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Non-specific activities of the major herbicide-resistance gene BAR

Nature Plants volume 3, pages 937–945 (2017)Cite this article

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Abstract

Bialaphos resistance (BAR) and phosphinothricin acetyltransferase (PAT) genes, which convey resistance to the broad-spectrum herbicide phosphinothricin (also known as glufosinate) via N-acetylation, have been globally used in basic plant research and genetically engineered crops1,2,3,4. Although early in vitro enzyme assays showed that recombinant BAR and PAT exhibit substrate preference toward phosphinothricin over the 20 proteinogenic amino acids1, indirect effects of BAR-containing transgenes in planta, including modified amino acid levels, have been seen but without the identification of their direct causes5,6. Combining metabolomics, plant genetics and biochemical approaches, we show that transgenic BAR indeed converts two plant endogenous amino acids, aminoadipate and tryptophan, to their respective N-acetylated products in several plant species. We report the crystal structures of BAR, and further delineate structural basis for its substrate selectivity and catalytic mechanism. Through structure-guided protein engineering, we generated several BAR variants that display significantly reduced non-specific activities compared with its wild-type counterpart in vivo. The transgenic expression of enzymes can result in unintended off-target metabolism arising from enzyme promiscuity. Understanding such phenomena at the mechanistic level can facilitate the design of maximally insulated systems featuring heterologously expressed enzymes.

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Fig. 1: Accumulation of acetyl-aminoadipate and acetyl-tryptophan in senescent leaves of Arabidopsis carrying the BAR transgene.
Fig. 2: BAR-dependent accumulation of acetyl-aminoadipate and acetyl-tryptophan is linked to nitrogen remobilization during senescence.
Fig. 3: In vitro enzyme kinetic assays of BAR against native and non-native substrates.
Fig. 4: Structural basis for amino acid N-acetylation catalysed by BAR and structure-guided engineering of BAR with reduced non-specific activities.

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Acknowledgements

We thank D.M. Sabatini, G.R. Fink, N. Amrhein and E. Martinoia for helpful discussions. We thank J.M. Cheeseman for providing the phosphinothricin-resistant Glycine max line. We thank J. Varberg for providing the phosphinothricin-resistant B. napus line and M. Rahman for providing conventional B. napus lines. This work is based on research conducted at the Northeastern Collaborative Access Team (NE-CAT) beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6 M detector on NE-CAT 24-ID-C beam line is funded by a NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This work was supported by the Swiss National Science Foundation (grant 31003A_149389 to S.H. and postdoctoral fellowship P2ZHP3_155258 to B.C.), the EU-funded Plant Fellows program (S.A.), the Pew Scholar Program in the Biomedical Sciences (J.K.W.) and the Searle Scholars Program (J.K.W.).

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  1. Ramon Hochstrasser

    Present address: Institute of Medical Microbiology, University of Zurich, 8006, Zurich, Switzerland

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, MA, 02142, USA

    Bastien Christ & Jing-Ke Weng

  2. Department of Plant and Microbial Biology, University of Zurich, 8008, Zurich, Switzerland

    Ramon Hochstrasser, Luzia Guyer, Rita Francisco, Sylvain Aubry & Stefan Hörtensteiner

  3. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA

    Jing-Ke Weng

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Contributions

B.C., S.A., S.H. and J.K.W. designed experiments; B.C., R.H., L.G., R.F. and S.A. performed experiments; B.C., R.H., L.G. and J.K.W. analysed data; B.C., S.H., S.A. and J.K.W. wrote the manuscript.

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Correspondence to Stefan Hörtensteiner or Jing-Ke Weng.

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Competing interests

B.C. and J.K.W. have filed a patent application on BAR and PAT mutants described in this paper that show altered acetyltransferase activity.

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Christ, B., Hochstrasser, R., Guyer, L. et al. Non-specific activities of the major herbicide-resistance gene BAR . Nature Plants 3, 937–945 (2017). https://doi.org/10.1038/s41477-017-0061-1

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