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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Nature Communications Open Access 11 May 2022
Orthogonal control of gene expression in plants using synthetic promoters and CRISPR-based transcription factors
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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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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.
All authors declare no competing interests.
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Supplementary Figs 1–21 and Tables 1–3.
Complete parts list with shorthand nomenclature and sequence data.
Expression strengths of all TF/promoter pairs characterized with the trans-element library.
Complete list of all cis-element sequences used in promoter design.
Minimal promoter library with sequences used.
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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