A GRF–GIF chimeric protein improves the regeneration efficiency of transgenic plants

Abstract

The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4–GIF1 transgenic plants were fertile and without obvious developmental defects. Moreover, GRF4–GIF1 induced efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. We also combined GRF4–GIF1 with CRISPR–Cas9 genome editing and generated 30 edited wheat plants with disruptions in the gene Q (AP2L-A5). Finally, we show that a dicot GRF–GIF chimera improves regeneration efficiency in citrus, suggesting that this strategy can be applied to dicot crops.

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Fig. 1: The GRF4–GIF1 chimera and its effects on regeneration frequency.
Fig. 2: The GRF4–GIF1 chimera induces embryogenesis in the absence of cytokinins.
Fig. 3: High frequency of genome-edited plants using combined GRF4–GIF1 and CRISPR–Cas9 technology.

Data availability

Accession numbers and gene names are available in the phylogenetic tree in Supplementary Fig. 1. All wheat gene names are based on genome release RefSeq v1.0. The raw data for the different experiments are available in Supplementary Tables 3, 4, 6 and 7. The steps for the generation of the different vectors and the transformation protocols are described in the Methods. The following vectors will be available through Addgene (http://www.addgene.org/): JD553-wheat GRF4–GIF1 in pDONR, JD633-wheat GRF4–GIF1 in the CRISPR vector, JD630-Vitis GRF4–GIF1 in pDONR, JD638-Vitis miR396-resistant GRF4–GIF1 in pDONR, JD689-Citrus GRF4–GIF1 in pDONR, JD690-Citrus GRF4–GIF1 in pGWB14, JD631-Vitis GRF4–GIF1 in pGWB14 and JD639-Vitis miR396-resistant GRF4–GIF1 in pGWB14.

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Acknowledgements

This project was supported by the Howard Hughes Medical Institute, NRI Competitive Grant 2017-67007-25939 from the USDA National Institute of Food and Agriculture (NIFA) and the International Wheat Partnership Initiative (IWYP). J.F.P. acknowledges support from the Argentinean Research Council (CONICET); Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación; and International Centre for Genetic Engineering and Biotechnology, grant ICGEB/ARG17-01. P.R. was supported by NIH grant GM122968. S.H. acknowledges support from the Biotechnology and Biological Sciences Research Council Genes in the Environment Institute Strategic Programme BB/P013511/1. J.M.D. was supported by a fellowship (LT000590/2014-L) from the Human Frontier Science Program. M.F.E. is a Latin American Fellow in the Biomedical Sciences, supported by the Pew Charitable Trusts. We thank Y. Wang (Chinese Academy of Sciences, Beijing) for the pYP25F binary vector, M. Padilla, R. Rasia, G. Rabasa, B. Van Bockern and M. Smedley for excellent technical support and C. Uauy for coordinating the testing of the GRF4–GIF1 chimera at the John Innes Centre.

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Contributions

J.M.D. contributed to the investigation, methodology, formal analysis, writing and editing. D.M.T. contributed to the investigation, supervision, methodology, project administration, funding acquisition, writing and editing. J.F.P. contributed to study conceptualization, writing and editing. M.F.E. contributed to the experiments involving rice, writing and editing. S.H. performed wheat transformation experiments at the John Innes Centre. P.R. supervised the experiments involving rice and contributed to writing and editing. J.D. contributed to study conceptualization, formal analysis, supervision, project administration, funding acquisition, writing and editing.

Corresponding author

Correspondence to Jorge Dubcovsky.

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Competing interests

J.F.P. and J.M.D. are co-inventors in patent US2017/0362601A1 that describes the use of chimeric GRF–GIF proteins with enhanced effects on plant growth (Universidad Nacional de Rosario Consejo Nacional de Investigaciones Científicas y Técnicas). J.F.P., J.D., D.M.T. and J.M.D. are co-inventors in UC Davis provisional patent application 62/873,123 that describes the use of GRF–GIF chimeras to enhance regeneration efficiency in plants. Vectors are freely available for research, but commercial applications may require a paid nonexclusive license. There is a patent application from KWS/BASF (WO 2019/134884 A1) for improved plant regeneration using Arabidopsis GRF5 and grass GRF1 homologs. None of the authors of this manuscript is part of the KWS/BASF patent or is related to these companies. The KWS/BASF patent focuses on a different cluster of GRF genes than the one described in our study and does not incorporate the GIF1 cofactor or the generation of GRF–GIF chimeras.

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Debernardi, J.M., Tricoli, D.M., Ercoli, M.F. et al. A GRF–GIF chimeric protein improves the regeneration efficiency of transgenic plants. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-0703-0

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