title: Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens.
abstract: Plant-targeted pCB302 plasmids containing sequences encoding gfp fusions with a microtubule-binding domain; gfp with the fimbrin actin-binding domain 2; and gfp with AtRACK1C from Arabidopsis thaliana, all harbored in Agrobacterium tumefaciens, were used to assay heterologous expression on three different clades of the photosynthetic dinoflagellate, Symbiodinium. Accessibility to the resistant cell wall and through the plasma membrane of these dinoflagellates was gained after brief but vigorous shaking in the presence of glass beads and polyethylene glycol. A resistance gene to the herbicide Basta allowed appropriate selection of the cells expressing the hybrid proteins, which showed a characteristic green fluorescence, although they appeared to lose their photosynthetic pigments and did not further divide. Cell GFP expression frequency measured as green fluorescence emission yielded 839 per every 106 cells for Symbiodinium kawagutii, followed by 640 and 460 per every 106 cells for Symbiodinium microadriaticum and Symbiodinium sp. Mf11, respectively. Genomic PCR with specific primers amplified the AtRACK1C and gfp sequences after selection in all clades, thus revealing their presence in the cells. RT-PCR from RNA of S. kawagutii co-incubated with A. tumefaciens harboring each of the three vectors with their respective constructs, amplified products corresponding to the heterologous gfp sequence while no products were obtained from three distinct negative controls. The reported procedure shows that mild abrasion followed by co-incubation with A. tumefaciens harboring heterologous plasmids with CaMV35S and nos promoters can lead to expression of the encoded proteins into the Symbiodinium cells in culture. Despite the obvious drawbacks of the procedure, this is an important first step towards a stable transformation of Symbiodinium.
authors.affiliation: Unidad Académica de Sistemas Arrecifales, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Prol. Avenida Niños Héroes S/N, Puerto Morelos, Quintana Roo, 77580, México; Posgrado en Ciencias del Mar y Limnología, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Circuito Exterior S/N, Ciudad Universitaria, Delegación Coyoacán, Distrito Federal, 04510, México.
authors.forename: Mario Fernando
authors.initials: MF
authors.lastname: Ortiz-Matamoros
authors.affiliation: Unidad Académica de Sistemas Arrecifales, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Prol. Avenida Niños Héroes S/N, Puerto Morelos, Quintana Roo, 77580, México.
authors.forename: Tania
authors.initials: T
authors.lastname: Islas-Flores
authors.affiliation: Institut für Zellulär und Molekulare Botanik Universität Bonn, Kirschallee 1, Bonn, D-53115, Germany.
authors.forename: Boris
authors.initials: B
authors.lastname: Voigt
authors.affiliation: Institut für Zellulär und Molekulare Botanik Universität Bonn, Kirschallee 1, Bonn, D-53115, Germany.
authors.forename: Diedrik
authors.initials: D
authors.lastname: Menzel
authors.affiliation: Institut für Zellulär und Molekulare Botanik Universität Bonn, Kirschallee 1, Bonn, D-53115, Germany.
authors.forename: František
authors.initials: F
authors.lastname: Baluška
authors.affiliation: Unidad Académica de Sistemas Arrecifales, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Prol. Avenida Niños Héroes S/N, Puerto Morelos, Quintana Roo, 77580, México.
authors.forename: Marco A
authors.initials: MA
authors.lastname: Villanueva
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D056893:Alveolata / Q000235:genetics / Q000378:metabolism*; D018744:DNA, Plant / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence
chemical_list: D018744:DNA, Plant; D049452:Green Fluorescent Proteins