A Noncanonical Auxin-Sensing Mechanism Uncovered by Screening the Auxin Response Factor 3 Interacting Proteins in Tomato

Within the canonical auxin signaling pathway, Auxin Response Factors (ARFs) are transcriptionally repressed by AUX/IAA proteins under low auxin conditions, and this repression is alleviated as auxin concentrations increase. By contrast, ARF3 functions as a central regulator of gynoecium morphogenesis in Arabidopsis via a non-canonical auxin-sensing mechanism that relies on dose-dependent modulation of its protein-protein interaction network. To investigate whether an analogous regulatory mechanism operates in tomato (<i>Solanum lycopersicum</i>), we identified the tomato ARF3 homolog (SlARF3) and utilized it as bait in a yeast two-hybrid (Y2H) screen. This screening approach yielded 137 positive clones, corresponding to 118 putative interacting proteins. Notably, all of these interactions were abolished in the presence of 3-indoleacetic acid (IAA), indicating that SlARF3 engages in auxin-sensitive protein-protein interactions and thereby mediates auxin-dependent signal transduction. Among these, we identified an auxin-sensitive interaction between SlARF3 and TM29, a central regulator of parthenocarpy, underscoring its critical role in this developmental pathway. Functional analyses further demonstrated that silencing <i>SlARF3</i> induces parthenocarpic fruit formation. Taken together, these findings define a previously uncharacterized SlARF3-centered interaction network and provide a conceptual framework for elucidating non-canonical auxin signaling pathways underlying tomato development.