Abstract
Genetic engineering of cis-regulatory elements in crop plants is a promising strategy to ensure food security. However, such engineering is currently hindered by our limited knowledge of plant cis-regulatory elements. Here, we adapted self-transcribing active regulatory region sequencing (STARR-seq)-a technology for the high-throughput identification of enhancers-for its use in transiently transformed tobacco (Nicotiana benthamiana) leaves. We demonstrate that the optimal placement in the reporter construct of enhancer sequences from a plant virus, pea (Pisum sativum) and wheat (Triticum aestivum), was just upstream of a minimal promoter and that none of these four known enhancers was active in the 3' untranslated region of the reporter gene. The optimized assay sensitively identified small DNA regions containing each of the four enhancers, including two whose activity was stimulated by light. Furthermore, we coupled the assay to saturation mutagenesis to pinpoint functional regions within an enhancer, which we recombined to create synthetic enhancers. Our results describe an approach to define enhancer properties that can be performed in potentially any plant species or tissue transformable by Agrobacterium and that can use regulatory DNA derived from any plant genome.
Highlights
In a time of climate change and increasing human population, crop plants with higher yields and improved response to abiotic stresses will be required to ensure food security
As many of the beneficial traits in domesticated crops are caused by mutations in cis-regulatory elements, especially enhancers, genetic engineering of such elements is a promising strategy for improving crops (Swinnen et al, 2016; Scheben et al, 2017)
Enhancers serve as binding sites for transcription factors that interact with the basal transcription machinery to increase its rate of recruitment, transcription initiation, and/or elongation (Weber et al, 2016; Marand et al, 2017; Andersson and Sandelin, 2020)
Summary
In a time of climate change and increasing human population, crop plants with higher yields and improved response to abiotic stresses will be required to ensure food security. As many of the beneficial traits in domesticated crops are caused by mutations in cis-regulatory elements, especially enhancers, genetic engineering of such elements is a promising strategy for improving crops (Swinnen et al, 2016; Scheben et al, 2017). A minimal promoter is the DNA sequence necessary and sufficient to define a transcription start site and recruit the basal transcription machinery. Such minimal promoters generally lead to low levels of expression (Andersson and Sandelin, 2020). Enhancers can interact with minimal promoters that are several kilobases away, with such long-distance interactions assembled by chromatin loops that bring the enhancer and minimal promoter into close proximity (Amano et al, 2009; Studer et al, 2011; Weber et al, 2016; Ricci et al, 2019)
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