Isolation of uracil auxotroph mutants of coral symbiont alga for symbiosis studies

Coral reef ecosystems rely on stable symbiotic relationship between the dinoflagellate Symbiodiniumspp. and host cnidarian animals. The collapse of such symbiosis could cause coral‘ bleaching’ and subsequent host death. Despite huge interest on Symbiodinium, lack of mutant strains and readily available genetic tools have hampered molecular research. A major issue was the tolerance to marker antibiotics. Here, we isolated Symbiodiniummutants requiring uracilfor growth, and hence, useful in transformation screening. We cultured Symbiodiniumspp. cells in the presence of 5-fluoroorotic acid(5FOA), which inhibits the growth of cells expressing URA3encoding orotidine-5′- monophosphatedecarboxylase, and isolated cells that require uracilfor growth. Sequence analyses and genetic complementation tests using yeast demonstrated that one of the mutant cell lines had a point mutation in URA3, resulting in a splicing error at an unusual exon–intron junction, and consequently, loss of enzyme activity. This mutant could maintain a symbiotic relationship with the model sea anemone Exaiptasia pallidaonly in sea water containing uracil. Results show that the URA3mutant will be a useful tool for screening Symbiodiniumtransformants, both ex and in hospite, as survival in the absence of uracilis possible only upon successful introduction of URA3.