# Plant Synthetic Promoters
## 5 Orthogonal Expression Systems  
Recently developed orthogonal expression systems built from over 500 elements could be employed to introduce complex, multi-gated logic principles such as \"or\", \"nor\" or \"kill\" into genetic circuits. Such a broad range or regulatory effect was obtained after combining five concatenated cis-active elements from yeast (Gal4, MCM1, ata1, Mata2, Gat1, Yap1) with the WUS minimal promoter (Figure 2) [28]. Such yeast cis-active elements were used to construct plant synthetic promoters of low homology to already existing systems. The presented cis-active motifs were recognized by DBD from yeast or bacterial trans-factors. These DBDs are joined to plant or virus TAD and NLS sequences, to build an orthogonal trans-factor, not existing in nature, recognizing only cis-active motifs within the synthetic promoter (Figure 2). Although the expression of a synthetic trans-factor was controlled by native plant promoter, the prepared expression system enabled the decoupling of the transcriptional regulation of transgenes from those endogenous to the genome, which is characteristic of orthogonal systems [28]  
The regulatory properties of synthetic trans-factors were extended by the repression activity achieved by combining the yeast DBD from Gal4 with the SRDX repression domain (Figures 3 and 4) [28]. The expression of synthetic trans-factors was controlled by an endosperm-specific At2S3 or phosphate-responsive AtPht1.1. promoter. Interestingly, the MCM1 and Yap1 containing solely DBDs, without the addition of TAD, preserved some  
Figure 2: Structure of an orthogonal trans-factor. NLS—nuclear localization signal, TAD—trans-activating domain, containing plant- or virus-derived elements, and DBD—DNA-binding domain not existing in plants and originating from the yeast of bacteria. trans-activity properties, suggesting that DBDs may have inherent activation functions that may be interrupted by the introduction of a TAD [28]. Synthetic transcription regulators were also created from bacterial TF-derived DBDs, combined with the TAD domain from VP16 or _Arabidopsis_ ethylene response factor 2 (ERF2) and the SV40 nuclear localization signal (NLS). An orthogonal set of plant synthetic promoters was created by fusing a core plant promoter, encompassing positions \(-66\) to \(+18\) of the CaMV35S, with the six copies of the DNA sequence (operator) bound by these TFs. The repressor activity was mediated also by synthetic regulators composed exclusively of DNA binding domains and NLS sequences, localized \(3^{\prime}\) downstream to the core promoter (Figure 4). The obtained direct and layered logic gates are used to qualitatively modify the expression of the root-specific promoters SOMBRERO (proSMB) and PIN-FORMED 4 (proPIN4) [29]. An interesting approach to developing plant synthetic promoters controlled by orthogonal regulators is the application of transcription activator-like effectors (dTALEs) [198]. In addition, synthetic dTALE activated promoters (STAPs) can be prepared; these are composed of a 19-base-long degenerate sequence, followed by an 18-base-long TALE-site, a TATA-box, a 43-base-long degenerate sequence, and the ATG start codon (Figure 5). This is consistent with the observation that locating the TALE site within approximately \(-55\) or \(-40\) of the gene is sufficient to confer TALE-mediated inducibility. Forty-three of these promoters were tested in transient assays in _N. benthamiana_ using a _GUS_ reporter gene; the results found their expression strength to range from around \(5\%\) to almost \(100\%\) of viral CAMV35S promoter activity. Moreover, these STAPs were neatly utilized to transiently express three genes for the production of a plant diterpenoid in _N. benthamiana_[201]. Figure 4: Synthetic trans-factors that can negatively regulate the orthogonal expression system are produced by combining the yeast DBD from Gal4 with the SRDX repression domain.