Ramos Báez, Román, Lanctot, Amy, Moss, Britney L (March 2025) Building AuxInYeast Synthetic Biology Strains for Biochemical Characterization of Maize Auxin Hormone Signaling Components. Cold Spring Harbor Protocols. ISSN 1940-3402
Abstract
The AuxInYeast system is a synthetic biology tool that facilitates complex biochemical analysis of the plant auxin hormone signaling pathway. As a plant synthetic biology chassis, Saccharomyces cerevisiae yeast offers rapid growth, well-established genetic and biochemical tools, and core eukaryotic cellular machinery compatible with heterologous plant gene expression. The AuxInYeast system for maize consists of yeast cells containing the minimal necessary set of plant auxin signaling parts: a receptor (ZmTIR1/AFB), repressor (ZmIAA), corepressor (REL2), transcription factor (ZmARF), and auxin response cis-element (auxRE). In plants, auxin binding to a receptor:repressor complex triggers ubiquitination and degradation of the repressor, preventing it from binding to transcription factors on auxin response elements. Thus, auxin-induced repressor degradation allows for the activation of auxin-inducible transcriptional responses. Tagging various auxin signaling components with fluorescent protein reporters then enables quantitative measurement of signaling dynamics via high-throughput approaches such as flow cytometry. As these signaling proteins each belongs to large gene families, AuxInYeast users can build strains with defined components to study their behaviors in isolation or various combinations. Such strains enable researchers to dissect auxin sensitivity, the dynamics of auxin repressor degradation and transcriptional activation, and promoter architecture. It also allows a head-to-head comparison of maize components with orthologs from other plant species to test the evolutionary conservation of component interactions. This protocol describes the construction of such strains. Finally, this protocol and the AuxInYeast approach can also be adapted to assay other multicomponent maize biochemical pathways in yeast.
| Item Type: | Paper |
|---|---|
| Subjects: | bioinformatics bioinformatics > genomics and proteomics > genetics & nucleic acid processing bioinformatics > genomics and proteomics organism description > plant > maize bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > auxin organism description > plant bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types |
| CSHL Authors: | |
| Communities: | CSHL labs > Lippman lab |
| SWORD Depositor: | CSHL Elements |
| Depositing User: | CSHL Elements |
| Date: | 12 March 2025 |
| Date Deposited: | 14 Mar 2025 18:59 |
| Last Modified: | 14 Mar 2025 18:59 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/41820 |
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