Engineering key components in a synthetic eukaryotic signal transduction pathway

Abstract

Signal transduction underlies how living organisms detect and respond to stimuli. A goal of synthetic biology is to rewire natural signal transduction systems. Bacteria, yeast, and plants sense environmental aspects through conserved histidine kinase (HK) signal transduction systems. HK protein components are typically comprised of multiple, relatively modular, and conserved domains. Phosphate transfer between these components may exhibit considerable cross talk between the otherwise apparently linear pathways, thereby establishing networks that integrate multiple signals. We show that sequence conservation and cross talk can extend across kingdoms and can be exploited to produce a synthetic plant signal transduction system. In response to HK cross talk, heterologously expressed bacterial response regulators, PhoB and OmpR, translocate to the nucleus on HK activation. Using this discovery, combined with modification of PhoB (PhoB-VP64), we produced a key component of a eukaryotic synthetic signal transduction pathway. In response to exogenous cytokinin, PhoB-VP64 translocates to the nucleus, binds a synthetic PlantPho promoter, and activates gene expression. These results show that conserved-signaling components can be used across kingdoms and adapted to produce synthetic eukaryotic signal transduction pathways.

Figure 1

Comparison of HK signal transduction systems from plants and bacteria. Ligands bind to the extracellular domain of transmembrane HKs and activate a cytoplasmic kinase domain. A phospho-relay (His → Asp in bacteria or His → Asp → His → Asp in plants) transmits the signal to DNA. Both systems can be defined as perceiving an input stimulus (input layer), transmitting the signal (transmission layer), and bringing about a response (response layer), but use different numbers of components. In simple bacterial systems (right panel), two proteins (HK and RRs) function in three layers. In plants (left panel), cytokinin responses involve multiple components that are each encoded by multigene families in these three layers. AHK, arabidopsis histidine kinase; AHP, arabidopsis histidine phosphotransfer protein; CK, cytokinin; HK, histidine kinase; H, histidine residue; D, aspartate residue; P, phosphate group; CRF, cytokinin response factor; ARR, Arabidopsis response regulator; A, bacterial HK ligand.

Figure 2

Bacterial RR PhoB translocates to plant nuclei in root cells in response to HK activation with exogenous cytokinin. (A, B) Cellular localization of PhoB-GFP in roots of transgenic Arabidopsis plants. (A) Before cytokinin treatment, PhoB-GFP fluorescence appears diffused and throughout the cells. (B) After exogenous cytokinin treatment, the same root shows PhoB-GFP accumulation in sub-cellular compartments. (C–H) Detail views of roots (D, G) before and (C, E, F, H) after treatment with cytokinin showing that before cytokinin is applied, GFP fluorescence is diffused; after cytokinin exposure, the compartments in which PhoB-GFP accumulates (C, E) also stain with DAPI (F, H), indicating that they are nuclei (arrowheads). −CK, tissue before cytokinin treatment; +CK, tissue after cytokinin treatment; DAPI, tissues treated with DAPI to stain DNA. Scale bars, 50 μm in (A–C, F); scale bars, 10 μm in (D–E, G–H).

Figure 3

PhoB also translocates to plant nuclei in leaf and crown cells in response to HK activation with exogenous cytokinin. (A–D) Localization of PhoB-GFP in leaves. Leaf (A) before and (B) after exogenous cytokinin treatment. (C) Close-up view of leaf showing punctate PhoB-GFP. (D) DAPI staining of the same area showing that the punctate compartments are nuclei. (E–H) PhoB-GFP localization in the Arabidopsis crown, a stem-like region. Crown (E) before and (F) after cytokinin treatment. (G) Close-up view of (F) showing punctate GFP localization. (H) DAPI staining of area shown in (G), indicating that punctate GFP compartments are nuclei. Arrowheads point to nuclei. −CK, tissues before cytokinin treatment; +CK same tissue after cytokinin treatment; DAPI, tissues treated with DAPI to stain DNA. Scale bars, 50 μm in (A, B, E, F); scale bars, 10 μm in (C, D, G, H).

Figure 4

Analysis of signal-dependent nuclear translocation of PhoB. (A–D) Confocal microscope images of PhoB-GFP protein in roots (A) before and (B–D) after cytokinin treatment. (C) Detail view of the boxed area in (B) shows PhoB-GFP accumulation in nuclei. (D) Detail view of the area boxed in (C), showing a single nucleus with PhoB-GFP accumulation throughout. (E–H) Cellular localization of PhoB-GFP-GUS fusion protein in roots of transgenic Arabidopsis plants (E) before and (F–H) after cytokinin treatment showing compartmentalized accumulation. (G) Detail view of a root treated with cytokinin, showing compartments (arrowheads) that also stain with (H) DAPI, indicating that they are nuclei (arrowheads). −CK, plants before cytokinin treatment; +CK, same plant tissue after cytokinin treatment; DAPI, same tissues treated with DAPI to stain DNA. Scale bars, 50 μm in (A, B, E, F); scale bars, 10 μm in (C, D, G, H).

Figure 5

Cellular localization of mutagenized PhoB^D53A-GFP in roots of transgenic Arabidopsis plants. (A) Fluorescence from PhoB^D53A-GFP is diffused in an untreated root. (B) The same root showing PhoB^D53A-GFP localization after cytokinin treatment. (C, D) Detailed view of a root showing that nuclear localization of PhoB^D53A-GFP is variable and sporadic (arrowheads point to nuclei). (E, F) Detail view of another root showing that PhoB^D53A-GFP accumulates at the base of cortical cells (arrows). Some nuclear localization of PhoB^D53A-GFP can be seen in the root vascular tissue (arrowheads). −CK, tissues before cytokinin treatment; +CK same tissue after cytokinin treatment; DAPI, tissues treated with DAPI to stain DNA. Scale bars, 50 μm in (A, B); scale bars, 10 μm in (C–F).

Figure 6

Design and function of the synthetic eukaryotic signal transduction system. (A) Diagram of PlantPho promoter, showing four Pho boxes fused to a minimal plant promoter, the −46 region of the CaMV35S promoter, with the nucleotide sequence of one Pho box indicated below. (B) Average GUS activity (nmoles 4-MU mg⁻¹ protein h⁻¹) in transgenic plants, containing the PlantPho system as a function of cytokinin (t-zeatin) concentration. Error bars indicate±one standard error. (C) Linear increase in GUS activity (nmoles 4-MU mg⁻¹ protein h⁻¹) with t-zeatin concentration. 4-MU, 4-methylumbelliferone.