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Engineered RNA-binding protein for transgene activation in non-green plastids

Abstract

Non-green plastids are desirable for the expression of recombinant proteins in edible plant parts to enhance the nutritional value of tubers or fruits, or to deliver pharmaceuticals. However, plastid transgenes are expressed at extremely low levels in the amyloplasts of storage organs such as tubers1,2,3. Here, we report a regulatory system comprising a variant of the maize RNA-binding protein PPR10 and a cognate binding site upstream of a plastid transgene that encodes green fluorescent protein (GFP). The binding site is not recognized by the resident potato PPR10 protein, restricting GFP protein accumulation to low levels in leaves. When the PPR10 variant is expressed from the tuber-specific patatin promoter, GFP accumulates up to 1.3% of the total soluble protein, a 60-fold increase compared with previous studies2 (0.02%). This regulatory system enables an increase in transgene expression in non-photosynthetic plastids without interfering with chloroplast gene expression in leaves.

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Fig. 1: PPR10 binding site system for regulating gene expression in potato amyloplasts.
Fig. 2: Leaf and tuber phenotypes of potato plants illuminated with tungsten or ultraviolet light.
Fig. 3: GFP expression in transplastomic potato plants expressing the PPR10GG protein.

Data availability

The nucleotide sequences are deposited in GenBank with accession numbers MK482729 and MK482730. Correspondence and requests for chloroplast transformation vectors and transplastomic plants should be addressed to P.M. (maliga@waksman.rutgers.edu). Requests for plasmids encoding the PPR10 variants, and for transgenic plants expressing the PPR10 variants should be addressed to A.B. (abarkan@uoregon.edu). Biological materials will be made available pending the execution of a Materials Transfer Agreement with Rutgers University and/or the University of Oregon, as applicable.

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Acknowledgements

We thank A. Ioannou and T. Tungsuchat Huang (Rutgers University) for plastid transformation vectors pAI3 and pAI5, R. Williams-Carrier (University of Oregon) for the PPR10 antibody and Agrobacterium binary vector carrying PPR10GG under the control of a patatin promoter and F. Ludewig (University of Erlangen-Nuremberg) for the patatin promoter, T. Osumi (Simplot Plant Sciences) for sterile shoot cultures of potato cv. Desire 2-24 and R. Williams-Carrier and M. Rojas (University of Oregon) for reading the manuscript and suggestions. This research was supported by USDA NIFA Foundational Program Award Number 2014-67013-21600 to A.B. and P.M.

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A.B. and P.M. designed the experiments. P.M. designed the plastid constructs. A.B. designed the Agrobacterium binary vector. Q.Y. transformed the potato plastid and nuclear genomes, regenerated the plants and characterized chloroplast and nuclear gene expression. Q.Y. and P.M. interpreted the results and all authors contributed to the preparation of the manuscript.

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Correspondence to Pal Maliga.

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Yu, Q., Barkan, A. & Maliga, P. Engineered RNA-binding protein for transgene activation in non-green plastids. Nat. Plants 5, 486–490 (2019). https://doi.org/10.1038/s41477-019-0413-0

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