Speaker
Description
The protein interactome comprises the full repertoire of molecular partners of a given protein, including proteins and small molecules, within a defined cellular and physiological context. Because these interactions are dynamic and condition-dependent, interactome analysis can reveal protein function and the regulatory networks that control it. In this sense, our project focuses on Auxin Response Factors (ARFs), a family of plant-specific transcription factors that regulate auxin-responsive gene expression and thereby shape key developmental processes, including cell elongation and root architecture. Although ARF molecular structure and DNA-binding properties have been characterized, the relationship between ARF molecular structure, interaction partners and phenotypical effects remain incompletely understood.
To characterize ARF-associated complexes in vivo, we will use Nicotiana benthamiana, a tractable model for transient expression and Agrobacterium-mediated transformation. By employing biotin proximity labelling, we aim to capture transient and short-lived ARF interactions in both cytoplasmic and nuclear compartments. In parallel, we are establishing a correlative imaging workflow that exploits complementary instrumentation available at the ALBA synchrotron to investigate ARF localization in cellulo. The workflow combines cryogenic 3D structured illumination microscopy (cryo-3D SIM) of fluorescently tagged ARFs with cryogenic soft X-ray tomography (cryo-SXT) at the MISTRAL beamline. Cryo-SXT will further enable characterization of the cellular phenotype and the consequences of perturbing these interactions in protoplasts. Finally, correlation with hard X-ray microtomography at the FaXToR beamline will extend this analysis to the tissue scale, enabling structural assessment within intact organs.
The project has already achieved several key milestones, including the cloning of an ARF for interactome profiling and fluorescent labeling, the establishment of a complete workflow for protoplast preparation and cryo-soft X-ray tomography imaging, and the acquisition of preliminary hard X-ray microtomography data from seeds. Together, these achievements lay the foundation for the proposed characterization of ARF interactions while establishing a versatile platform for future studies in plant structural biology.