Abstract
Developmental programs sculpt plant morphology to meet environmental challenges, and these same programs have been manipulated to increase agricultural productivity (Doebley et al., 1997; Khush, 2001). Hormones coordinate these programs, creating chemical circuitry (Vanstraelen and Benková, 2012) that has been represented in mathematical models (Refahi et al., 2016; Prusinkiewicz et al., 2009); however, model-guided engineering of plant morphology has been limited by a lack of tools (Parry et al., 2009; Voytas and Gao, 2014). Here, we introduce a novel set of synthetic and modular hormone activated Cas9-based repressors (HACRs) in Arabidopsis thaliana that respond to three hormones: auxin, gibberellins and jasmonates. We demonstrate that HACRs are sensitive to both exogenous hormone treatments and local differences in endogenous hormone levels associated with development. We further show that this capability can be leveraged to reprogram development in an agriculturally relevant manner by changing how the hormonal circuitry regulates target genes. By deploying a HACR to re-parameterize the auxin-induced expression of the auxin transporter PIN-FORMED1 (PIN1), we decreased shoot branching and phyllotactic noise, as predicted by existing models (Refahi et al., 2016; Prusinkiewicz et al., 2009).
Highlights
The body plans of plants are inherently plastic, making them amenable to optimization for a wide range of natural or artificial environments
The deactivated Cas9 (dCas9) associates with a guide RNA that targets the hormone activated Cas9-based repressors (HACRs) to a promoter with sequence complementarity where it can repress transcription
The degron sequence targets the HACR for ubiquitination and subsequent proteasomal degradation
Summary
The body plans of plants are inherently plastic, making them amenable to optimization for a wide range of natural or artificial environments. There is a need for tools that can predictably alter how a specific hormone regulates a gene of interest to facilitate re-wiring plant development (Brophy et al, 2017) To date, such efforts have been largely limited to reducing or increasing expression of components of the native hormone signaling machinery (Voytas and Gao, 2014), an approach ill-suited for tuning the strength of connections within a network and confounded by redundancy and buffering within a network. Of hormone regulation on specific genes, in regard to the impact of transcriptional feedback loops on differentiation and morphogenesis For all of these reasons, the potential predictive power of mathematical models has not been fully leveraged in the engineering of morphologies of agronomic interest. To facilitate more sophisticated interventions in plant developmental programs, we designed a set of synthetic and modular hormone-activated Cas9-based repressors (HACRs, pronounced ‘hackers’)
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