Synthetic Switches and Regulatory Circuits in Plants

Abstract

Synthetic biology is an established but ever-growing interdisciplinary field of research currently revolutionizing biomedicine studies and the biotech industry. The engineering of synthetic circuitry in bacterial, yeast, and animal systems prompted considerable advances for the understanding and manipulation of genetic and metabolic networks; however, their implementation in the plant field lags behind. Here, we review theoretical-experimental approaches to the engineering of synthetic chemical- and light-regulated (optogenetic) switches for the targeted interrogation and control of cellular processes, including existing applications in the plant field. We highlight the strategies for the modular assembly of genetic parts into synthetic circuits of different complexity, ranging from Boolean logic gates and oscillatory devices up to semi- and fully synthetic open- and closed-loop molecular and cellular circuits. Finally, we explore potential applications of these approaches for the engineering of novel functionalities in plants, including understanding complex signaling networks, improving crop productivity, and the production of biopharmaceuticals.

Figure 1.

Illustration of the natural bacterial tetracycline resistance mechanism and synthetic tetracycline-based gene expression systems. A, In the absence of tetracycline (tet), the tet repressor (TetR) is bound to its cognate tet operator (tetO) DNA-binding motif, repressing the expression of the tet resistance-mediating tetA gene. Upon increasing cellular levels of tet, tet binding induces a conformational change of TetR, leading to its dissociation from the operator sequence, and expression of tetA ensues. B, The tet-OFF system designed for use in mammalian cells is based on a synthetic switch comprising the natural TetR fused to the activating domain of VP16 of the herpes simplex virus and a synthetic promoter with a series of repeats of the tetO motif placed upstream of a minimal promoter (e.g. human cytomegalovirus minimal promoter). The system is constitutively active and is turned OFF in the presence of the antibiotic. Implementation of a reversed TetR mutant (rTetR) generates a tet-ON system: tet induces the binding of rTetR to the target sequence, which in turn induces gene expression (tet can be replaced by other antibiotics of the tetracycline family like doxycycline). Replacement of VP16 by a transrepressor such as KRAB inverts the effect of the switch (not depicted here). GOI, Gene of interest. (Adapted from Gossen et al., 1995.)

Figure 2.

Optogenetic switches. Molecular principles of light-induced signaling and optogenetic tools are illustrated. A, Natural red light-inducible signaling mediated by the plant photoreceptor phytochrome B (phyB) and optogenetic tools developed based on it. A1, The red/far-red light-perceiving photoreceptor phyB remains in its inactive Pr conformation in the dark. Upon absorption of a red light photon, the photoreceptor undergoes a conformational change, converting to its active Pfr conformation. The active form can interact with several transcription factors like the bHLH transcription factors of the PHYTOCHROME INTERACTING FACTOR (PIF) family. This interaction triggers light-signaling responses. In contrast, illumination with far-red light reconverts phyB to its inactive Pr form, abolishing the interaction with PIFs (Rockwell and Lagarias, 2006). Several optogenetic approaches make use of the red light/far-red light switchable interaction of phyB and PIFs. A2, Selective activation of intracellular signaling pathways with light. Red light illumination induces the recruitment of the cytoplasmic fusion protein consisting of a PIF, C-terminally fused to the fluorescent protein YFP and the catalytic domain of the SOS protein (SOS^cat), to the membrane-bound, RFP-tagged phyB. When recruited to the membrane, SOS^cat is capable of activating the Ras-signaling cascade and inducing nuclear transport of BFP-Erk and subsequent Erk pathway signaling. (Adapted from Toettcher et al., 2013.) A3, Construction of a phyB-PIF-based, red light-inducible split-transcription factor system. A truncated PIF6 was N-terminally fused to the tetracycline repressor (TetR), and the synthetic protein is bound to the tetracycline operator motif tetO of a synthetic reporter construct (as in Fig. 1). In the absence of light or under far-red light illumination (740 nm), there is no expression from the minimal promoter, P_CMVmin. Upon illumination with red light, phyB, C-terminally fused to the VP16 transactivation domain, interacts with the PIF. The spatial proximity of the transactivator recruits the transcriptional machinery to the minimal promoter. Only in this condition is the expression of the secreted human alkaline phosphatase (SEAP) reporter gene activated. (Adapted from Müller et al., 2013a.) An adaptation of this system was engineered in Arabidopsis and tobacco (Nicotiana tabacum) cells and the moss Physcomitrella patens (Müller et al., 2014; Ochoa-Fernandez et al., 2016). A4, Reversible red light-inducible nuclear transport of phyB fusion proteins. phyB was C-terminally fused to the fluorescent protein mCherry and a nuclear export sequence (NES), while PIF3, containing an intrinsic nuclear localization sequence (NLS), was C-terminally fused to enhanced GFP (EGFP). Upon illumination with red light, the nucleocytoplasmic shuttling PIF induces nuclear transport of phyB, while far-red radiation reversed the translocation of the photoreceptor-mediated by the NES. (Adapted from Beyer et al., 2015.) B, Natural blue light-induced signal transduction mediated by the plant photoreceptor phototropin1 and the light-sensitive bacterial transcription factor EL222. A synthetic approach based on blue light-triggered conformational change of EL222 and the LOV2 domain for the dual-controlled optogenetic down-regulation of proteins in animal cells was used. B1, EL222 is a light-sensitive transcription factor from the gram-negative bacterium Erythrobacter litoralis. It contains a blue light-sensitive LOV domain and a helix-turn-helix (HTH)-DNA-binding domain. In the dark, the HTH domain is docked to the LOV core. Upon illumination with blue light, the interaction of LOV and the HTH domain is disrupted, enabling homodimerization of the protein via the HTH and subsequent binding to the C120-DNA motif (Nash et al., 2011). B2, Schematic illustration of light-induced signal transduction via the blue light plant photoreceptor phototropin1. In the dark, the kinase domain is bound to the LOV domain, inhibiting its phosphorylation activity. Under blue light, the kinase domain is released, inducing protein phosphorylation and downstream signal transduction. (Adapted from Kimura et al., 2006.) B3, The dual optogenetic system for targeted degradation and repression of expression of a protein of interest (POI) consists of a synthetic reporter module comprising the P_SV40 promoter, for constitutive expression of a POI fused to the B-LID (Bonger et al., 2014) module, and the C120₅-DNA-binding motif of the EL222 protein. EL222 is fused to the transrepressor KRAB. In the dark, the degron (peptide RRRG) is docked to the LOV domain of the B-LID, and KRAB-EL222 is not able to bind to the C120 motif on the reporter plasmid. In this case, the POI accumulates. Upon illumination with blue light, the degron is exposed, triggering proteasomal degradation of the POI-B-LID fusion protein. Simultaneously, KRAB-EL222 dimerizes binding to the C120 motif, repressing transcription of the POI-B-LID. (Adapted from Baaske et al., 2018.)

Figure 3.

Molecular principle of a natural and synthetic oscillator. A, Simplified molecular model of the circadian clock in Arabidopsis (natural oscillator). The core oscillator feedback loop consists of TOC1, CCA1, and LHY. In this core oscillator, LHY and CCA1 repress the transcription of TOC1; TOC1 in turn is a positive regulator of CCA1 and LHY. In a second loop, LHY and CCA1 are also positive regulators of three TOC1 paralogs (PRR5, PRR7, and PRR9), which in turn are negative regulators of CCA1 and LHY. In a third loop, CCA1 and LHY positively regulate GI, ELF3, ELF4, and LUX; these in turn regulate CCA1 and LHY. The circadian oscillator of Arabidopsis is illustrated here in a simplified form; for clarity, several other components involved were not included. (Adapted from McClung, 2006.) B, Scheme of a synthetic oscillator engineered by Stricker et al. (2008). This synthetic oscillator comprises positive and negative feedback loops. The araC, lacI, and yemGFP (as a readout) genes are all under the control of the hybrid synthetic promoter P_lac/ara-1, comprising the activation operator site from the araBAD promoter and the repression operator site from the lacZYA promoter (Lutz and Bujard, 1997). In the presence of arabinose, the AraC protein activates the hybrid promoter and, thus, the gene expression of araC, lacI, and yemGFP, which results in two feedback loops: a positive feedback loop mediated by the produced AraC and the resulting activation of the hybrid promoter, and a negative feedback loop due to the production of the LacI protein. In the absence of IPTG, LacI negatively regulates the expression of all three genes under the control of the hybrid promoter. Both engineered feedback loops together constitute the synthetic oscillator. (Adapted from Stricker et al., 2008.)

Figure 4.

Natural and synthetically built AND NOT (NOT IF) Boolean logic gates. An AND NOT gate generates an output when only one specific single input signal is present, but not when there is no input signal, nor a second input, nor both signals. A, Truth table and scheme of the regulatory region of the Lac operon as an AND NOT (NOT IF) gate. This AND NOT gate only generates an output when lactose is the only single input available. If Glc and lactose are available in the cell, the lac operon is OFF because the catabolite activator protein, CAP, is not bound. The same is true when Glc, but no lactose, is available. In this case, the lac repressor is bound. In the case when there is neither Glc nor lactose, the lac operon is OFF because even though CAP is bound, the lac repressor prevents transcriptional initiation. Only when there is lactose, but no Glc, available is the lac operon ON. In the absence of Glc, CAP can bind, and because of the availability of lactose, the lac repressor is not bound. Both actions are necessary for transcriptional initiation of the lac operon. (Adapted from Phillips et al., 2009.) B, An example of an AND NOT (NOT IF) gate in synthetic biology. In this synthetic system, the transactivator SCA (transactivator of the streptogramin-responsive gene regulation system) and the transrepressor PIP-KRAB are constitutively expressed along with a reporter plasmid containing a chimeric SCA- and PIP-specific promoter. The absence of SCB1 [racemic 2-(1V-hydroxy-6-methylheptyl)-3-(hydroxymethyl)butanolide] enables the binding of the transactivator SCA to its corresponding promoter region. The presence of the transrepressor pristinamycin (PI) in turn prevents the binding of PIP-KRAB to its promoter. Thus, this engineered AND NOT gate generates an output only in the presence of pristinamycin and the absence of SCB1. (Adapted from Kramer et al., 2004a.)

Figure 5.

Cell-cell communication in bacteria and synthetic cell-cell communication networks. A, Simplified illustration of the natural homoserine lactone (HSL) quorum-sensing network in V. fischeri. Quorum sensing describes the ability of bacteria to assess the cell density of a population by sensing chemical signals that are produced by surrounding cells (Davis et al., 2015). HSLs, in the case of V. fischeri AHL, bind to the LuxR protein. LuxR then binds to its cognate operator, inducing the transcription of LuxI, which catalyzes the synthesis of AHL. AHL is able to diffuse out of the cell, accumulating in the external milieu and entering surrounding cells, thus activating the circuit in those cells. B, Engineered cell-cell communication networks in mammalian cells. Engineered cell-cell signaling via two synNotch ligand-receptor pairs was used to manipulate cell adhesion, differentiation, and the production of new cell-cell signals (Toda et al., 2018). Upon binding of the ligand to the synNotch receptor, an orthogonal transcription factor is cleaved from the cytoplasmic tail of the receptor, migrates to the nucleus, and then drives gene expression of the output proteins. These genes include fluorescent proteins as cellular markers for differentiation, several cadherins as morphological outputs, and two synNotch ligand-receptor pairs as input signals. In this way, the outputs are propagated to the next generation. (Adapted from Toda et al., 2018.)

Figure 6.

Natural and engineered combinatorial T-cells. A, Natural T-cell with its T-cell receptor, targeting only single antigens. This single-antigen recognition without any further control machinery can lead to off-target tissue damage. B, An engineered synthetic T-cell with new types of receptors specific for detecting given combinations of antigens. Upon binding of antigen A to the synNotch receptor, an orthogonal transcription factor is cleaved from the cytoplasmic tail of the receptor, which in turn activates CAR transcription. If a second antigen, antigen B, is recognized by the newly synthesized CAR receptor, the T-cell is activated. (Adapted from Roybal et al., 2016; Roybal and Lim, 2017.)

Figure 7.

Natural and engineered open-loop regulatory circuits. A, GA₃-induced degradation of DELLA proteins suppresses the repression of PHYTOCHROME INTERACING FACTORs (PIFs). The PIFs subsequently bind to G-box cis regulatory elements in the promotors of response genes, promoting growth responses. In parallel, transcription of PIFs is inhibited by the red light-induced active conformer of phytochrome B, modulating the growth promotion in response to the light conditions. (Adapted from Havko et al., 2016.) B, Schematic overview of a synthetic device for detection of inflammation signals in mammalian systems. Detection of inflammatory signals through the NF-kB-responsive element of the sensor module leads to expression of the transcriptional regulator GAL4 fused to the VP16 transactivation domain (GAL4-VP16). GAL4-VP16 subsequently binds to the UAS motif in the amplifier and effector modules, increasing the abundance of GAL4-VP16 through a self-activating positive feedback loop from the amplifier module. This triggers production of anti-inflammatory proteins via the effector module. Additionally, the system is equipped with a thresholder device, constitutively expressing GAL4 lacking the transactivation domain. GAL4 competes for binding the UAS motifs with the activating GAL4-VP16, thereby restricting the initiation of the expression of the therapeutic output. A fifth module constitutively expresses the doxycycline-inducible reversed tetracycline repressor protein (rTetR) fused to the inhibitory KRAB domain. Exogenous application of doxycycline inhibits the activation of the sensor, amplifier, and effector modules by binding to their upstream tetO motifs, thus deactivating the system. (Adapted from Smole et al., 2017.)

Figure 8.

Natural and engineered closed-loop regulatory circuits. A, Simplified model of the homeostatic regulation of GA₃ metabolism and signaling in Arabidopsis. In the absence of the phytohormone GA, the regulator DELLA proteins accumulate. Through transcriptional control of GA metabolism and catabolism, DELLAs boost the level of GA and subsequently of the GA receptor GID1 proteins. Accumulation of the GID1 proteins and of GA eventually leads to GID1-mediated DELLA degradation. These feedback loops ensure GA homeostasis. (Adapted from Hedden and Thomas, 2012.) B, Schematic overview of a synthetic autoregulatory gene circuit for adjusting insulin resistance in mammalian systems. Upon binding of insulin to the insulin receptor of the designer cell, the intracellular β-subunit of the receptor is autophosphorylated. This leads to further phosphorylation of Tyr residues of the insulin receptor substrate 1 (IRS-1), among other proteins, triggering their interaction with several signaling components. Induced by this interaction, the GTPase Ras and the MAPK are activated, triggering nuclear import of the MAPK. In the nucleus, the MAPK phosphorylates the ELK1 domain of the synthetic regulator protein, consisting of the tet repressor (TetR) and the regulated activation domain of the transcription factor ELK1, expressed under the control of the constitutive human cytomegalovirus immediate early promoter (P_hCMV). The hybrid transcription factor binds to the tet operator motif (tetO) in a synthetic effector device; however, the activation domain remains inactive. It gets activated and initiates the expression of the therapeutic Fc-adiponectin protein only upon MAPK-induced phosphorylation of the ELK1 domain. Subsequent secretion of Fc-adiponectin increases the sensitivity for insulin in other tissues (e.g. muscle cells), leading to a decreased insulin production of pancreatic β-cells. (Adapted from Ye et al., 2017.)