Rational Design of Minimal Synthetic Promoters for Plants

Cai, Yuyang; Kallam, Kalyani; Tidd, Henry; Gendarini, Giovanni; Salzman, Amanda; Patron, Nicola J.

doi:10.1101/2020.05.14.095406

Cited by 9 publications

(5 citation statements)

References 71 publications

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“…Appropriate gene expression is the critical function of a nucleus, and understanding how gene regulatory networks control transcription has become a major focus in a variety of fields, from medicine to plant breeding. Owing to the massive output of information from next-generation sequencing, CREs have been identified as key players in gene transcription, and even small mutations in these regions have been shown to cause dramatic changes in phenotype (Cai et al, 2020;Rodr ıguez-Leal et al, 2017). These regulatory elements represent an exciting new area of exploration in which levels of gene product can be controlled with high sensitivity, without affecting the coding sequence of the gene or needing to introduce RNA to a cell, as with RNAi, and are ideal sequences to be modified by CRISPR/Cas9 mediated genome editing techniques to achieve fine tuning of gene expression.…”

Section: Discussionmentioning

confidence: 99%

Open chromatin in grapevine marks candidate CREs and with other chromatin features correlates with gene expression

et al. 2021

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Vitis vinifera is an economically important crop and a useful model in which to study chromatin dynamics. In contrast to the small and relatively simple genome of Arabidopsis thaliana, grapevine contains a complex genome of 487 Mb that exhibits extensive colonization by transposable elements. We used Hi-C, ChIP-seq and ATAC-seq to measure how chromatin features correlate to the expression of 31 845 grapevine genes. ATAC-seq revealed the presence of more than 16 000 open chromatin regions, of which we characterize nearly 5000 as possible distal enhancer candidates that occur in intergenic space > 2 kb from the nearest transcription start site (TSS). A motif search identified more than 480 transcription factor (TF) binding sites in these regions, with those for TCP family proteins in greatest abundance. These open chromatin regions are typically within 15 kb from their nearest promoter, and a gene ontology analysis indicated that their nearest genes are significantly enriched for TF activity. The presence of a candidate cis-regulatory element (cCRE) > 2 kb upstream of the TSS, location in the active nuclear compartment as determined by Hi-C, and the enrichment of H3K4me3, H3K4me1 and H3K27ac at the gene are correlated with gene expression. Taken together, these results suggest that regions of intergenic open chromatin identified by ATAC-seq can be considered potential candidates for cis-regulatory regions in V. vinifera. Our findings enhance the characterization of a valuable agricultural crop, and help to clarify the understanding of unique plant biology.

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Section: Discussionmentioning

confidence: 99%

Open chromatin in grapevine marks candidate CREs and with other chromatin features correlates with gene expression

et al. 2021

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“…To date, the basic gene construct design has largely included synthetic sequences upstream of a core promoter sequence such as the −46 35S promoter. Recently, a minimal synthetic promoter was designed for constitutive response by various arrangement of CREs at different positions to TATA box area (Cai et al ., 2020). In our study, no downstream gene expression was observed in synthetic promoters including three copies of single CREs (Figure S3).…”

Section: Discussionmentioning

confidence: 99%

Rational design and testing of abiotic stress‐inducible synthetic promoters from poplar cis‐regulatory elements

Yang

Lee

Poindexter

et al. 2021

Plant Biotechnology Journal

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Abiotic stress resistance traits may be especially crucial for sustainable production of bioenergy tree crops. Here, we show the performance of a set of rationally designed osmotic-related and salt stress-inducible synthetic promoters for use in hybrid poplar. De novo motif-detecting algorithms yielded 30 water-deficit (SD) and 34 salt stress (SS) candidate DNA motifs from relevant poplar transcriptomes. We selected three conserved water-deficit stress motifs (SD18, SD13 and SD9) found in 16 co-expressed gene promoters, and we discovered a well-conserved motif for salt response (SS16). We characterized several native poplar stress-inducible promoters to enable comparisons with our synthetic promoters. Fifteen synthetic promoters were designed using various SD and SS subdomains, in which heptameric repeats of five-to-eight subdomain bases were fused to a common core promoter downstream, which, in turn, drove a green fluorescent protein (GFP) gene for reporter assays. These 15 synthetic promoters were screened by transient expression assays in poplar leaf mesophyll protoplasts and agroinfiltrated Nicotiana benthamiana leaves under osmotic stress conditions. Twelve synthetic promoters were induced in transient expression assays with a GFP readout. Of these, five promoters (SD18-1, SD9-2, SS16-1, SS16-2 and SS16-3) endowed higher inducibility under osmotic stress conditions than native promoters. These five synthetic promoters were stably transformed into Arabidopsis thaliana to study inducibility in whole plants. Herein, SD18-1 and SD9-2 were induced by waterdeficit stress, whereas SS16-1, SS16-2 and SS16-3 were induced by salt stress. The synthetic biology design pipeline resulted in five synthetic promoters that outperformed endogenous promoters in transgenic plants.

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“…However, when applied to genome-scale collections of TFs, insufficient expression was obtained for several hundred proteins (91). We expressed TGA TFs, known to regulate expression of defence related genes (93-97) as well as the CaMV 35s promoter (74,(98)(99)(100)(101)(102). We also expressed wheat homologues of TFs that regulate circadian rhythms in Arabidopsis including homeologues of LUX ARRHYTHMO (LUX) from each of the three wheat sub-genomes (103).…”

Section: Discussionmentioning

confidence: 99%

Biofoundry-assisted expression and characterisation of plant proteins

Dudley

Cai

Kallam

et al. 2021

Preprint

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Many goals in synthetic biology, including the elucidation and refactoring of biosynthetic pathways and the engineering of regulatory circuits and networks, require knowledge of protein function. In plants, the prevalence of large gene families means it can be particularly challenging to link specific functions to individual proteins. However, protein characterisation has remained a technical bottleneck, often requiring significant effort to optimise expression and purification protocols. To leverage the ability of biofoundries to accelerate design-built-test-learn cycles, we present a workflow for automated DNA assembly and cell-free expression of plant proteins that accelerates optimisation and enables rapid progression to characterisation. First, we developed a phytobrick-compatible Golden Gate DNA assembly toolbox containing plasmid acceptors for cell-free expression using E. coli or wheat germ lysates as well as a set of N- and C-terminal tag parts for detection, purification, and improved expression/folding. We next optimised automated assembly of miniaturised cell-free reactions using an acoustic liquid handling platform and then compared tag configurations to identify those that increase expression. We additionally developed a luciferase-based system for rapid quantification that requires a minimal 11 aa tag and demonstrate facile removal of tags following synthesis. Finally, we show that several functional characterisation experiments can be performed with cell-free protein synthesis reactions without the need for protein purification. Together, the combination of automated assembly of DNA parts and cell-free expression reactions should significantly increase the throughput of experiments to test and understand plant protein function and enable the direct reuse of DNA parts in downstream plant engineering workflows.

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Rational Design of Minimal Synthetic Promoters for Plants

Cited by 9 publications

References 71 publications

Open chromatin in grapevine marks candidate CREs and with other chromatin features correlates with gene expression

Open chromatin in grapevine marks candidate CREs and with other chromatin features correlates with gene expression

Rational design and testing of abiotic stress‐inducible synthetic promoters from poplar cis‐regulatory elements

Biofoundry-assisted expression and characterisation of plant proteins

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