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. 2008 Jun;53(6):381-8.
doi: 10.1007/s00294-008-0189-7. Epub 2008 Apr 12.

A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii

Ning Shao  1 , Ralph Bock
Affiliations

A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii

Ning Shao et al. Curr Genet. 2008 Jun.

Abstract

The unicellular green alga Chlamydomonas reinhardtii has emerged as a superb model species in plant biology. Although the alga is easily transformable, the low efficiency of transgene expression from the Chlamydomonas nuclear genome has severely hampered functional genomics research. For example, poor transgene expression is held responsible for the lack of sensitive reporter genes to monitor gene expression in vivo, analyze subcellular protein localization or study protein-protein interactions. Here, we have tested the luciferase from the marine copepod Gaussia princeps (G-Luc) for its suitability as a sensitive bioluminescent reporter of gene expression in Chlamydomonas. We show that a Gaussia luciferase gene variant, engineered to match the codon usage in the Chlamydomonas nuclear genome, serves as a highly sensitive reporter of gene expression from both constitutive and inducible algal promoters. Its bioluminescence signal intensity greatly surpasses previously developed reporters for Chlamydomonas nuclear gene expression and reaches values high enough for utilizing the reporter as a tool to monitor responses to environmental stresses in vivo and to conduct high-throughput screenings for signaling mutants in Chlamydomonas.

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Figures

Fig. 1
Fig. 1
Codon usage optimization of the G. princes luciferase and construction of expression cassettes for the transformation of Chlamydomonas reinhardtii. a Adaptation of the G-Luc gene to the codon usage in the nuclear genome of Chlamydomonas reinhardtii. The relative frequencies of the individual codons of the native G-Luc gene (GenBank accession number AY015993) in the Chlamydomonas nuclear genome are indicated by grey bars with the most frequently used triplet in Chlamydomonas set to 100%. The synthetic G-Luc gene (diamonds) was optimized by changing all codons to the most frequently used ones in Chlamydomonas reinhardtii. bG-Luc and R-Luc expression cassettes. To comparatively assess constitutive expression levels, the G-Luc (dark grey box) and R-Luc (hatched box) coding regions were inserted into the PsaD expression cassette (PsaD promoter and 5′ UTR shown as open box; Fischer and Rochaix 2001). Both the R-Luc (Fuhrmann et al. 2004) and the G-Luc genes are optimized with regard to the codon usage in the Chlamydomonas nuclear genome. For inducible expression, the Hsp70A promoter (from position −23 to −285 with respect to the translation initiation codon of Hsp70A) was fused to a gene fragment containing the 5′ UTR and the first three exons of the Hsp70B gene (Shao et al. 2007). The positions of the three heat shock elements (HSE, black boxes) within the promoter region are indicated. Light grey bars represent exons, introns are depicted as broken lines. The third exon of Hsp70B was fused to the coding region of R-Luc and G-Luc, respectively. The 3′ UTR of the reporter gene cassettes is derived from RBCS2, a nuclear gene for the small subunit of Rubisco (Shao et al. 2007)
Fig. 2
Fig. 2
Comparison of G-Luc and R-Luc activity in transgenic Chlamydomonas reinhardtii strains. a Expression of G-Luc and R-Luc under the control of the constitutive PsaD promoter. For each construct, bioluminescence assays with six independent transformants were performed using a luminescence counter. The bars represent the mean of three independent experiments. The standard deviation is indicated. b Heat induction of the G-Luc and R-Luc reporters under the control of the Hsp70A promoter. Expression of luciferase in transformants harboring the PHsp70A-Luc constructs was induced by a temperature shift from 23 to 40°C for 1 h. After a 1 h recovery phase at room temperature, luciferase activity was assayed and the induction factors were calculated by comparison with untreated samples. Four arbitrarily chosen transformants were assayed for each luciferase construct. The bars represent the mean of three independent experiments, the standard deviation is indicated. The basal expression levels of the transformants under non-inducing conditions were 105, 5, 133 and 2 LCPS μg−1 chlorophyll for the four R-Luc clones and 4, 5, 37 and 18 LCPS μg−1 chlorophyll for the four G-Luc clones
Fig. 3
Fig. 3
In vivo assay of Gaussia luciferase and Renilla luciferase activities in Chlamydomonas reinhardtii by visualizing luminescence with a photon-counting camera. Left panels show photographs of the algal colonies prior to luminescence imaging, right panels show the luminescence images. The clones with the highest luciferase expression levels in Fig. 2 were used. a For detection of luciferase activity in living algae, wild-type cells (WT) and transformants harboring the PpsaD R-Luc or PpsaD G-Luc constructs (strains R1 and G9 from Fig. 2a) were spotted in three replicas onto agar-solidified medium and grown under normal light conditions (55 μE m−2 s−1) for 4 days. The ratio of luciferase signal intensities of PpsaD G-Luc:PpsaD R-Luc was approximately 40 (40.4 ± 2.5); the signal intensity of PpsaD R-Luc was only slightly above background (PpsaD R-Luc: WT = 3.4 ± 1.5). b Luminescence of the PHsp70A-Luc transformants induced by heat shock. The cultures were shifted from 23 to 40°C for 1 h. After recovery at room temperature for 1 h, the luminescence image was taken with a photon-counting camera. Luminescence intensities are color-coded with the maximum set to 1.3 × 105
Fig. 4
Fig. 4
Monitoring the heat stress response in Chlamydomonas strains expressing luciferase constructs driven by the heat-inducible Hsp70A promoter. a Assessment of the thermostability of the Gaussia and Renilla luciferases. PpsaD-Luc transformants were subjected to 30 min of high temperature treatment as indicated followed by a 30 min recovery phase at room temperature. Luciferase activities were measured with a luminescence counter. Note the high temperature sensitivity of the Renilla enzyme with all activity being lost at temperatures above 46°C. In contrast, the Gaussia enzyme appears to be much more stable, facilitating its use as a sensitive reporter of heat stress-induced gene expression. b Visualization of the heat stress response with G-Luc. For luciferase imaging, wild-type cells (WT) and transformants harboring PHsp70A R-Luc, PHsp70A G-Luc or P∆HSEG-Luc (G-Luc fusion to a Hsp70A promoter lacking the HSE region; Shao et al. 2007) were spotted in three replicas on agar plates, grown under normal light conditions (55 μE m−2 s−1) for 4 days, photographed (left picture), and then shifted from 23 to 47°C for 15 min. After 3.5 h at room temperature (RT), the luminescence of the transformants upon substrate addition was recorded with a photon-counting camera (right picture). c Analysis of the kinetics of luciferase induction in a PHsp70A G-Luc transformant exposed to heat stress. Colonies of the PHsp70A G-Luc strain growing on a TAP agar plate were exposed to 40 or 47°C for 15 min. After 1, 3 or 5 h recovery at RT, the luminescence of the colonies was visualized using a photon-counting camera. d Comparison of the induction of G-Luc expression by heat stress with induction of the endogenous Hsp70A gene at the mRNA level. Expression from the Hsp70A promoter was induced by a 15-min incubation at 40 or 47°C, and then followed over time by incubation at room temperature (RT) for the time spans indicated. The induction kinetics of luciferase expression from the Hsp70A promoter parallels that of the endogenous Hsp70A gene
Fig. 5
Fig. 5
In vivo assay of Gaussia luciferase and Renilla luciferase activities in primary Chlamydomonas transformants by visualizing luminescence with a photon-counting camera. Co-transformation experiments were conducted using identical amounts of linearized DNA (500 ng Luc-containing plasmid + 100 ng ARG7-containing plasmid) and identical amounts of algal cells. Upper panels show photographs of the algal colonies prior to luminescence imaging, lower panels show the luminescence images. Luminescence intensities are color-coded with the maximum set to 1.3 × 105. a Luminescence of PpsaD-Luc transformants. The ratio of total-plate luminescence of PpsaD G-Luc:PpsaD R-Luc was approximately 36. b Luminescence of PHsp70A-Luc transformants induced by heat shock. The cultures were shifted from 23 to 40°C for 1 h. After recovery at room temperature for 1 h, the luminescence image was taken. The ratio of total-plate luminescence of PHsp70A G-Luc:PHsp70A R-Luc was approximately 16. Note that a large fraction of the non-luminescing colonies is not co-transformed (i.e., harbors the selectable marker gene but not the luciferase reporter)
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