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Design of orthogonal regulatory systems for modulating gene expression in plants


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 Once logged in, the plasmids and strains are listed under the JBEI Public Registry tab.


  1. 1.

    Shih, P. M., Liang, Y. & Loqué, D. Biotechnology and synthetic biology approaches for metabolic engineering of bioenergy crops. Plant J. 87, 103–117 (2016).

    PubMed  Google Scholar 

  2. 2.

    Hawkins, K. M. & Smolke, C. D. Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nat. Chem. Biol. 4, 564–573 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Potvin-Trottier, L., Lord, N. D., Vinnicombe, G. & Paulsson, J. Synchronous long-term oscillations in a synthetic gene circuit. Nature 538, 514–517 (2016).

    PubMed  PubMed Central  Google Scholar 

  4. 4.

    Brückner, K. et al. A library of synthetic transcription activator-like effector-activated promoters for coordinated orthogonal gene expression in plants. Plant J. 82, 707–716 (2015).

    PubMed  PubMed Central  Google Scholar 

  5. 5.

    Bashor, C. J. et al. Complex signal processing in synthetic gene circuits using cooperative regulatory assemblies. Science 364, 593–597 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Farzadfard, F., Perli, S. D. & Lu, T. K. Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. ACS Synth. Biol. 2, 604–613 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Lebar, T. & Jerala, R. Benchmarking of TALE- and CRISPR/dCas9-based transcriptional regulators in mammalian cells for the construction of synthetic genetic circuits. ACS Synth. Biol. 5, 1050–1058 (2016).

    CAS  PubMed  Google Scholar 

  8. 8.

    Gaber, R. et al. Designable DNA-binding domains enable construction of logic circuits in mammalian cells. Nat. Chem. Biol. 10, 203–208 (2014).

    CAS  PubMed  Google Scholar 

  9. 9.

    Khalil, A. S. et al. A synthetic biology framework for programming eukaryotic transcription functions. Cell 150, 647–658 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Haseloff, J. GFP variants for multispectral imaging of living cells. Methods Cell Biol. 58, 139–151 (1999).

    CAS  PubMed  Google Scholar 

  11. 11.

    Laplaze, L. et al. GAL4-GFP enhancer trap lines for genetic manipulation of lateral root development in Arabidopsis thaliana. J. Exp. Bot. 56, 2433–2442 (2005).

    CAS  PubMed  Google Scholar 

  12. 12.

    Gardner, M. J. et al. GAL4 GFP enhancer trap lines for analysis of stomatal guard cell development and gene expression. J. Exp. Bot. 60, 213–226 (2009).

    CAS  PubMed  Google Scholar 

  13. 13.

    Smale, S. T. & Kadonaga, J. T. The RNA polymerase II core promoter. Annu. Rev. Biochem. 72, 449–479 (2003).

    CAS  PubMed  Google Scholar 

  14. 14.

    Lubliner, S. et al. Core promoter sequence in yeast is a major determinant of expression level. Genome Res. 25, 1008–1017 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Pan, S., Czarnecka-Verner, E. & Gurley, W. B. Role of the TATA binding protein–transcription factor IIB interaction in supporting basal and activated transcription in plant cells. Plant Cell 12, 125–135 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Horikoshi, M. et al. Transcription factor TFIID induces DNA bending upon binding to the TATA element. Proc. Natl Acad. Sci. USA 89, 1060–1064 (1992).

    CAS  PubMed  Google Scholar 

  17. 17.

    Hampsey, M. Molecular genetics of the RNA polymerase II general transcriptional machinery. Microbiol. Mol. Biol. Rev. 62, 465–503 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Sadowski, I., Ma, J., Triezenberg, S. & Ptashne, M. GAL4-VP16 is an unusually potent transcriptional activator. Nature 335, 563–564 (1988).

    CAS  Google Scholar 

  19. 19.

    Taylor, I. C., Workman, J. L., Schuetz, T. J. & Kingston, R. E. Facilitated binding of GAL4 and heat shock factor to nucleosomal templates: differential function of DNA-binding domains. Genes Dev. 5, 1285–1298 (1991).

    CAS  PubMed  Google Scholar 

  20. 20.

    Kiran, K. et al. The TATA-box sequence in the basal promoter contributes to determining light-dependent gene expression in plants. Plant Physiol. 142, 364–376 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Joshi, C. P. An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucleic Acids Res 15, 6643–6653 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Vaillant, I., Schubert, I., Tourmente, S. & Mathieu, O. MOM1 mediates DNA‐methylation‐independent silencing of repetitive sequences in Arabidopsis. EMBO Rep. 7, 1273–1278 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Okamoto, H. & Hirochika, H. Silencing of transposable elements in plants. Trends Plant Sci. 6, 527–534 (2001).

    CAS  PubMed  Google Scholar 

  24. 24.

    Matzke, M. A., Mette, M. F. & Matzke, A. J. Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Mol. Biol. 43, 401–415 (2000).

    CAS  PubMed  Google Scholar 

  25. 25.

    Kooter, J. M., Matzke, M. A. & Meyer, P. Listening to the silent genes: transgene silencing, gene regulation and pathogen control. Trends Plant Sci. 4, 340–347 (1999).

    CAS  PubMed  Google Scholar 

  26. 26.

    Mutalik, V. K. et al. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat. Methods 10, 354–360 (2013).

    CAS  PubMed  Google Scholar 

  27. 27.

    Jensen, P. R. & Hammer, K. The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl. Environ. Microbiol. 64, 82–87 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Levo, M. & Segal, E. In pursuit of design principles of regulatory sequences. Nat. Rev. Genet. 15, 453–468 (2014).

    CAS  PubMed  Google Scholar 

  29. 29.

    Harcum, S. W. & Bentley, W. E. Heat-shock and stringent responses have overlapping protease activity in Escherichia coli: implications for heterologous protein yield. Appl. Biochem. Biotechnol. 80, 23–38 (1999).

    CAS  PubMed  Google Scholar 

  30. 30.

    Denancé, N., Sánchez-Vallet, A., Goffner, D. & Molina, A. Disease resistance or growth: the role of plant hormones in balancing immune responses and fitness costs. Front. Plant Sci. 4, 155 (2013).

    PubMed  PubMed Central  Google Scholar 

  31. 31.

    Tian, D., Traw, M. B., Chen, J. Q., Kreitman, M. & Bergelson, J. Fitness costs of R-gene-mediated resistance in Arabidopsis thaliana. Nature 423, 74–77 (2003).

    CAS  PubMed  Google Scholar 

  32. 32.

    Fordyce, P. M. et al. De novo identification and biophysical characterization of transcription-factor binding sites with microfluidic affinity analysis. Nat. Biotechnol. 28, 970–975 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Baker, C. R., Tuch, B. B. & Johnson, A. D. Extensive DNA-binding specificity divergence of a conserved transcription regulator. Proc. Natl Acad. Sci. USA 108, 7493–7498 (2011).

    CAS  PubMed  Google Scholar 

  34. 34.

    Baker, C. R., Booth, L. N., Sorrells, T. R. & Johnson, A. D. Protein modularity, cooperative binding, and hybrid regulatory states underlie transcriptional network diversification. Cell 151, 80–95 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Goff, S. A., Cone, K. C. & Fromm, M. E. Identification of functional domains in the maize transcriptional activator C1: comparison of wild-type and dominant inhibitor proteins. Genes Dev. 5, 298–309 (1991).

    CAS  PubMed  Google Scholar 

  36. 36.

    Dingwall, C. & Laskey, R. A. Nuclear targeting sequences—a consensus? Trends Biochem. Sci. 16, 478–481 (1991).

    CAS  PubMed  Google Scholar 

  37. 37.

    Li, T., Stark, M. R., Johnson, A. D. & Wolberger, C. Crystal structure of the MATa1/MATα2 homeodomain heterodimer bound to DNA. Science 270, 262–269 (1995).

    CAS  PubMed  Google Scholar 

  38. 38.

    Hiratsu, K., Matsui, K., Koyama, T. & Ohme-Takagi, M. Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. Plant J. 34, 733–739 (2003).

    CAS  PubMed  Google Scholar 

  39. 39.

    Mead, J. et al. Interactions of the Mcm1 MADS box protein with cofactors that regulate mating in yeast. Mol. Cell. Biol. 22, 4607–4621 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Tan, S. & Richmond, T. J. Crystal structure of the yeast MATα2/MCM1/DNA ternary complex. Nature 391, 660–666 (1998).

    CAS  PubMed  Google Scholar 

  41. 41.

    Elble, R. & Tye, B. K. Both activation and repression of a-mating-type-specific genes in yeast require transcription factor Mcm1. Proc. Natl Acad. Sci. USA 88, 10966–10970 (1991).

    CAS  PubMed  Google Scholar 

  42. 42.

    Fagard, M. & Vaucheret, H. (Trans)gene silencing in plants: how many mechanisms? Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 167–184 (2000).

    CAS  PubMed  Google Scholar 

  43. 43.

    Morel, J. B., Mourrain, P., Béclin, C. & Vaucheret, H. DNA methylation and chromatin structure affect transcriptional and post-transcriptional transgene silencing in Arabidopsis. Curr. Biol. 10, 1591–1594 (2000).

    CAS  PubMed  Google Scholar 

  44. 44.

    Matzke, M. A., Primig, M., Trnovsky, J. & Matzke, A. J. Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J. 8, 643–649 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Sunilkumar, G., Mohr, L., Lopata-Finch, E., Emani, C. & Rathore, K. S. Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP. Plant Mol. Biol. 50, 463–474 (2002).

    CAS  PubMed  Google Scholar 

  46. 46.

    Liu, W. et al. Computational discovery of soybean promoter cis-regulatory elements for the construction of soybean cyst nematode-inducible synthetic promoters. Plant Biotechnol. J. 12, 1015–1026 (2014).

    CAS  PubMed  Google Scholar 

  47. 47.

    Shih, P. M. et al. A robust gene-stacking method utilizing yeast assembly for plant synthetic biology. Nat. Commun. 7, 13215 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Patron, N. J. et al. Standards for plant synthetic biology: a common syntax for exchange of DNA parts. N. Phytol. 208, 13–19 (2015).

    CAS  Google Scholar 

  49. 49.

    Ham, T. S. et al. Design, implementation and practice of JBEI-ICE: an open source biological part registry platform and tools. Nucleic Acids Res 40, e141 (2012).

    PubMed  PubMed Central  Google Scholar 

  50. 50.

    Lohr, D., Venkov, P. & Zlatanova, J. Transcriptional regulation in the yeast GAL gene family: a complex genetic network. FASEB J. 9, 777–787 (1995).

    CAS  PubMed  Google Scholar 

  51. 51.

    Johnston, M. A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae. Microbiological Rev. 51, 458–476 (1987).

    CAS  Google Scholar 

  52. 52.

    Sparkes, I. A., Runions, J., Kearns, A. & Hawes, C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protoc. 1, 2019–2025 (2006).

    CAS  PubMed  Google Scholar 

  53. 53.

    Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    CAS  PubMed  Google Scholar 

  54. 54.

    Jefferson, R. A. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Report. 5, 387–405 (1987).

    CAS  Google Scholar 

  55. 55.

    Liu, P.-P., Koizuka, N., Martin, R. C. & Nonogaki, H. The BME3 (Blue Micropylar End 3) GATA zinc finger transcription factor is a positive regulator of Arabidopsis seed germination. Plant J. 44, 960–971 (2005).

    CAS  PubMed  Google Scholar 

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This work was part of the Department of Energy Early Career Award and the Department of Energy JBEI ( 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.

Author information




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).

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