Inspiration and signaling cascade
Our plant-based detection system was inspired by the work of Antunes et al. 2011 in June Medford’s lab at Colorado State University, where a TNT (2,4,6-trinitrotoluene) detection system in plants was developed, that utilizes a modified bacterial signaling cascade to detect TNT[1] . For our plant-based detection of chemical weapon precursors, a similar signaling cascade is used (figure 1). It features a computationally re-designed periplasmic binding protein (PBP), which should be capable to bind certain chemical degradation products. The PBP originates from bacteria and is responsible for chemotaxis[2]. It initiates our cascade upon binding the respective ligand that is located in the plant's apoplast, which is the space outside of the cell walls in between further cells, to the export of the expressed PBP is enabled by a plant secretory sequence (ss), which is fused to the PBPs N-Terminus[1]. The signal transduction is facilitated by a transmembrane protein, which interacts with the ligand-bound PBPs. Therefore, the transmembrane fusion protein of the bacterial chemotactic response regulator Trg and the bacterial response regulator PhoRis activated by phosphorylation. Trg is located at the extracellular section of the protein that binds the activated PBP and PhoR is an intracellular histidine kinase. PhoR is known for its role in phosphate sensing by signal transduction to the response regulator PhoB. According to Antunes et al. 2011, the phosphate sensing domain of PhoR should be replaced by a fusion of the the DHP8 region (dimerization, histidine phosphotransfer) and HAMP domain (central signal converter), enabling the best signal transmission among Trg and PhoR to PhoB while also facilitating the best ligand dependent induction [3]. To ensure a proper localization of the transmembrane protein, a N-terminal signal peptide (FLS2) is added to the transmembrane protein [4]. Since this signaling cascade is adapted from bacteria, the transcription factor that is activated through the PhoR histidine kinase needs to be modified to enable gene expression in plants. Through addition of the eukaryotic transcriptional activator VP64, the transcription factor is capable of binding the synthetic PlantPho promoter that in return regulates the desired reporter gene [1].
It has been shown that the binding specificity of the PBPs can be altered for other ligands by modification within its binding pocket [5,6] . In our project we created a variety of modified PBPs to detect degradation products of chemical weapons and established the RUBY reporter system as output signal for the detection. RUBY is comprised of three enzymes which facilitate the synthesis of betalain, red tyrosine-derived pigments which are visible with the bare eye [7]. Our plant-based detection system for chemical weapon degradation products, is comprised of a periplasmic binding protein (PBP) collection, a signaling cascade and a reporter system to facilitate not only a detection of those chemicals, but also the visible and lasting signal of a red plant. With this system we would create a versatile and cost-effective tool for monitoring large areas for harmful chemicals with an output that can be assessed at first glance without additional tools or assays.
