Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica

Abstract

The sea slug Elysia chlorotica acquires plastids by ingestion of its algal food source Vaucheria litorea. Organelles are sequestered in the mollusc's digestive epithelium, where they photosynthesize for months in the absence of algal nucleocytoplasm. This is perplexing because plastid metabolism depends on the nuclear genome for >90% of the needed proteins. Two possible explanations for the persistence of photosynthesis in the sea slug are (i) the ability of V. litorea plastids to retain genetic autonomy and/or (ii) more likely, the mollusc provides the essential plastid proteins. Under the latter scenario, genes supporting photosynthesis have been acquired by the animal via horizontal gene transfer and the encoded proteins are retargeted to the plastid. We sequenced the plastid genome and confirmed that it lacks the full complement of genes required for photosynthesis. In support of the second scenario, we demonstrated that a nuclear gene of oxygenic photosynthesis, psbO, is expressed in the sea slug and has integrated into the germline. The source of psbO in the sea slug is V. litorea because this sequence is identical from the predator and prey genomes. Evidence that the transferred gene has integrated into sea slug nuclear DNA comes from the finding of a highly diverged psbO 3' flanking sequence in the algal and mollusc nuclear homologues and gene absence from the mitochondrial genome of E. chlorotica. We demonstrate that foreign organelle retention generates metabolic novelty ("green animals") and is explained by anastomosis of distinct branches of the tree of life driven by predation and horizontal gene transfer.

Fig. 1.

Laboratory culturing of E. chlorotica. (A) Free-swimming E. chlorotica veliger larvae. (Scale bar, 100 μm.) Under laboratory conditions, the veliger larvae develop and emerge from plastid-free sea slug–fertilized eggs within approximately 7 days. The green coloring in the digestive gut is attributable to planktonic feeding and not to the acquisition of plastids at this stage. Metamorphosis of the larvae to juvenile sea slugs requires the presence of filaments of V. litorea. (B) Metamorphosed juvenile sea slug feeding for the first time on V. litorea. (Scale bar, 500 μm.) The grayish-brown juveniles lose their shell, and there is an obligate requirement for plastid acquisition for continued development. This is fulfilled by the voracious feeding of the juveniles on filaments of V. litorea. (Also see Movie S1). (C) Young adult sea slug 5 days after first feeding. (Scale bar, 500 μm.) By a mechanism not yet understood, the sea slugs selectively retain only the plastids in cells that line their highly branched digestive tract. (D) Adult sea slug. (Scale bar, 500 μm.) As the sea slugs further develop and grow in size, the expanding digestive diverticuli spread the plastids throughout the entire body of the mollusc, yielding a uniform green coloring. (Also see Movie S2.) From these controlled rearing studies, we were able to conclude that the only source of plastids in our experimental sea slugs was V. litorea.

Fig. 2.

Map of the ptDNA of V. litorea. Genes on the outside are transcribed in the clockwise direction, whereas genes on the inside are transcribed in the counterclockwise direction. Genes are color coded according to their function as shown. tRNAs are listed by the one-letter amino acid code followed by the anticodon. The only gene with an intron (L-uaa) is indicated by an asterisk.

Fig. 3.

Expression of psbO in V. litorea and E. chlorotica. (A) RT-PCR using heterologous primers to psbO amplified a 452-bp fragment from both algal and adult sea slug cDNA. Water served as the negative control. Standards were a 1-kb Plus DNA ladder (Invitrogen). Vli, Vaucheria litorea, Ecl, Elysia chlorotica. (B) Northern blot analysis employing the cloned V. litorea 452 bp psbO product as probe hybridized with a 1.2-kb transcript for V. litorea and E. chlorotica as well as a 1.6-kb transcript in the sea slug. RNA Millennium Size Markers (Ambion) were run to estimate transcript size. (C) Homologous primers were designed from the RACE-amplified sequence of the V. litorea psbO fragment in A. By PCR, these primers amplified a 963-bp product from genomic DNA of V. litorea and E. chlorotica eggs and adult tissue as well as E. chlorotica adult cDNA by RT-PCR. (D) Translation of the psbO sequences obtained from the 963-bp products in B, for both V. litorea and E. chlorotica, yielded an identical amino acid sequence with a putative tripartite targeting signal for MSP. The signal sequence is in red, the transit peptide is in blue, and the thylakoid targeting domain is in green. Note the highly conserved phenylalanine residue at the cleavage site of the signal sequence.

Fig. 4.

Map of the mtDNA of the sacoglossan mollusc E. chlorotica. Genes transcribed clockwise are shown on the outside of the circle, whereas those transcribed counterclockwise are shown to the inside of the circle. Names of tRNA genes are indicated by the three-letter amino acid code with the two leucine and two serine tRNAs differentiated by + and − signs, recognizing codons UAG and UAA for leucine and AGN and UCN for serine, respectively.