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Synthetic memory circuits for programmable cell reconfiguration in plants

James P B Lloyd, Florence Ly, Patrick Gong, Jahnvi Pflüger, Tessa Swain, Christian Pflüger, Muhammad Adil Khan, Brendan Kidd, Ryan Lister

Preprint posted on February 12, 2022 https://www.biorxiv.org/content/10.1101/2022.02.11.480167v1

and

Synthetic genetic circuits enable reprogramming of plant roots

Jennifer A. N. Brophy, Katie J. Magallon, Kiril Kniazev, José R. Dinneny

Preprint posted on February 03, 2022 https://www.biorxiv.org/content/10.1101/2022.02.02.478917v1

Synthetic gene circuits – a promising way to create ideotypical plant architecture

Selected by Gwendolyn K. Kirschner

Background:

A central idea of plant breeding research is the creation of crop ideotypes that are adapted perfectly to their environment and growth conditions and therefore outperform in production 1. However, it is difficult to verify the impact of proposed phenotypic adaptations to environmental conditions in the field, because manipulating genes on the whole-plant level often has pleiotropic effects that cannot be untangled. Even after the discovery of economic trait regulators, it is challenging to fine tune their expression spatially and timely because of the lack of native promoters that drive the desirable strength and expression pattern. A possible solution are synthetic genetic circuits, which integrate customizable input signals through processing units in the form of promoters to produce a specific and tunable output gene expression pattern. These synthetic genetic circuits are well-established in bacterial, yeast and mammalian cells, having been introduced over 10 years ago, but are relatively novel in the plant world2.

In these preprints, the authors design synthetic gene circuits in Arabidopsis and tobacco for expressing genes fine-tuned in specific tissues and demonstrate the application in changing root architecture.

Key findings:

Brophy et al., 2022, designed a collection of synthetic transcriptional regulators interacting with synthetic promoters. They tested the output of these combinations transiently in tobacco leaves, where they expressed mCherry constitutively from a ubiquitous promoter, while GFP was expressed as output of the transcription factor (TF)/promoter combination, so that the ratio of mCherry to GFP served as quantification of the activity of these circuits. In this way, they created different logical gates in which two TFs interact with the promoter in a unique way. They optimised the output of the circuits by introducing different plant elements to the synthetic TFs, changing the number of TF binding sites in the promoters, or by moving the location of operators. The authors then applied the system in Arabidopsis, where they used the well established SOMBRERO and PINFORMED4 promoters in the root to express the gain of function mutant gene solitary root exclusively in the lateral root stem cells, to avoid pleiotropic effects of the mutation on the whole-plant level. They were able to fine tune the expression by introducing BUFFER gates with a variable number of operators in the promoter, resulting in an expression level gradient correlating with the promoter strength, and thereby in a gradient of lateral root densities.

Lloyd et al., 2022, designed recombinase-based gene circuits, in which a transcriptional terminator was inserted between promoter and coding sequence (CDS), flanked by recombination sites that enable removal upon recombinase expression, which creates a positive output. Vice versa, a negative output was created by flanking a CDS or promoter with recombination sites so that a removal deactivates the circuit. The authors quantified the circuit activity in Arabidopsis leaf mesophyll protoplasts where they expressed a Renilla luciferase as circuit output and a Firefly luciferase for normalisation, so that the ratio between both serves as measure for the output. By that, they optimised the repressive sequences, as well as the promoter driving the recombinase expression and tested different recombinases. They created multicomponent gates by introducing terminators with different recombination sites, and added another layer of regulation by using split recombinases, which are only activated when both halves are expressed. The authors then tested the applicability in Arabidopsis roots, where they activated target gene expression upon chemical induction in a specific cell layer. The distinctive feature of this system as opposed to the one presented by Brophy et al. is that recombinase-based gates keep the memory of the input signal in cells because the sequences flanked by recombination sites are permanently removed from the genes.

A) Schematic view of an AND gate, in which the expression of the output gene (nuclear localized GFP) is inhibited, unless in both recombinases (Flp and B3) are expressed in the same cell and remove the terminators between the 35S promoter and GFP. Flp expression is driven by a DEX inducible promoter, while B3 is under control of a cortex-specific promoter. B) Schematic view of an AND gate with the output of Renilla luciferase (Rluc) expression. C) Optical cross sections of Arabidopsis roots expressing the construct from A), either without induction (DMSO) or with induction of Flp (DEX). Magenta channel shows the cell walls and the nuclei in focus (propidium iodide and nuclear localized mCherry), cyan channel shows GFP, which is only expressed in the cortex (c), but not in the epidermis (ep) or in the endodermis (end). Scale bars 20 µm. Figure modified from Lloyd et al. 2022.

 

Importance:

The preprints by Brophy et al. and Lloyd et al. present a first important step towards the sophisticated design of ideotypical crops. Synthetic gene circuits circumvent the tedious search for promoters with desired expression patterns and allow for adapting gene expression in a spatial and timely manner to bypass problems that arise with ubiquitous overexpression or pleiotropic effects of mutations. To date, the most common system for inducible gene expression is to couple promoters with inducible elements, but this involves promoter engineering, and can only promote the output promoter activation; the systems presented in these studies integrate and combine signals from two promoters, leading to specific expression patterns and controlled activation or repression of a target. While the system presented by Brophy et al. requires a constant input signal to keep the circuit running, the recombinase-based system by Lloyd et al. acts as an irreversible switch that requires the input signal to be present only once. Both systems are useful under different conditions. For example, for a circuit in a crop that prepares for an incoming stress it is more favorable if a continuous treatment with the input signal is not necessary.

Future directions:

Brophy et al. have already demonstrated the applicability of synthetic genetic circuits for modifying lateral root branching density, which is an essential trait for water and nutrient uptake by increasing soil space coverage; however, it is clear that the field of synthetic genetic circuits in plants is still in its infancy. The studies pointed out that the elements of the circuits have to be optimised for each plant species and tissue and that there is no “one fits all” design. Furthermore, application in the field involves creation of transgenic crop plants, a process that is time-consuming and tedious, and the optimization of the pathway components that are less well researched in crop plants. However, the studies are an important step towards the goal of creating crop ideotypes.

 

Questions for the authors

 

1) Besides lateral root density, which other traits do you have in mind that could be easily modified by fine-tuning gene expression of a single gene like IAA14?

 

2) Which challenges do you expect when transferring the concept of synthetic gene circuits from Arabidopsis to monocot crop plants? Do you see any way to test the circuits for functionality before stably transforming the target plants, given that transformation of monocots is very tedious?

 

References:

  1. Donald, C. M. The breeding of crop ideotypes. Euphytica 17, 385–403 (1968).
  2. Nevozhay, D., Zal, T. & Balázsi, G. Transferring a synthetic gene circuit from yeast to mammalian cells. Nat. Commun. 4, 1–11 (2013).

Tags: arabidopsis, root ideotypes

Posted on: 28th April 2022

doi: https://doi.org/10.1242/prelights.31880

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