Isolation of uracil auxotroph mutants of coral symbiont alga for symbiosis studies
2018
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.
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