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Causes and consequences of experimental variation in Nicotiana benthamiana transient expression
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  • Published: 14 February 2026

Causes and consequences of experimental variation in Nicotiana benthamiana transient expression

  • Sophia N. Tang  ORCID: orcid.org/0000-0003-1921-78861,2,3,
  • Matthew J. Szarzanowicz1,2,4,
  • Amy Lanctot1,2,4,
  • Sasilada Sirirungruang1,2,4,
  • Liam D. Kirkpatrick1,2,4,
  • Krista Drako  ORCID: orcid.org/0009-0007-6171-37591,2,3,
  • Simon Alamos  ORCID: orcid.org/0000-0003-2867-38401,2,4,
  • Lyurui Cheng  ORCID: orcid.org/0009-0005-2553-05981,2,5,
  • Lucas M. Waldburger1,2,6,7,
  • Shuying Liu1,2,
  • Lena Huang  ORCID: orcid.org/0009-0003-1719-96421,2,
  • Sami Kazaz  ORCID: orcid.org/0000-0001-8891-31351,2,
  • Emine Akyuz Turumtay1,
  • Edward Baidoo1,
  • Aymerick Eudes1,2,
  • Mitchell G. Thompson  ORCID: orcid.org/0000-0002-1490-80741,2 &
  • …
  • Patrick M. Shih  ORCID: orcid.org/0000-0002-2119-33451,2,4,8 

Nature Communications , Article number:  (2026) Cite this article

We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.

Subjects

  • Molecular engineering in plants
  • Plant molecular biology
  • Synthetic biology

Abstract

Infiltration of Agrobacterium tumefaciens into Nicotiana benthamiana has become a foundational technique in plant biology, enabling efficient delivery of transgenes in planta with technical ease, robust signal, and relatively high throughput. Despite transient expression’s prevalence in disciplines such as synthetic biology, little work has been done to describe and address the variability inherent in this system, a concern for experiments that rely on highly quantitative readouts. In a comprehensive analysis of N. benthamiana agroinfiltration experiments, we model sources of variability that affect transient expression. Our findings emphasize the need to validate normalization methods under the specific conditions of each study, as distinct normalization schemes do not always reduce variation either within or between experiments. Using a dataset of 1915 plants collected over three years, we develop a model of variation in N. benthamiana transient expression, using power analysis to determine the number of individual plants required for a given effect size. Drawing on our longitudinal data, these findings inform practical guidelines for minimizing variability through strategic experimental design and power analysis, providing a foundation for more robust and reproducible use of N. benthamiana in quantitative plant biology and synthetic biology applications.

Data availability

All raw data related to this study are publicly available on GitHub (https://github.com/shih-lab/benthi_variation/tree/main/01-data) and Zenodo (DOI: 10.5281/zenodo.1800400555). Prior data reused for this work is available in the Source Data file. All plasmid sequences have been deposited to NCBI and are available under GenBank accession numbers PX927304-PX927337 (available at https://www.ncbi.nlm.nih.gov/genbank/)— see Supplementary Table 1 for individual accession codes. Source data are provided with this paper.

Code availability

Mixed effects model and Monte Carlo simulations were run using R (v4.2.0) and the following packages: tidyverse (v2.0.0)56, transport (v0.14.6), car (v3.1.3), lme4 (v1.1.31)57, and performance (v0.13.0)58. All other analyses and figures were generated with Python (v3.11.4) and the following packages: jupyterlab (v4.0.3)59, pandas (v2.3.3), numpy (v2.3.3)60, seaborn (v0.13.2), matplotlib (v3.10.6), scipy (v1.16.1)61, and statsmodels (v0.14.5). All code related to this study is publicly available on GitHub (github.com/shih-lab/benthi_variation/tree/main/02-code) and Zenodo (DOI: 10.5281/zenodo.1800400555).

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Acknowledgements

We would like to thank Dr. Tyler Backman and Dr. Hector Garcia-Martin for their invaluable advice on statistics, and to all members of the Shih lab for their helpful discussions. We also thank Konami for facilitating the discussion of expression cassette orientation via Dance Dance Revolution artwork. BioRender was used to generate Fig. 1 (https://BioRender.com/i2ej92v) and 2 (https://BioRender.com/0573uvz), as well as Supplementary Figs. S1 (https://biorender.com/u99vtsf), S5 (https://biorender.com/4fpcf4p), S9 (https://BioRender.com/4ila5ts), and S10 (https://biorender.com/w0f87at). This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE 2146752 (received by S.N.T.). This work was part of the DOE JBEI (https://www.jbei.org) supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, supported by the U.S. Department of Energy, Energy Efficiency and Renewable Energy, Bioenergy Technologies Office, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy (received by S.N.T., M.J.S., A.L., S.S., L.D.K., K.D., S.A., L.C., L.M.W., S.L., L.H., S.K., E.A.T., E.B., A.E., M.G.T., and P.M.S). The funders had no role in manuscript preparation or the decision to publish. The views and opinions of the authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, expressed or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. The United States Government retains, and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).

Author information

Authors and Affiliations

  1. Joint BioEnergy Institute, Emeryville, CA, USA

    Sophia N. Tang, Matthew J. Szarzanowicz, Amy Lanctot, Sasilada Sirirungruang, Liam D. Kirkpatrick, Krista Drako, Simon Alamos, Lyurui Cheng, Lucas M. Waldburger, Shuying Liu, Lena Huang, Sami Kazaz, Emine Akyuz Turumtay, Edward Baidoo, Aymerick Eudes, Mitchell G. Thompson & Patrick M. Shih

  2. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Sophia N. Tang, Matthew J. Szarzanowicz, Amy Lanctot, Sasilada Sirirungruang, Liam D. Kirkpatrick, Krista Drako, Simon Alamos, Lyurui Cheng, Lucas M. Waldburger, Shuying Liu, Lena Huang, Sami Kazaz, Aymerick Eudes, Mitchell G. Thompson & Patrick M. Shih

  3. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

    Sophia N. Tang & Krista Drako

  4. Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA

    Matthew J. Szarzanowicz, Amy Lanctot, Sasilada Sirirungruang, Liam D. Kirkpatrick, Simon Alamos & Patrick M. Shih

  5. Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA, USA

    Lyurui Cheng

  6. Department of Bioengineering, University of California, Berkeley, CA, USA

    Lucas M. Waldburger

  7. Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Lucas M. Waldburger

  8. Innovative Genomics Institute, Berkeley, CA, USA

    Patrick M. Shih

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Contributions

P.M.S. and M.G.T. conceptualized the initial study and are the corresponding authors. M.G.T., S.A., L.M.W., and S.N.T. designed experiments. S.N.T., K.D., and L.C. generated plasmids and strains. S.K., E.A.T., A.E., and E.B. performed the PDC extractions and metabolomics, and S.N.T, S.S., L.D.K., S.L., M.G.T., M.J.S., K.D., A.L., and L.H. performed all other experiments. M.J.S. performed mixed-effect modeling and Monte Carlo simulations, and S.N.T. completed all other data analysis. S.N.T. wrote the draft manuscript. All authors discussed the results, reviewed the article, and approved the final article.

Corresponding authors

Correspondence to Mitchell G. Thompson or Patrick M. Shih.

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Competing interests

M.G.T., M.J.S., and P.M.S. have a financial interest in BasidioBio. P.M.S. also has a financial interest in Totality Biosciences. M.G.T., M.J.S., and P.M.S. have no other, non-financial competing interests. All other authors declare that there are no competing interests.

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Tang, S.N., Szarzanowicz, M.J., Lanctot, A. et al. Causes and consequences of experimental variation in Nicotiana benthamiana transient expression. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69458-1

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  • Received: 16 June 2025

  • Accepted: 02 February 2026

  • Published: 14 February 2026

  • DOI: https://doi.org/10.1038/s41467-026-69458-1

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