Risk assessment of plant-to-bacterium transgene flow associated with novel small synthetic genome (minisynplastome) platforms for plastid genetic engineering

Novel cutting-edge technologies for plastid genetic engineering have a great potential in agriculture. Genetic engineering of the plastid genome (plastome) can be performed using both conventional homologous recombination vectors, and novel episomal platforms that rely on synthetic plastomes (minisynplastomes) to express transgenes from a nonintegrating plasmid. Evaluating the potential risk of horizontal gene transfer (HGT) is an important step for regulatory approval of environmental release of these novel genetic engineering tools. In particular, the endosymbiotic origin of plastids from a prokaryotic progenitor may increase the probability of HGT to the environmental microbial community. In this study, the naturally competent soil bacterium Acinetobacter baylyi has been used to test the probability of plant-to-bacterium HGT under laboratory conditions. While plant-to-bacterium HGT can be detected in vitro as a low probability event, the minisynplastome does not show an increased HGT compared to conventional transformation platforms. After a comprehensive evaluation of minisynplastome elements affecting plasmid persistence in bacteria (plastid origin of replications, plastomic regions containing rRNA genes, and regulatory elements for transgene expression), optimized minisynplastome (Gen3) platforms with no residual activity in bacteria and with undetectable HGT were characterized. This study represents a valuable resource for designing minisynplastome transformation platforms with improved environmental biosafety in agriculture.