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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