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 partners evolve into an organism with a new organelle is still mostly unknown and requires investigation into modern symbioses. Dinotoms, dinoflagellates with evolutionarily intermediate diatom plastids, are considered excellent models for studying organellogenesis as they remain at three successive but distinct stages. Efforts to understand the host dinoflagellate-endosymbiotic diatom relationship has been limited by the lack of genetic transformation methods for either member of the symbiosis. To address this absence, we modified existing diatom biolistic and conjugation transformation methods and cryopreservation protocols for the diatom Nitzschia captiva, an essential prey for the kleptoplastic dinotom Durinskia capensis. Through the use of Phaeodactylum tricornutum, Cylindrotheca fusiformis, and native Nitzschia captiva diatom designed plasmids, we successfully express and target EGFP to the cytosol, mitochondria, and plastids of N. captiva, and visualize these organelles inside D. capensis in vivo, allowing specific labeling and tracking of organelles and proteins after ingestion. Furthermore, we attempt to utilize CRISPR/Cas9 to target the introduced EGFP gene but find no evidence of successful gene editing.