While photoautotrophic organisms utilize inorganic nitrogen as the nitrogen source, heterotrophic organisms utilize organic nitrogen and thus do not generally have an inorganic nitrogen assimilation pathway. Here we focused on the nitrogen metabolism ofRapaza viridis, a unicellular eukaryote exhibiting kleptoplasty. Although belonging to the lineage of essentially heterotrophic flagellates,R. viridisexploits the photosynthetic products of the kleptoplasts and was therefore suspected to

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... ly utilize inorganic nitrogen. From the transcriptome data ofR. viridis, we identified the gene RvNaRL, which had sequence similarity to nitrate reductases found in plants. Phylogenetic analysis revealed that RvNaRLwas acquired by a horizontal gene transfer event. To verify its function of the protein productRvNaRL, we established a RNAi mediated knockdown and a CRISPR-Cas9-mediated knockout experiments for the first time inR. viridisand applied them to this gene. The RvNaRLknockdown and knockout cells exhibited significant growth only when ammonium was supplied but, in contrast to the wild-type cells, no substantial growth when nitrate was supplied. Such arrested growth in absence of ammonium was attributed to impaired amino acid synthesis due to the deficiency of nitrogen supply from the nitrate assimilation pathway; this in turn resulted in the accumulation of excess photosynthetic products in the form of cytosolic polysaccharide grains as observed. These results indicate thatRvNaRL is certainly involved in nitrate assimilation byR. viridis. Thus, we infer thatR. viridisachieved its advanced kleptoplastic strategy owing to a posteriori acquisition of the nitrate assimilation pathway the horizontal gene transfer.