Su, D.; Chen, S.-A.; Hammer, P.; Chacko, E.; Beilinson, V.; Kinev, A.; Onishi, M.

Most proteins targeted to the organelles of endosymbiotic origin are encoded in the nuclear genome, placing them under the regulatory dominance of the nucleus. For photosynthetic eukaryotes, nuclear-encoded chloroplast proteins arise via two routes: First, genes of cyanobacterial origin were relocated to the nucleus through endosymbiotic gene transfer (EGT). Second, proteins of eukaryotic origin emerged to support chloroplast function and structure. These proteins are reimported into the chloroplast via an import machinery. Reversing the transfer of such genes from the nucleus to the chloroplast genome may offer insights into chloroplast regulation and evolution. In this study, we established a highly efficient and accessible electroporation protocol for chloroplast transformation in the green alga Chlamydomonas reinhardtii, and used it to reverse-transfer two nuclear-encoded genes encoding proteins arising via the two routes described above: the cyanobacteria-derived chloroplast division protein FtsZ1 and the Rubisco-linker EPYC1 of eukaryotic origin. Regardless of origin, both chloroplast-encoded FtsZ1 and EPYC1 showed proper localization and functionality comparable to their nuclear-encoded counterparts. Together, our study provides a robust protocol for chloroplast transformation, a platform for investigating the evolutionary drivers of EGT, and a foundation for advancing chloroplast bioengineering. SIGNIFICANCE STATEMENTO_LIEndosymbiotic gene transfer has resulted in the mass migration of genes from the chloroplast genome to the nuclear genome. Reversing the gene transfer could reveal the evolutionary significance of genome partitioning. C_LIO_LIUsing the green alga Chlamydomonas reinhardtii, this study developed an efficient, electroporation-based protocol for chloroplast transformation. Relocating the genes encoding two chloroplast-targeted proteins, FTSZ1 and EPYC1, to the chloroplast genome showed that the proteins maintained normal localization and function. C_LIO_LIThe established transformation protocol facilitates systematic testing of reverse gene transfer to elucidate the potential evolutionary advantages of genome partitioning and opens new avenues for chloroplast bioengineering. C_LI