Genetic Transformation Methods for Diatom Nitzschia captiva: New Tools to Better Understand Dinotom Endosymbiosis

Endosymbiosis is a widespread and ecologically significant phenomenon in the marine environment. How these endosymbiotic relationships evolve into one organism with a new organelle is still mostly unknown and requires investigation into modern symbioses. Dinotoms, dinoflagellates with evolutionarily intermediate diatom plastids, remain at three successive but distinct early stages of organellogenesis and they has been considered to be excellent models for this topic. Efforts to understand the host dinoflagellate-endosymbiotic diatom relationship has been restricted due to the absence of genetic transformation methods for either member of the symbiosis and their hosts. Through the use of Phaeodactylum tricornutum, Cylindrotheca fusiformis, and native Nitzschia captiva diatom plasmids, we have developed biolistic and conjugation transformation methods and a cryopreservation protocol for the diatom N. captiva, an essential prey for the kleptoplastic dinotom Durinskia capensis. We successfully express and target EGFP to the cytosol, mitochondria, and plastids of N. captiva, and visualize these organelles inside D. capensis in vivo. Furthermore, we attempt to utilize CRISPR/Cas9 to target the introduced EGFP gene but find no evidence of successful gene editing.