Total RNA from algae was prepared using either the InnuPREP Plant RNA Kit (Analytik Jena; used for N. gaditana, P. parvum, and T. pseudonana), the RNeasy Mini Kit (Qiagen; for E. siliculosus and H. akashiwo), the High Pure RNA Isolation Kit (Roche; for A. carterae), or TRIzol reagent (58) (Thermo Fisher Scientific; for P. minimum and P. tricornutum). Algal material (100 mg) was homogenized in liquid nitrogen by mortar and pestle (N. gaditana, P. parvum, T. pseudonana, and E. siliculosus) and then transferred to the kit-specific buffer or suspended directly in buffer and homogenized either in a Precellys Glass/Ceramic kit SK38/2-ml tube (Bertin Instruments) using a Mini-Beadbeater-1 (BioSpec) for 3 × 20 s at 5000 rpm (H. akashiwo and A. carterae), or by brief vortexing in a test tube containing micro glass beads of 0.2- to 0.25-μm diameter (P. minimum and P. tricornutum). Complementary DNA (cDNA) was synthesized from total RNA using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche Life Science, Mannheim, Germany) and an anchored-oligo(dT)18 primer. Total RNA isolation of N. oceanica and cDNA synthesis was carried out as previously described (26). Cloning of genes and gene fragments for expression constructs was performed by polymerase chain reaction (PCR) using Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Carlsbad, USA), with templates and gene-specific primers as listed in data file S3. Accuracy of cloned sequences was confirmed by comparison with reference sequences and with directly sequenced PCR products to ensure that deviations from reference sequences were caused by strain-specific single-nucleotide polymorphisms (see data file S4) and not by PCR errors.

Recombinant VDL and VDE proteins for in vitro assays were generated by heterologous expression in E. coli Rosetta (Novagen) using pET-44a (Novagen). Gene fragments encoding the mature proteins without targeting sequences were placed in-frame between the Nde I and Xho I sites of pET-44a by either conventional restriction and ligation or using In-Fusion Cloning HD (Takara) (see data file S3 for further details on PCR templates and primers, cloning strategies, and protein fragments encoded in the final expression constructs). Expression, preparation, and renaturation of the recombinant proteins were performed as previously described (27). Expression efficiency in E. coli and purity of the recombinant proteins in the inclusion body (IB) preparations were checked by SDS–polyacrylamide gel electrophoresis (59). The concentrations of recombinant protein in the in vitro assays were estimated from determination of the protein concentration in the respective IB preparation and calculation of the IB dilution in the renaturation buffer. Ten microliters of the tris-buffered IB suspension was diluted with 990 μl of A280-buffer (10 mM tris, pH 6.8; 2% SDS, w/v) and boiled for 2 min. Protein concentration was determined from the absorbance at 280 nm against A280 buffer, using extinction coefficient and molecular weight of the respective VDL protein sequence as calculated by the ExPASy ProtParam tool (

Transient expression of algal VDL genes in tobacco leaves was achieved by Agrobacterium-mediated transformation of leaves of N. benthamiana using pPZP200BAR-based (60) expression constructs. For transient expression of VDL from N. oceanica, pCAMBIA1300_35Su (61) was used instead. Gene fragments encoding the mature proteins were placed in-frame behind a gene fragment encoding the N-terminal targeting sequence of the VDE from N. tabacum (tpNtVDE = amino acid positions 1 to 134) for targeting of the expression products to the thylakoid lumen of tobacco plastids. In the case of the VDL1 from P. tricornutum, the gene fragment was also placed behind a gene fragment encoding the targeting sequence of the zeaxanthin epoxidase from A. thaliana (tpAtZEP) for targeting of the protein to the plastid stroma as previously described (27). Further details on PCR templates and primers, cloning strategies, and protein fragments encoded in the final expression constructs are given in data file S3. For transient expression in N. benthamiana, leaves of the upper part of 6- to 8-week-old plants were infiltrated with Agrobacterium transformants harboring the respective expression constructs as previously described (27). Four days after infiltration, infiltrated and untreated control leaves were harvested, one to three leaf discs of 7-mm diameter were taken per leaf, immediately frozen in liquid nitrogen, lyophilized, and stored at −20°C until further analysis.

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