Cell wall digestion of the dinoflagellate Breviolum minutum.

The mitigation of coral bleaching requires adaptation of its endosymbionts to rising ocean temperatures, acidity, and ultra-violet radiation. While genetic transformation may offer a means for enhancing endosymbiont resilience to these environmental stressors, the opportunity for doing so in dinoflagellates is likely hampered, in part, by their armored cortex, which may present a formidable barrier to intracellular delivery. Here, using Breviolum minutum (Clade B Symbiodiniaceae) as a model, we show that this barrier may be moderated by first disrupting the outer plasma membrane with the detergent octyl β-D-glucopyranoside, followed by enzymatic digestion of the underlying cellulose with Cellulase RS and Macerozyme R-10. Treatment using this new protocol results in 61% reduction in calcofluor-based cell wall staining (i.e., 25% staining for protocol vs. 64% for control), thus demonstrating the ability for considerable cell wall digestion. Furthermore, protoplasts isolated thereby exhibit rapid cell wall recovery, as well as comparable PSII activity and cell growth as the control, thus suggesting that the protocol minimally affects acute- and long-term cellular function. Finally, the isolated protoplasts also show a potentially slight increase in permeability to a model exogenous cargo after electroporation with a non-optimized protocol. Collectively, this constitutes the first known successful generation of viable B. minutum protoplasts, and thus, serves as a foundation for future studies seeking to lower the barrier for transformation of these and possibly other Symbiodiniaceae. [ABSTRACT FROM AUTHOR]