Abstract
The ability to conduct reverse genetic studies in symbiotic systems is enabled by transgene expression and transformation of at least one partner. The symbiotic relationship between the yellow spotted salamander, Ambystoma maculatum, and the green alga, Oophila amblystomatis, is a unique model of vertebrate-algae symbiosis. Despite over 130 years of scientific study, there are still many open questions in this symbiosis. Transgene expression in one partner will accelerate research into the symbiotic relationship. In this paper we describe a tool and method for expression of foreign DNA in, and presumed transformation of, the alga O. amblystomatis. We successfully introduced heritable antibiotic resistance to algal cultures, and observed expression of a green fluorescent reporter protein in all transfected and presumably transformed algal populations. The outcomes of this work enable genetic manipulation of the symbiotic alga O. amblystomatis, allowing direct testing of hypotheses derived from gene expression or genomic studies that will usher in a deeper understanding of the A. maculatum-O. amblystomatis symbiotic system.
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Acknowledgements
We thank Dr. Cory Bishop for sharing his unpublished draft genomes of the algae O. amblystomatis, Chlamydomonas nasuta, Chlamydomonas moewusii, and Chlamydomonas pseudogloegama. Comparative analyses of those genomes enabled promoter region selection.
Funding
This work was funded by the Gordon and Betty Moore Foundation grant GBMF5604 and by NSF OIA-1826734.
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13199_2022_861_MOESM1_ESM.pdf
Supplementary file1 (PDF 131 KB) Figure S1: Promoter selection using alignments of genomic DNA and coding sequences of target genes from related species. Cnas/C_nasuta = Chlamydomonas nasuta; Cpsued/C_pseudo = Chlamydomonas pseudogloegama; Cmoe = Chlamydomonas moewusii; Oamb/O_ambyst = Oophila amblystomatis
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Supplementary file3 (PNG 771 KB) Figure S3: Oophila amblystomatis cells are sensitive to paromomycin. Oophila amblystomatis cultures in AF6 media 7 days after treatment with 20 and 40 µg/mL paromomycin
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Supplementary file4 (PNG 1737 KB) Figure S4: Fluorescence micrographs of a brightly fluorescent cell (A) from population pRPS4-i-GFP-APHVIII-A and from representative cells (B) from transformations with each plasmid. Green fluorescence is not easily visible by the naked eye, but can be bright occasionally (A), and on average cells from transformed lines have brighter green fluorescence than untransformed, wild-type cells, as shown by flow cytometry in Figure 5 of the main text
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Supplementary file5 (TXT 18 KB) Supplementary data: Plasmid sequences (.txt files) created for this study. Sequenced regions are in upper case
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Genot, B., Burns, J.A. Transformation of the symbiotic alga Oophila amblystomatis: a new tool for animal-algae symbiosis studies. Symbiosis 87, 143–151 (2022). https://doi.org/10.1007/s13199-022-00861-0
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DOI: https://doi.org/10.1007/s13199-022-00861-0