A library of synthetic transcription activator-like effector-activated promoters for coordinated orthogonal gene expression in plants

Abstract

A library of synthetic promoters containing the binding site of a single designer transcription activator-like effector (dTALE) was constructed. The promoters contain a constant sequence, consisting of an 18-base long dTALE-binding site and a TATA box, flanked by degenerate sequences of 49 bases downstream and 19 bases upstream. Forty-three of these promoters were sequenced and tested in transient assays in Nicotiana benthamiana using a GUS reporter gene. The strength of expression of the promoters ranged from around 5% to almost 100% of the viral 35S promoter activity. We then demonstrated the utility of these promoters for metabolic engineering by transiently expressing three genes for the production of a plant diterpenoid in N. benthamiana. The simplicity of the promoter structure shows great promise for the development of genetic circuits, with wide potential applications in plant synthetic biology and metabolic engineering.

Keywords: Nicotiana benthamiana; metabolic engineering; orthogonality; plants; synthetic biology; synthetic promoters; technical advance; terpenoid; transcription activator-like effectors; transient assays.

Figure 1

Design and cloning procedure of the synthetic transcription activator-like effector-activated promoters (STAPs).(a) general sequence of the STAPS. The constant designer transcription activator-like effector (dTALE)-binding site (EBE002) is highlighted in blue and the TATA box in green. The atg triplet at the end of the promoter corresponds to the translation start codon. The degenerate sequences upstream and downstream of the constant region are indicated by (n)₁₉ and (n)₄₃ respectively.(b) The STAPs cloning procedure. The library was synthesized using two degenerate oligonucleotides overlapping the constant region and cloned into a level –1 Golden Gate vector. Subsequent cloning into a level 0 vector allowed elimination of aberrant products (e.g. dimers) and false positives. The level 0 vectors can then be used for assembly into transcription units.

Figure 2

Testing the synthetic transcription activator-like effector-activated promoters (STAPs) in a transient assay with a GUS reporter gene.(a) Overview of the constructs used for the transient assay. The designer transcription activator-like effector (dTALE), which binds to the constant EBE002 sequence of the STAPs, is under the control of the constitutive and moderate Act2 promoter. The STAPs control the expression of a GUS transgene with introns (represented by white bars within the blue coding sequence) to prevent expression in Agrobacterium tumefaciens (which is used for the infiltration of Nicotiana benthamiana leaves).(b) Distribution of the GUS activity values for the STAPs promoters with or without the dTALE, expressed in percentage of 35S:GUS expression.

Figure 3

Testing selected synthetic transcription activator-like effector-activated promoters (STAPs) with GFP.Eight different STAPs displaying different levels of expression with the GUS reporter gene were tested with GFP. Nicotiana benthamiana leaves were infiltrated with Agrobacterium tumefaciens strains carrying different STAP:GFP constructs with (left side) or without (right side) the designer transcription activator-like effector (dTALE). Pictures of the leaves were taken in normal light (first and third columns) or under UV illumination (second and fourth columns) to visualize GFP.

Figure 4

Using selected synthetic transcription activator-like effector-activated promoters (STAPs) for metabolic engineering of a plant diterpene.(a) overview of the pathway for cembratrienols (CBTols) including the methylerythritol-phosphate (MEP) pathway, highlighting the genes that were over-expressed in Nicotiana benthamiana. Pyr, pyruvate; GA3P, glyceraldehyde-3-phosphate; DXP, 1-deoxyxylulose 5-phosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GGPP, geranylgeranyl diphosphate; SlDXS2, tomato 1-deoxyxylulose 5-phosphate synthase; NtGGPPS2, tobacco GGPP synthase; CBTS2a, tobacco cembratrienol synthase.(b) Overview of the constructs used for the overexpression of SlDXS2, NtGGPPS2 and CBTS2a using STAPs. The numbers of the STAPs correspond to the numbers in Figures2 and 3.(c) Comparison of transcription activator-like effector (TALE)-driven expression and p35S-driven expression in N. benthamiana leaves agro-infiltrated with p19 and CBTS2a,DXS2 and GGPPS2 in different combinations. Two leaves in three individual plants were infiltrated for each construct combination (n = 6). Mean amounts of CBTol are expressed as equivalents of the internal standard (IS, sclareol) ± SE. Groups indicated by different letters (a1/a2, and b1/b2) differ significantly from each other regarding their CBTol values (P < 0.05; Student’s t-test and two-way anova). No CBTol was detected in leaves expressing the empty T-DNA vector as a control.

Figure 5

Transcription activator-like effector (TALE)- and 35S-driven expression of the CBTS2a,DXS2 and GGPPS2 genes in agro-infiltrated Nicotiana benthamiana leaves.Transcript levels were quantified by RT-PCR in three selected biological replicates from samples used in Figure4 (rep1-3) from agro-infiltrated leaves co-expressing p19,CBTS2a,DXS2 and GGPPS2 driven by synthetic transcription activator-like effector-activated promoters (TALE) or the 35S promoter (35S). Mean expression values of three technical replicates ± SD and the corresponding CBTol amounts for each sample are given.