# Plant Synthetic Promoters
## 5 Orthogonal Expression Systems
Alternatively, an STF containing only NLS and DBD could recognize the DNA sequence downstream of the core promoter to create a steric hindrance, protecting against RNA polymerase II preinitiation complex formation. Figure 3: Synthetic _trans_-factor (STF) positively regulates the orthogonal expression system. The STAPs developed by [PERSON] et al. for application in dioct plants served as the starting point to assemble related systems (STAP1 and STAP2) in a monocot plant, _O. sativa_[197,201]. To ensure the spatial regulation of the _GUS_ reporter gene, the TALE-dependent expression was driven by the bundle sheath cell-specific _Zoysia japonica PHOS-PHOENOLPYRIVATE CARBOXYKINASE_ (_ZjPCK_) promoter (_ZjPCKpro_) and two _PHOS-PHOENOLPYRIVATE CARBOXYLASE_ promoters (_PEPCpro_), maintaining direct and strong mesophyll-specific gene expression in rice. The tissue gene expression mediated by the STAP1 and STAP2 systems was heritable and scalable enough to efficiently regulate up to four different genes in one genetic construct. Moreover, _dTALE1_ expression was characterized by relatively low off-target activity, as indicated by a comparison of RNAseq data from wild-type and T2 plants. Only 139 upregulated and eight downregulated genes could be attributable to _dTALE1_ expression [197]. TALES could be exercised not only for the direct regulation of STAPS but also indirectly for the control of orthogonal TF expression [186]. DNA sequences recognized and bound by TALEs, known as effector binding elements (EBEs), could be predicted in silico and exercised to build repeatedly repeated fragments, controlling the expression of reporter genes with significant biological functions. One such example is the _avrGf2_ gene from the bacterial pathogen _Xanthomonas citri_. The gene can induce plant cell death, protecting against the development of the pathogen [239]. Artificial trans-factors (ATF) are typically created by fusing the deactivated form of the Cas9 protein (dCas9) to the transcriptional activator domain VP64. The ATF dCas9:VP64 upregulates the expression of reporter genes via specific guide RNAs (gRNAs) that target the promoter region upstream from them (Figure 6). The expression of _ATF_ is usually controlled by CAMV35S, while the gRNA transcription rate is regulated by Pol III (U6) [196]. The presented system is known as the first generation of dCas9-dependent ATFs and was further developed to achieve the effective modulation of target gene expression. The novel form, known as the second generation of dCas9-dependent ATFs, contains the modified gRNA, which contains two aptamer loops to allow the attachment of the viral MS2 protein (Figure 7). The MS2 protein is bound by activators (VPR), transcriptional repressors (SRDX), or epigenetic regulators such as the catalytic core domain of _Homo sapiens_ p300, H3K27 histone acetyltransferase, H3K9 histone methyltransferase, the SET domain of KRYPTONITE (KYP), or H3K9 methyltransferase from _Arabidopsis_[202,203]. In addition to constitutive CAMV35S, the expression of dCas9:EDLL and MS2:VPR could also be regulated by a copper-inducible promoter containing a cis-active motif named copper-binding site (CBS), recognized by the copper-responsive factor CUP2, fused to the yeast Gal4 domain [199]. The presented system enabled a 2600-fold increase in endogenous _N. benthamian DFR_ transcription, and a 245-fold increase in _PAL2_ gene expression, with only trace expression observed in the absence of copper ions [200]. Another approach to introducing inducibility into the orthogonal expression systems was proposed by [PERSON] et al. [240]. The authors developed an inducible, tissue-specific expression system based on the chimeric transcription factor LhG4 fused to the ligand binding domain of the rat corticoid receptor (GR), localized under the control of well-characterized, cell-type-specific promoters. In resting conditions, the GR domain is bound by the cytosolic HSP90, handling the transcription factor outside of the nucleus.