A quantitative autonomous bioluminescence reporter system with a wide dynamic range for Plant Synthetic Biology

Abstract

Plant Synthetic Biology aims to enhance the capacities of plants by designing and integrating synthetic gene circuits (SGCs). Quantitative reporting solutions that can produce quick, rich datasets affordably are necessary for SGC optimization. In this paper, we present a new, low-cost, and high-throughput reporter system for the quantitative measurement of gene expression in plants based on autonomous bioluminescence. This method eliminates the need for an exogenous supply of luciferase substrate by exploiting the entire Neonothopanus nambi fungal bioluminescence cyclic pathway to build a self-sustained reporter. The HispS gene, the pathway's limiting step, was set up as the reporter's transcriptional entry point as part of the new system's design, which significantly improved the output's dynamic range and brought it on par with that of the gold standard FLuc/RLuc reporter. Additionally, transient ratiometric measurements in N. benthamiana were made possible by the addition of an enhanced GFP as a normalizer. The performance of new NeoLuc/eGFP system was extensively validated with SGCs previously described, including phytohormone and optogenetic sensors. Furthermore, we employed NeoLuc/eGFP in the optimization of challenging SGCs, including new configurations for an agrochemical (copper) switch, a new blue optogenetic sensor, and a dual copper/red-light switch for tight regulation of metabolic pathways.

Keywords: Nicotiana benthamiana; Autonomous bioluminescence; ratiometric reporter system; transcriptional regulation.

Figure 1

A quantitative bioluminescence reporter system for N. benthamiana. (a) Schematic representation of the N. nambi fungal bioluminescence pathway (FBP). (b) Scheme of T‐DNA construct used for the dual reporter system, including the transcriptional units for the constitutive expression of the FBP genes (Luz, H3H, HispS, CPH), the enhanced GFP gene (eGFP) and the p19 silencing suppressor gene. (c) Schematic representation of the experimental procedure used for luminescence and fluorescence measurements. (d) NeoLuc signal (a.u.) and (e) eGFP fluorescence signal (a.u.) of N. benthamiana leaf discs transiently expressing the reporter module depicted in (b) (P35S) or an empty vector (EV). (f) NeoLuc/eGFP ratios calculated with the luminescence and fluorescence values plotted in (d) and (e). Error bars indicate SD (n = 18).

Figure 2

Optimization of the FBP/eGFP reporter employing five constitutive promoters. (a) Relative transcriptional activities (RTA) of four different constitutive promoters (PNtA11, PNtA1, PMTB and P35S) transiently expressed in N. benthamiana, calculated as FLuc/RLuc ratios for each promoter and normalized with the FLuc/RLuc ratios conferred by the constitutive PNOS promoter. (b) NeoLuc/eGFP ratios and (c) FBP[Luz]‐RTA values of N. benthamiana leaf discs transiently expressing the FBP reporter with the Luz gene driven by four different constitutive promoters. (d) NeoLuc/eGFP ratios and (e) FBP[HispS]‐RTA values of N. benthamiana leaf discs transiently expressing the FBP reporter with HispS gene driven by four constitutive promoters. (f) Correlation of normalized FLuc/RLuc ratios (FLuc‐RTA) depicted in (a) with FBP[Luz]‐RTA and FBP[HispS]‐RTA values depicted in (c) and (e), respectively. An empty vector (EV) was used as a negative control in all experiments. Error bars indicate SD (a, b and d) and SE (c and e): n = 9. Statistical analyses were performed using One‐way ANOVA (Tukey's multiple comparisons test, P‐value ≤0.05). Variables within the same statistical groups are marked with the same letters.

Figure 3

Phytohormone sensors with the FBP[HispS]/eGFP reporter. (a) Schematic representation of HispS gene activation driven by the ABA inducible promoter PMAPKKK18 (ABA sensor). (b) NeoLuc/eGFP ratios and (c) FBP[HispS]‐RTA values of the ABA sensor in N. benthamiana leaf discs treated with different ABA concentrations. (d) Schematic representation of an IAA mediated AcrIIA4 protein depletion switch for the regulation of the dCasEV2.1‐mediated reporter activation. (e) NeoLuc/eGFP ratios and (f) FBP[HispS]‐RTA values of the IAA‐mediated depletion switch in N. benthamiana leaves expressing dCasEV2.1 with an unspecific gRNA (C−) or the specific gRNA gDFR without (C+) or with mAID:AcrIIA4 treated with different IAA concentrations. (g) FBP[HispS]‐RTA values of the IAA‐mediated depletion switch in N. benthamiana leaves expressing dCasEV2.1 with an unspecific gRNA (C−) or the specific gRNA gDFR with mAID:AcrIIA4. Samples were either left untreated (0 mm), or treated with 10 mm IAA at different time points. Error bars indicate SD (b and e) and SE (c, f and g): n = 6 for (b, c and g) and n = 5 for (e, f). Statistical analyses were performed using one‐way ANOVA (Tukey's multiple comparisons test, P‐value ≤0.05). Variables within the same statistical groups are marked with the same letters.

Figure 4

Alternative designs for a copper switch assayed with the FBP[HispS]/eGFP reporter. (a) Schematic representation of HispS gene activation driven by the copper inducible transcription factor CUP2:GAL4AD. (b) FBP[HispS]‐RTA values of the simple copper switch depicted in (a) treated with different CuSO4 concentrations or (c) incubating with 0.1 mm CuSO₄ at different time points. EV represents control experiments where the sensor CUP2:GAL4AD was substituted by an empty vector. (d) Schematic representation of HispS gene activation driven by a copper inducible dCasEV2.1. In this circuit both protein components of dCasEV2.1 (dCas9:EDLL and MS2:VPR) are regulated by copper, while the gRNA (gDFR) is constitutively expressed. (e) FBP[HispS]‐RTA values of the copper swich represented in (d) with or without CUP2:GAL4AD and treated with different CuSO4 concentrations. (f) Schematic representation of HispS gene activation controlled by a recombinase (FLP or PhiC31) and a copper sensor. In this design the minimal DFR promoter is disrupted by the octopine synthase terminator flanked by recombination sites (FRT or att). The presence of both components (CUP2:GAL4AD and recombinase) is required for HispS expression. (g) FBP[HispS]‐RTA values of the copper switch depicted in (f) with or without CUP2:GAL4AD and the corresponding recombinase (FLP or PhiC31) either driven by a PNOS or by the controlled by copper (CBS). Samples were treated with 0.1 mm CuSO₄ (+) or let untreated (−). EV represents an empty vector. Error bars indicate SE: n = 9 for (b) and (e); n = 6 for (c) and (g). Statistical analyses were performed using one‐way ANOVA (Tukey's multiple comparisons test, P‐value ≤0.05). Variables within the same statistical groups are marked with the same letters.

Figure 5

Dual optogenetic and chemical control of the fungal bioluminescence pathway (FBP). (a) Schematic representation of the CRY2:EBD‐CIB1:VPR blue light (BL)‐dependent HispS activation. (b) Schematic representation of the PIF6:EBD‐PHYB:VP16 red light (RL)‐dependent HispS activation. (c) NeoLuc/eGFP ratios and (d) FBP[HispS]‐RTA values of the BL switch depicted in (a) expressed in N. benthamiana leaves, with or without CRY2:EBD and CIB1:VPR. Two minimal promoters driving HispS were assayed: mDFR and mCMV. Blue bars are BL treatments; black bars are dark treatments. (e) NeoLuc/eGFP ratios and (f) FBP[HispS]‐RTA values of the RL switch depicted in (b) assayed in N. benthamiana, with or without PIF6:EBD and PHYB:VP16. Red bars are RL treatments; black bars are dark treatments. (g) Schematic representation of the two alternative configurations assayed for the dual RL and copper control of the FBP. (h) NeoLuc/eGFP ratios and (i) FBP[HispS]‐RTA values of the circuits depicted in (g) in Configurations I and II. (−) are water treatments and (+) represents 0.1 mm CuSO₄ treatments. CBS:HISPS‐Etr:H3H represents Configuration I of the circuit. Etr:HISPS‐CBS:H3H represents Configuration II. CUP2 + PIF‐PHYB are samples containing the entire circuit. PIF‐PHYB+EV and CUP2 + EV represent control samples were one of the sensor modules was missing and substituted by an empty vector. PNOS:HISPS is a normalizing control. EV samples were agroinfiltrated with an empty vector. Error bars indicate SD (c, e and h) and SE (d, f and i): n = 9 for (c), (d), (e) and (f) and n = 6 for (h) and (i)). Statistical analyses were performed using one‐way ANOVA (Tukey's multiple comparisons test, P‐value ≤0.05). Variables within the same statistical groups are marked with the same letters.

Figure 6

N. benthamiana plants expressing components of the FBP reduce the size of the plasmids delivered via agroinfiltration. Schematic representation of the FBP genes incorporated in the plant and those delivered via agroinfiltration in (a) the [∆HISPS] plants and (b) the [∆HISPS, ∆H3H] plants. NeoLuc/eGFP ratios of (c) seven independent T0 [∆HISPS] and (d) eight independent T0 [∆HISPS, ∆H3H] N. benthamiana plants transiently expressing either an empty vector (EV) or the missing components of the FBP reporter in each plant. (e) NeoLuc/eGFP ratios and (f) FBP[HispS]‐RTA values of T0 [∆HISPS] line 1 transiently expressing either an EV or the HispS gene driven by the PNOS or P35S promoters. Error bars indicate SD (e) and SE (f): n = 9. Statistical analyses were performed using one‐way ANOVA (Tukey's multiple comparisons test, P‐value ≤0.05). Variables within the same statistical groups are marked with the same letters.