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Engineered PPR proteins as inducible switches to activate the expression of chloroplast transgenes

Abstract

The engineering of plant genomes presents exciting opportunities to modify agronomic traits and to produce high-value products in plants. Expression of foreign proteins from transgenes in the chloroplast genome offers advantages that include the capacity for prodigious protein output, the lack of transgene silencing and the ability to express multicomponent pathways from polycistronic mRNA. However, there remains a need for robust methods to regulate plastid transgene expression. We designed orthogonal activators that boost the expression of chloroplast transgenes harbouring cognate cis-elements. Our system exploits the programmable RNA sequence specificity of pentatricopeptide repeat proteins and their native functions as activators of chloroplast gene expression. When expressed from nuclear transgenes, the engineered proteins stimulate the expression of plastid transgenes by up to ~40-fold, with maximal protein abundance approaching that of Rubisco. This strategy provides a means to regulate and optimize the expression of foreign genes in chloroplasts and to avoid deleterious effects of their products on plant growth.

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Fig. 1: Experimental strategy.
Fig. 2: Quantitative relationship between PPR10GG expression and the expression of plastid GFP reporters with the GG cis-element.
Fig. 3: Specificity of PPR10GG and PPR10AA for plastid transgenes harbouring the cognate binding site.
Fig. 4: Ethanol-inducible expression of a plastid GFP reporter in a dicistronic context.
Fig. 5: Effects of PPR10GG on RNA transcripts from the monocistronic and dicistronic chloroplast reporters harbouring the GG cis-element.

Data availability

All essential data supporting the findings of this study are available within the paper and its Supplementary Information. Data for additional replicates are available from the corresponding author on request. Nucleotide sequences of the plastid transformation vectors are available under GenBank accessions MK482730 (pAI5), MK482731 (PQY1) and MK482728 (pQY3). Correspondence, requests for plasmids encoding the PPR10 variants and requests for transgenic plants expressing the PPR10 variants should be addressed to A.B. Requests for chloroplast transformation vectors and transplastomic plants lacking the PPR10 transgene should be addressed to P.M. Biological materials will be made available pending on the execution of a Materials Transfer Agreement with the University of Oregon and/or Rutgers University, as applicable.

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Acknowledgements

We are grateful to S. Belcher (University of Oregon) for assistance propagating tobacco and for help preparing figures, and to A. Ioannou and T. Tungsuchat Huang (Rutgers University) for their contributions to preparing plastid reporter constructs. We also appreciate the generous gifts of vectors from S. Strauss (Oregon State University) and D. Weigel (Max Planck Institute for Developmental Biology). This research was supported by the USDA NIFA Foundational Program Award number 2014-67013-21600 to A.B. and P.M.

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A.B. and P.M. conceived the study, designed the strategy and supervised the experiments. M.R., Q.Y. and R.W.-C. performed the experiments and interpreted the data. A.B. wrote the manuscript, which was edited by all co-authors.

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Correspondence to Alice Barkan.

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Rojas, M., Yu, Q., Williams-Carrier, R. et al. Engineered PPR proteins as inducible switches to activate the expression of chloroplast transgenes. Nat. Plants 5, 505–511 (2019). https://doi.org/10.1038/s41477-019-0412-1

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