Functional relocation of the maize chloroplast atpB gene to the nucleus restores photosynthetic competence to a gene-edited non-photosynthetic mutant

We present a novel approach to photosynthetic gene engineering in maize using a nuclear-encoded, chloroplast-targeted TALE-cytidine deaminase enzyme to create non-photosynthetic knockout mutants of the chloroplast rbcL gene. An off-target mutation in the adjacent atpB gene, encoding the {beta} subunit of ATP synthase, was consistently found in all edited lines, identified as pigment-deficient in tissue culture. These double mutants, carrying mutations in both genes, were purified to homoplasmy using unique leaf-base regeneration techniques. To test mutation complementation and identify the causal gene, nuclear transgenic lines expressing chloroplast-targeted RbcL and AtpB proteins were generated. The results show that nuclear expression of AtpB restores chlorophyll accumulation and supports wild-type growth in tissue culture.