Abstract
Coral reef ecosystems rely on stable symbiotic relationship between the dinoflagellate Symbiodinium spp. 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 Symbiodinium mutants requiring uracil for growth, and hence, useful in transformation screening. We cultured Symbiodinium spp. cells in the presence of 5-fluoroorotic acid (5FOA), which inhibits the growth of cells expressing URA3 encoding orotidine-5′-monophosphate decarboxylase, and isolated cells that require uracil for 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 pallida only in sea water containing uracil. Results show that the URA3 mutant will be a useful tool for screening Symbiodinium transformants, both ex and in hospite, as survival in the absence of uracil is possible only upon successful introduction of URA3.
Highlights
IntroductionThe dinoflagellate Symbiodinium spp. are known to sustain a stable symbiotic relationship with cnidarian animals (e.g. coral, sea anemone, jellyfish) by endosymbiosis in the gastroderm (endoderm) cells of host cnidarian animals[1]
The dinoflagellate Symbiodinium spp. are known to sustain a stable symbiotic relationship with cnidarian animals by endosymbiosis in the gastroderm cells of host cnidarian animals[1]
The area ratio of T01 in the uracil-replete condition increased gradually compared to the wild type in uracil-lacking artificial seawater (ASW) (Supplementary Fig. S4), and the rate of increase in the area ratio seemed to reflect the growth ability of the free-living cells (Figs 3 and 5C). These results clearly demonstrate that stable symbiotic relationship between E. pallida and T01 was dependent on the availability of uracil in the environment, which is critical for the growth of the algal symbiont (Fig. 5C)
Summary
The dinoflagellate Symbiodinium spp. are known to sustain a stable symbiotic relationship with cnidarian animals (e.g. coral, sea anemone, jellyfish) by endosymbiosis in the gastroderm (endoderm) cells of host cnidarian animals[1]. Previous studies suggested that the specificity of the Symbiodinium-cnidarian symbioses was dependent on the size of the algal symbiont[5], and that the symbiont specificity of corals increased (i.e. fewer Symbiodinium types can be associated with corals) as the host coral grew[6]. Two independent studies on gene delivery into the Symbiodinium cells have been published. Further elaboration of methods for gene delivery into Symbiodinium cells is clearly needed: No follow-up studies have been published using the methods developed by ten Lohuis and Miller[9] and, it was shown that transient gene introduction methods used for land plants could be applicable to Symbiodinium, no stable transformant lines have been reported[12]. We show that the cell growth could be switched on and off by replacing the media, and that the growth switching was inducible in and ex hospite
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