Higher plant plastids contain active homologous recombination (HR) machinery that has been harnessed for plastid transformation in a limited number of plant species. Stable plastid transformation requires homoplasmic plants in which all plastid genome (plastome) copies are transformed to avoid segregation of the plastid-encoded trait. In plant species where plastid transformation has been demonstrated to date, homoplasmy is achieved through a stochastic process by which plastome copies segregate randomly, occasionally enriching for the transformed plastome. It is unknown why this process can result in homoplasmy in dicots but not monocots, but current plastid transformation and selection mechanisms cannot generate homoplasmic monocot plastid transformants. The present project describes a novel plastid selection mechanism that will harness the native plastid HR machinery to drive plastid transformants to homoplasmy. Double-stranded break (DSB) induction actively recruits DNA repair machinery to the DSB site. This project will actively create DSBs in wild-type copies of the plastome. These DSBs will drive the plastid transformant toward homoplasmy by two complementary mechanisms: DSB induction will stimulate HR at the site of the DSBs, resulting in recombination between the transformed and the wild-type plastome that will actively incorporate the transgene(s) into all copies of the plastome. DSBs in untransformed plastome copies will remove necessary plastid genes. This will actively select against the untransformed plastome and slow its replication relative to the intact transformed plastome. Successful demonstration of the proposed plastid transformation and selection scheme will allow for the practical application of plastid transformation to all crop plants. Given the benefits of plastid relative to nuclear transformation, this represents a significant market opportunity for Benson Hill Biosystems.
