Engineered RNA-Binding Protein for Gene Regulation in Non-Green Plastids

Non-green plastids are desirable for the expression of recombinant proteins in edible plant parts to enhance the nutritional value of tubers or fruits. However, plastid transgenes are expressed at extremely low levels in the amyloplasts of storage organs such tubers. Here we report a two-component regulatory system consisting of an engineered PPR10^GG RNA binding protein and a cognate BS^GG binding site upstream of a GFP reporter gene in the plastid genome. The BS^GG binding site is not recognized by the resident potato PPR10 protein, restricting GFP protein accumulation to low levels in leaves. When the engineered PPR10^GG protein is expressed from the tuber-specific patatin promoter, GFP accumulation is enhanced 20-fold, from 0.06% to about 1.3% of total soluble protein in tubers while having little impact on GFP accumulation in leaves. The two-component regulatory system enables high-level transgene expression in non-photosynthetic plastids without interfering with chloroplast gene expression in leaves.