Transient transformation of cultured photosynthetic dinoflagellates (Symbiodinium spp.) with plant-targeted vectors

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Mario Fernando Ortiz-Matamoros
Marco A Villanueva
Tania Islas-Flores

Abstract

Reproducible and reliable genetic transformation methods are a key tool for understanding the physiology and cell biology of Symbiodinium. Nevertheless, transformation methods previously applied to cells such as microalgae, including those utilizing glass beads, have not been tested on these microorganisms. Here, we report a simple, transient transformation method, which allowed plasmid incorporation into three distinct clades of cultured Symbiodinium cells with the plant-targeted plasmid pCB302 harboring sequences encoding a fusion of green fluorescent protein (gfp) with RACK1C from Arabidopsis thaliana (AtRACK1C). Accessibility of the plasmid to the resistant cell wall and through the plasma membrane of the dinoflagellates was achieved through vigorous shaking in the presence of glass beads and polyethylene glycol. A resistance gene to the herbicide Basta allowed appropriate selection in the photosynthetic cells. The transformation frequency per every 106 cells was 107 ± 7 transformants for Symbiodinium kawagutii, 74 ± 8 for Symbiodinium microadriaticum ssp. microadriaticum, and 65 ± 5 for Symbiodinium sp. Mf11. Moreover, Symbiodinium pulchrorum cultures were successfully transformed with a different vector (pCAMBIA-FABD2-gfp) under the same conditions, further validating our procedure. The observation of green fluorescent emission from the cell cytoplasm in all performed transformations indicated that the procedure allowed the heterologous plasmids to enter and undergo expression in the Symbiodinium cells. The success of this transient transformation method opens interesting possibilities for functional genomics studies in Symbiodinium spp. 

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