Biology subjects

Leydon, A. R.

Publications and source records attributed to Leydon, A. R..

6 recordsLinked to original sources

bioRxiv2025

Reprogramming feedback strength in gibberellin biosynthesis highlights conditional regulation by the circadian clock and carbon dioxide.

The phytohormone gibberellin (GA) is an important regulator of plant morphology and reproduction, and the biosynthesis and distribution of GA in planta is agriculturally relevant to past and current breeding efforts. Tools like biosensors, extensive molecular genetic resources in reference plants and mathematical models have greatly contributed to current understanding of GA homeostasis; however, these tools are difficult to tune or repurpose for engineering crop plants. Previously, we showed that a GA-regulated Hormone Activated CAS9-based Repressor (GAHACR) functions in planta. Here, we use GAHACRs to modulate the strength of feedback on endemic GA regulated genes, and to directly test the importance of transcriptional feedback in GA signaling. We first adapted existing mathematical models to predict the impact of targeting a GAHACR to different nodes in the GA biosynthesis pathway, and then implemented a perturbation predicted by the model to lower GA levels. Specifically, we individually targeted either the biosynthetic gene GA20 oxidase (GA20ox) or the GA receptor GID1, and characterized primary root length, flowering time and the transcriptome of these transgenic lines. Using this approach, we identified a strong connection between GA signaling status and the circadian clock, which can be largely attenuated by elevated carbon dioxide levels. Our results identify a node in the GA signaling pathway that can be engineered to modulate plant size and flowering time. Our results also raise concerns that rising atmospheric CO2 concentration are likely to reverse many of the gains of Green Revolution crops.

synthetic biology↗

bioRxiv2024

A Hot-Swappable Genetic Switch: Building an inducible and trackable functional assay for the essential gene MEDIATOR 21

Essential genes, estimated at approximately 20% of the Arabidopsis genome, are broadly expressed and required for reproductive success. They are difficult to study, as interfering with their function leads to premature death. Transcription is one of the essential functions of life, and the multi-protein Mediator complex coordinates the regulation of gene expression at nearly every eukaryotic promoter. In this study, we focused on a core Mediator component called MEDIATOR21 (MED21), which is required for activation of transcription. Our previous work has also shown a role for MED21 in repression of gene expression through its interaction with a corepressor protein. Here, we sought to differentiate the role MED21 plays in activation versus repression using the model plant Arabidopsis. As mutations in MED21 lead to embryo lethal phenotypes, we constructed a set of synthetic switches using PhiC31 serine integrases to create an "on-to-off" inducible loss of function MED21 in a non-essential tissue. Our technology, which we call Integrase Erasers, made it possible for med21 mutant plants to survive into adulthood by ablating protein expression selectively in lateral root primordia, allowing quantification and characterization of med21 mutant phenotypes in a post-embryonic context. In addition, we engineered chemical induction of the Integrase Eraser to ablate MED21 expression in whole seedlings at a user-specified timepoint. Finally, we extended this technology to build a hot swappable Integrase Isoform Switch where expression of the integrase toggled cells from expressing wildtype MED21 to expressing MED21 sequence variants. Our analysis of the entire set of new integrase-based tools demonstrates that this is a highly efficient and robust approach to the study of essential genes.

synthetic biology↗

bioRxiv2024

A conserved function of corepressors is to nucleate assembly of the transcriptional preinitiation complex.

The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we have leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5 and SPT6 as necessary for repression with the SPT4 subunit acting as a bridge connecting TPL to SPT5 and SPT6. We also discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved in early transcription initiation events. These findings were validated in yeast and plants through multiple assays, including a novel method to analyze conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate rapid onset of transcription once repression is relieved.

molecular biology↗

bioRxiv2022

A single helix repression domain is functional across eukaryotes

The corepressor TOPLESS (TPL) and its paralogs coordinately regulate a large number of genes critical to plant development and immunity. As in many members of the larger pan-eukaryotic Tup1/TLE/Groucho corepressor family, TPL contains a Lis1 Homology domain (LisH), whose function is not well understood. We have previously found that the LisH in TPL--and specifically the N-terminal 18 amino acid alpha-helical region (TPL-H1) --can act as an autonomous repression domain. We hypothesized that homologous domains across diverse LisH-containing proteins could share the same function. To test that hypothesis, we built a library of H1s that broadly sampled the sequence and evolutionary space of LisH domains, and tested their activity in a synthetic transcriptional repression assay in Saccharomyces cerevisiae. Using this approach, we found that repression activity was highly conserved and likely the ancestral function of this motif. We also identified key residues that contribute to repressive function. We leveraged this new knowledge for two applications. First, we tested the role of mutations found in somatic cancers on repression function in two human LisH-containing proteins. Second, we validated function of many of our repression domains in plants, confirming that these sequences should be of use to synthetic biology applications across eukaryotes.

plant biology↗

bioRxiv2020

Structure-function analysis of Arabidopsis TOPLESS reveals conservation of repression mechanisms across eukaryotes

The plant corepressor TOPLESS (TPL) is recruited to a large number of loci that are selectively induced in response to developmental or environmental cues, yet the mechanisms by which it inhibits expression in the absence of these stimuli is poorly understood. Previously, we had used the N-terminus of Arabidopsis thaliana TPL to enable repression of a synthetic auxin response circuit in Saccharomyces cerevisiae (yeast). Here, we leveraged the yeast system to interrogate the relationship between TPL structure and function, specifically scanning for repression domains. We identified a potent repression domain in Helix 8 located within the CRA domain, which directly interacted with the Mediator middle domain subunits Med21 and Med10. Interactions between TPL and Mediator were required to fully repress transcription in both yeast and plants. In contrast, we found that multimer formation, a conserved feature of many corepressors, had minimal influence on the repression strength of TPL.

plant biology↗

bioRxiv2018

Accelerating structure-function mapping using the ViVa webtool to mine natural variation

Thousands of sequenced genomes are now publicly available capturing a significant amount of natural variation within plant species; yet, much of this data remains inaccessible to researchers without significant bioinformatics experience. Here, we present a webtool called ViVa (Visualizing Variation) which aims to empower any researcher to take advantage of the amazing genetic resource collected in the Arabidopsis thaliana 1001 Genomes Project (http://1001genomes.org). ViVa facilitates data mining on the gene, gene family or gene network level. To test the utility and accessibility of ViVa, we assembled a team with a range of expertise within biology and bioinformatics to analyze the natural variation within the well-studied nuclear auxin signaling pathway. Our analysis has provided further confirmation of existing knowledge and has also helped generate new hypotheses regarding this well studied pathway. These results highlight how natural variation could be used to generate and test hypotheses about less studied gene families and networks, especially when paired with biochemical and genetic characterization. ViVa is also readily extensible to databases of interspecific genetic variation in plants as well as other organisms, such as the 3,000 Rice Genomes Project (http://snp-seek.irri.org/) and human genetic variation (https://www.ncbi.nlm.nih.gov/clinvar/).

bioinformatics↗