Inducible gene expression from the plastid genome by a synthetic riboswitch

Abstract

Riboswitches are natural RNA sensors that regulate gene expression in response to ligand binding. Riboswitches have been identified in prokaryotes and eukaryotes but are unknown in organelles (mitochondria and plastids). Here we have tested the possibility to engineer riboswitches for plastids (chloroplasts), a genetic system that largely relies on translational control of gene expression. To this end, we have used bacterial riboswitches and modified them in silico to meet the requirements of translational regulation in plastids. These engineered switches were then tested for functionality in vivo by stable transformation of the tobacco chloroplast genome. We report the identification of a synthetic riboswitch that functions as an efficient translational regulator of gene expression in plastids in response to its exogenously applied ligand theophylline. This riboswitch provides a novel tool for plastid genome engineering that facilitates the tightly regulated inducible expression of chloroplast genes and transgenes and thus has wide applications in functional genomics and biotechnology.

Fig. 1.

Secondary structural models for synthetic riboswitches tested in this study. Nucleotides changed with respect to the original sequence are shown in red. SD sequences are boxed. Restriction sites (BamHI, NsiI) are underlined. The start codons are part of NsiI restriction sites. All switches are designed as translational “on” switches and are shown in the proposed off mode (i.e., in the absence of the metabolite). In this mode the SD sequence is masked and, therefore, inaccessible to the ribosome. (A) A synthetic glycine riboswitch (s.gly-RS) containing an anti-Shine-Dalgarno (ASD) sequence designed to allow translational regulation by base-pairing with the SD sequence. The switch is derived from the glycine riboswitch (gly-RS) from Bacillus subtilis (8) (Fig. S1A). (B) A synthetic adenine riboswitch (s.ade-RS) designed as translational “on” switch. The switch is derived from the ydhL adenine riboswitch (ade-RS) from Bacillus subtilis (9) (Fig. S1B). (C) The synthetic theophylline responsive riboswitch (s.theo-RS) based on helix slipping (12). The one-nucleotide theophylline-induced slipping in the secondary structure is indicated by the red arrow. The 5^′UTR contains two possible SD sequences (boxed).

Fig. 3.

Generation of plastid transformants with vectors carrying gfp gene constructs under the control of riboswitch elements. (A) Physical map of the targeting region in the plastid genome. (B) Structure of plastid transformation vectors of the pAV series harboring riboswitch-containing GFP expression cassettes. Relevant restriction sites are marked. The transgenes are targeted to the intergenic region between the trnfM and trnG genes (37). The GFP expression cassette consists of the ribosomal RNA operon promoter (Prrn) fused to the riboswitch (RS) element (see Fig. 1 and Fig. S1) and the 3^′UTR from the plastid rps16 gene (Trps16). The expected sizes of DNA fragments in restriction fragment length polymorphism analyses with the enzyme BglII are indicated. The location of the RFLP probe is shown as a black bar. The selectable marker gene aadA is driven by a chimeric ribosomal RNA operon promoter (Prrn) and fused to the 3^′UTR from the plastid psbA gene [(TpsbA (38)]. (C) RFLP analysis of transplastomic tobacco lines. Total cellular DNA was digested with BglII and hybridized to a radiolabeled probe detecting the region of the plastid genome that flanks the transgene insertion site. Fragment sizes for the wild-type and the transplastomic lines are indicated. Absence of a hybridization signal for the wild-type genome indicates homoplasmy of the transplastomic lines. Line Nt-pAV4-10A shows the hybridization signal for the wild-type genome suggesting that this line represents a spontaneous antibiotic-resistant mutant. The transplastomic lines harbor the following riboswitches: Nt-pAV1: gly-RS; Nt-pAV2: ade-RS; Nt-pAV3: tpp-RS; Nt-pAV4: s.gly-RS; Nt-pAV5: s.ade-RS; Nt-pAV6: s.theo-RS.

Fig. 2.

Test of metabolite dependence of GFP expression from riboswitch constructs in E. coli by western blotting. Cells were grown in minimal medium. An E. coli strain harboring a luciferase gene (51) instead of gfp was used as negative control. To allow for quantitative comparisons, a dilution series of purified GFP (10 ng, 5 ng, 2.5 ng) was included in all blots. (A) GFP accumulation in the absence (-) versus the presence (+) of the regulatory metabolite. In the upper panel, 3 μg of total soluble protein (TSP) were loaded for the gly-RS, ade-RS, tpp-RS, and the control strain (C). In the lower panel, 9 μg TSP were loaded for the s.gly-RS, s.ade-RS, s.theo-RS, and the C strain. Cells were grown to midexponential phase. For metabolite-induced switching, the medium was supplemented with 100 mM glycine, 5 mM adenine, 1 mM thiamine pyrophosphate, or 10 mM theophylline. (B) GFP expression controlled by the glycine riboswitch. In the upper panel, the GFP accumulation in dependence on the glycine concentration is shown. The lower panel shows the time course of GFP accumulation following addition of 100 mM glycine. 3 μg TSP were loaded in all lanes. (C) GFP expression controlled by the theophylline riboswitch. The upper panel shows the GFP accumulation in dependence on the theophylline concentration in the medium. The lower panel shows the time course of GFP accumulation following addition of 10 mM theophylline. 9 μg TSP were loaded in all lanes.

Fig. 4.

Metabolite-dependent mRNA and foreign protein accumulation in transplastomic plants. (A) GFP accumulation in transplastomic tobacco plants. Total soluble protein was extracted from pools of 10–20 seedlings (28 d old) grown on medium without inducers (-) or medium supplemented with 10 mM glycine, 5 mM adenine, 0.1 mM thiamine pyrophosphate, or 2.5 mM theophylline (+). 10 μg TSP were loaded for the gly-RS, tpp-RS, and s.ade-RS lines; 1 μg TSP for the ade-RS; and 20 μg TSP for the s.gly-RS and s.theo-RS lines. For comparison, a dilution series of purified GFP was included. Wt: wild-type. (B) GFP expression in transplastomic lines harboring the glycine riboswitch (Nt-pAV1) in dependence on the glycine concentration in the medium. 10 μg TSP were loaded in all lanes. As glycine in high concentrations is toxic to plants (and results in pale phenotypes), concentrations higher than 50 mM were not tested. (C) GFP expression in transplastomic lines harboring the synthetic theophylline riboswitch (Nt-pAV6). The upper panel shows the dependence of GFP accumulation on the theophylline concentration in the medium. Induction is detectable at 1 mM theophylline and peaks at 2.5 mM. Application of 5 mM theophylline is toxic to plants and results in aberrant growth and reduced induction of GFP expression. 20 μg TSP were loaded in all lanes. The lower panel shows the induction of GFP expression 24 h after spraying with a solution of 50 mM theophylline. (D) Analysis of metabolite dependence of mRNA accumulation in transplastomic lines harboring riboswitch constructs. Plants were grown on medium without inducer (-) medium or supplemented with 10 mM glycine, 5 mM adenine, 0.1 mM thiamine pyrophosphate, or 2.5 mM theophylline (+). Samples of 2 μg total RNA were blotted and hybridized to a gfp-specific probe. The size of the gfp transcripts varies slightly due to length differences of the riboswitches (Fig. S2). The major band represents monocistronic gfp mRNA and the upper band originates from read-through transcription, as observed before with transgene inserted into the same genomic location (52).