scalable-transfection-of-maize-mesophyll-protoplasts

Scalable Transfection of Maize Mesophyll Protoplasts

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation ...

University of Washington

Genetic engineering of -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 -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 () leaves. We demonstrate that the optimal placement in the reporter construct of enhancer sequences from a plant virus, pea () and wheat (), 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  and that can use regulatory DNA derived from any plant genome.

Identification of Plant Enhancers and Their Constituent Elements by STARR-seq in Tobacco Leaves.

Targeted engineering of plant gene expression holds great promise for ensuring food security and for producing biopharmaceuticals in plants. However, this engineering requires thorough knowledge of cis-regulatory elements to precisely control either endogenous or introduced genes. To generate this knowledge, we used a massively parallel reporter assay to measure the activity of nearly complete sets of promoters from Arabidopsis, maize and sorghum. We demonstrate that core promoter elements-notably the TATA box-as well as promoter GC content and promoter-proximal transcription factor binding sites influence promoter strength. By performing the experiments in two assay systems, leaves of the dicot tobacco and protoplasts of the monocot maize, we detect species-specific differences in the contributions of GC content and transcription factors to promoter strength. Using these observations, we built computational models to predict promoter strength in both assay systems, allowing us to design highly active promoters comparable in activity to the viral 35S minimal promoter. Our results establish a promising experimental approach to optimize native promoter elements and generate synthetic ones with desirable features.

Article	Year
Identification of Plant Enhancers and Their Constituent Elements by STARR-seq in Tobacco Leaves. The Plant cell\| PubMed ID: 32409318	2020
Synthetic promoter designs enabled by a comprehensive analysis of plant core promoters. Nature plants\| PubMed ID: 34083762	2021

Jackson Tonnies

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