec ID 85335789 (P.M.S.). P.M.S. was supported by grant no. 1K99AT009573/R00AT009573 from the National Center for Complementary and Integrative Health at the National Institutes of Health. M.S.B. was supported by a National Science Foundation Graduate Research Fellowship, fellow ID 2018262076.

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Contributions

P.M.S. and D.L. designed the project. M.S.B., K.M.V. and A.A.R. carried out transient expression experiments in N. benthamiana. K.M.V. and N.M. carried out stable Arabidopsis experiments. M.S.B. and A.Z. performed data analysis. M.S.B. and M.G.T. carried out qPCR experiments. M.S.B., A.Z. and P.M.S. wrote the paper. P.M.S., H.V.S. and D.L. edited the document and provided financial support and supervision.

Corresponding author

Correspondence to Patrick M. Shih.

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Supplementary information

Supplementary Information

Supplementary Figs 1–21 and Tables 1–3.

Reporting Summary

Supplementary Dataset 1

Complete parts list with shorthand nomenclature and sequence data.

Supplementary Dataset 2

Expression strengths of all TF/promoter pairs characterized with the trans-element library.

Supplementary Dataset 3

Complete list of all cis-element sequences used in promoter design.

Supplementary Dataset 4

Minimal promoter library with sequences used.

Supplementary Dataset 5

Summary of all parts, constructs and sequences synthesized and assembled for the Gal4 system to investigate CCE and minimal promoters.

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Belcher, M.S., Vuu, K.M., Zhou, A. et al. Design of orthogonal regulatory systems for modulating gene expression in plants. Nat Chem Biol 16, 857–865 (2020). https://doi.org/10.1038/s41589-020-0547-4

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