A molecular toolkit for the green seaweed Ulva mutabilis

Abstract

The green seaweed Ulva mutabilis is an ecologically important marine primary producer as well as a promising cash crop cultivated for multiple uses. Despite its importance, several molecular tools are still needed to better understand seaweed biology. Here, we report the development of a flexible and modular molecular cloning toolkit for the green seaweed U. mutabilis based on a Golden Gate cloning system. The toolkit presently contains 125 entry vectors, 26 destination vectors, and 107 functionally validated expression vectors. We demonstrate the importance of endogenous regulatory sequences for transgene expression and characterize three endogenous promoters suitable to drive transgene expression. We describe two vector architectures to express transgenes via two expression cassettes or a bicistronic approach. The majority of selected transformants (50%-80%) consistently give clear visual transgene expression. Furthermore, we made different marker lines for intracellular compartments after evaluating 13 transit peptides and 11 tagged endogenous Ulva genes. Our molecular toolkit enables the study of Ulva gain-of-function lines and paves the way for gene characterization and large-scale functional genomics studies in a green seaweed.

Figure 1

Overview of cloning strategy and transformation of Ulva mutabilis. A, Summary of GreenGate cloning strategy (Lampropoulos et al., 2013). Up to six entry modules (depicted with A–B, B–C, C–D, D–E, E–F, and F–G) can be assembled in one destination vector in one reaction generating an expression vector. All entry modules have an ampicillin/carbenicillin (bla) resistance cassette for bacterial selection, the destination vectors contain a kanamycin (aph) resistance cassette. Although any sequence can be cloned in all entry modules, the most common sequences in every module are depicted (e.g. promoter sequence in A–B module). BleR is a module containing the ble gene, encoding Bleomycin resistance protein, the promoter/5′ UTR, first intron and terminator of Rubisco SSU (see Oertel et al., 2015). See Supplemental Figure S6 for a cloning scheme of custom destination vectors. B, Overview of Ulva transformation and selection. Gametogenesis of mature Ulva is induced by fragmentation and changing medium to wash out sporulation inhibitors. After 72 h, the cells have differentiated into gametes that are released from the tissue after one more medium changes. Gametes are isolated using their phototactic properties and subsequently DNA can be transfected using PEG-mediated transformation (Oertel et al., 2015). Transformed gametes develop parthenogenically into new thalli and can be selected using phleomycin. Representative images of transformants at different developmental stages.

Figure 2

Functional validation of Ulva promoter and intron sequences. A, Twenty different regulatory promoter regions were cloned upstream of the resistance cassette (BleR-GFP; Oertel et al., 2015). To evaluate the relative strength of each promoter, the number of resistant individuals was counted relative to the number of resistant individuals for pRbcS (Oertel et al., 2015; Supplemental Figure S1). Visualization of BleR-GFP expression is included for constructs containing pRbcS, pUM140_0016 and pUM056_0044 (see Supplemental Figure S2 for other lines; scale bar: 50 µM). Each dot represents relative transformation efficiency for one independent experiment, the mean is indicated with a bar. n = 3–5. B, Promoter deletion experiment for three promoters (pRbcS, pUM140_0016 and pUM056_0044). The size of the promoter region is indicated for each independent line. Resistant individuals were counted and relative transformation efficiency was calculated as in (A). A control without promoter sequence was included. Each dot represents relative transformation efficiency for one independent experiment, the mean is indicated with a bar, error bars represent sd. n = 3. C, Effect of intron variants for transgene expression in Ulva. The Rbcs2 intron is originally an integral part of the BleR cassette (IBT; Oertel et al., 2015). The Rbcs2 intron was cloned as a separate module directly upstream of the TLSS (I_BT), incorporated in the BleR gene with a G4SGS protein linker (GSIBT) or incorporated in one of three promoter regions (pRbcS+I; pUM140_0016+I or pUM056_0044+I). The number of resistant individuals was counted and transformation efficiency was calculated relative to pRbcS_IBT. Each dot represents relative transformation efficiency for one independent experiment, the mean is indicated with a bar, error bars represent sd. n = 3.

Figure 3

Stable expression of transgenes in Ulva. A, Confocal imaging visualizing the expression YFP in transgenic Ulva individuals and untransformed control (WT). Ulva gametes were transformed with expression vectors containing two promoters (pRbcS-BleR-tRbcs and pUM140_0016-YFP-tNOS with RbcsI or not [−]), one promoter (pUM140_0016-BleR-2A-YFP-tRbcS and pRbcS-BleR-2A-YFP-tRbcS) or a direct fusion to BleR (pRbcS-BleR-YFP-tRbcS; -2A). The 2A sequence is E2A, F2A, P2A, or T2A. Eight primary transformants per construct are shown. See Supplemental Figure S4 for constructs containing mCherry or mTagBFP2. Scale bar: 5 mm. B, RT-qPCR analysis of relative YFP, mCherry, and mTagBFP2 expression in Ulva transgenic lines transformed with expression vectors containing two promoters (pRbcS-BleR-tRbcs and pUM140_0016-GOI-tNOS) or one promoter (pRbcS-BleR-2A-GOI-tRbcS) or a direct fusion to BleR (pRbcS-BleR-GOI-tRbcS; -2A) and untransformed control (WT). For each sample, at least 10 independent primary transformants were pooled. Relative expression values are calculated by normalization against two reference genes (EFLa and UBQ10) and are the mean of three technical replicates ± se. C, Immunoblot analysis on Ulva transgenic lines transformed with expression vectors containing two promoters (pRbcS-BleR-tRbcs and pUM140_0016-mCherry-tNOS) or one promoter (pRbcS-BleR-2A-mCherry-tRbcS), a direct fusion of BleR-mCherry (-2A), vector control (Neg.) and untransformed control (WT). For each sample, at least 10 independent primary transformants were pooled. The expected size of the fusion protein (BleR-mCherry) and the cleaved mCherry is indicated with blue and yellow arrows, respectively. Loading control for each line is represented with ponceau staining (P) and stain-free gel imaging (G). N.D., not determined. See Supplemental Figure S4 for constructs containing YFP or mTagBFP2.

Figure 4

Tagged Ulva transgenic lines. A, Visualization of Ulva transgenic lines expressing YFP targeted to different intracellular locations using TPs: mitochondria (AtpA and HSP70c), microbodies (PMS, CrMS, and RMS) and nucleus (SV40; 2xSV40 and N7). One transgenic line (psaD) shows free YFP signal. For each line a representative individual is shown, green indicates the YFP signal and red represents chlorophyll autofluorescence. Scale bar: 50 µM. See also Supplemental Figure S7. B, Visualization of Ulva transgenic lines expressing endogenous Ulva genes tagged with YFP targeted to different intracellular locations: chloroplast (UM120_0017.1), nucleus (UM003_0201.1 and UM001_0379.1), microbodies (UM079_0010.1), and vacuole (UM110_0052.1). One transgenic line (UM025_0020.1) shows YFP signal at the outer chloroplast membrane and cytosol. For each line a representative individual is shown, green indicates the YFP signal and red represents chlorophyll autofluorescence. Scale bar: 50 µM. See also Supplemental Figure S8.