Development of a genetic modification system for the dinoflagellate Amphidinium carterae

Abstract

Dinoflagellates are important marine algae, being an essential symbiont in corals. Loss of the dinoflagellates causes coral bleaching, and ultimately death of coral reefs. Current efforts to study dinoflagellates are greatly hindered by the lack of a reliable method of genetic transformation. The chloroplast genome of dinoflagellates is fragmented into multiple plasmid-like minicircles, each carrying at most a few genes. We have used ‘artificial’ minicircles, based on fusions between endogenous minicircles and E. coli plasmids to establish a transformation system and optimize parameters for it. We have determined the sensitivity of wild type dinoflagellate strains to possible selective agents. We have compared the performance of a number of transformation methods, including electroporation and particle bombardment, and find that electroporation is not effective for transformation, whereas particle bombardment (‘biolistics’) is. We have assessed different parameters for biolistics and subsequent selection. We have shown the successful maintenance of sequence from an artificial minicircle within a dividing cell population over a period of six months, as well as evidence (using RT-PCR) of transcription and the expected phenotype for the inserted gene. This represents a significant step forward in developing the genetic modification of Amphidinium carterae. We are currently testing the protocol with other dinoflagellates.