Engineering key components in a synthetic eukaryotic signal transduction pathway

Mauricio S Antunes¹, Kevin J Morey¹, Neera Tewari-Singh^1,a, Tessa A Bowen¹, J Jeff Smith^2,b, Colleen T Webb¹, Homme W Hellinga² & June I Medford¹

^aPresent address: Department of Pharmaceutical Sciences, University of Colorado Denver, Aurora, CO 80045, USA

^bPresent address: Precision BioSciences, 104 TW Alexander Drive, Building 7, PO Box 12292, Research Triangle Park, NC 27709, USA

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Synopsis

Signal transduction underlies how living organisms detect and respond to environmental stimuli. Synthetic biology aims to better understand natural signal transduction systems and also rewire them to produce controllable outputs. Bacteria have an array of HK or two-component signal transduction systems that are involved in perceiving environmental conditions and eliciting responses (Wolanin et al, 2002). In plants, HK systems are involved in hormone perception and response, notably cytokinins (Guo and Ecker, 2004; Chen et al, 2005; Ferreira and Kieber, 2005). HK signaling proceeds through a phospho-relay between protein components that are typically modular and conserved across different kingdoms. The high degree of sequence conservation among these proteins has allowed functional assays to be developed for plant HKs in bacteria and yeast (Urao et al, 1999; Suzuki et al, 2001). The presence of the eukaryotic nuclear membrane constitutes a major difference between HK signaling in bacteria and plants. Plant HK components involved in signal transduction are found in different parts of the cell, for example membrane-localized HKs, and cytoplasmic and nuclear-localized Arabidopsis RRs. Some components, such as Hpt proteins, are re-distributed from the cytoplasm to the nucleus in response to the HK phosphorylation signal. Cross talk between components of HK signaling is known to occur (Hass et al, 2004). To test whether conserved bacterial HK components are able to functionally interact with plant HK components, we expressed the E. coli RRs, PhoB and OmpR, in Arabidopsis and found that these proteins respond to phosphate signals from endogenous cytokinin-mediated HK signaling by accumulating in the plant nucleus (Figure 2). This is a remarkable observation given that bacterial cells do not have nuclei and these proteins are not involved in eukaryotic pathogenic infection. We show that translocation into the nucleus cannot readily be explained by passive diffusion of the bacterial RR by making a large fusion protein PhoB-GFP-GUS.

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 m in (A–C, F); scale bars, 10 m in (D–E, G–H).

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In E. coli, the RR PhoB is phosphorylated in response to external stimuli resulting in a protein conformational change that uncovers a DNA-binding domain. This domain has high affinity for a specific DNA sequence, the Pho box. Binding of phospho-PhoB to Pho boxes results in transcriptional activation of downstream genes (Makino et al, 1989; Ellison and McCleary, 2000; Blanco et al, 2002). We produced a signal-dependent eukaryotic transcriptional response system by using the properties of PhoB and its cytokinin-dependent nuclear translocation in plants. PhoB, fused to a eukaryotic transcriptional activation domain (VP64), translocates to the nucleus, binds a synthetic PhoB-responsive promoter, and activates transcription of the GUS reporter gene in Arabidopsis (Figure 6). These results show that conserved-signaling components can be used across kingdoms and adapted to provide key components of synthetic signal transduction pathways in eukaryotes.

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 indicateone standard error. (C) Linear increase in GUS activity (nmoles 4-MU mg^-1 protein h^-1) with t-zeatin concentration. 4-MU, 4-methylumbelliferone.

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