Stable transformation of Micractinium conductrix SAG 241.80: New tools for exploring photosymbiotic interactions

Endosymbiosis played a key role in the evolution of cellular complexity, but mechanisms underpinning establishment and maintenance remain unclear. Paramecium bursaria harbours Chlorellaceae algal endosymbionts and is a valuable model system for understanding photosymbiosis. However, there are a lack of molecular tools available for exploring the association. Here we report stable transformation of the P. bursaria endosymbiont Micractinium conductrix SAG 241.80. Bioluminescent reporter assays were used to identify endogenous regulatory sequences for transgene expression and to refine a protocol for delivery of exogenous DNA. The bleomycin resistance gene Shble was then identified as an effective selectable marker for isolating transformed cell lines. We demonstrate that endogenous introns enhance transgene expression and that isolation of transformants with desirable characteristics can be achieved without extensive screening by using a 2A peptide to couple expression of an upstream non-selectable transgene to expression of the Shble selectable marker. Finally, we demonstrate that M. conductrix SAG 241.80 transformants expressing fluorescent proteins can be introduced into host cells and observed in the absence of selection, enabling distinct endosymbiont genotypes to be unambiguously identified, compared and monitored within the host environment. The development of molecular tools reported here opens new avenues for addressing unresolved questions in photosymbiosis. Significance StatementO_LIParamecium bursaria is a single-celled, mixotrophic ciliate which forms photosymbiotic interactions with Chlorellaceae green algae and is a popular model system for symbiosis research. C_LIO_LIWe established a stable transformation protocol for the P. bursaria algal endosymbiont M. conductrix SAG 241.80 and identified properties of synthetic gene constructs which facilitate isolation of transformants with robust transgene expression that can be detected within the host environment. C_LIO_LIThis marks significant progress in model system development and opens new avenues for exploring molecular and cellular mechanisms underpinning photosymbiotic interactions. C_LI