Abstract: Phosphate starvation response (PHR) transcription factors are master regulators of the plant phosphate (Pi) starvation response (PSR), yet the mechanisms governing the dynamic control of their transcriptional activity remain elusive. Here, we report a dual regulatory module comprising the coactivator SlMED25 and the corepressor SlSPX2 that fine-tunes SlPHR3 activity in Solanum lycopersicum (tomato). Genetic and biochemical evidence collectively confirmed that SlPHR3 acts as the central regulator orchestrating tomato PSR. Specifically, the mediator subunit SlMED25 interacts with the N-terminal domain (NTD) of SlPHR3 to recruit RNA polymerase II (Pol II) to SlPHR3 target promoters in a SlPHR3-dependent fashion, whereas SlSPX2 binds to the same NTD of SlPHR3 to robustly suppress its transcriptional activity. Biochemical assays further demonstrated that SlSPX2 and SlMED25 compete for binding to SlPHR3, with SlSPX2 exhibiting higher binding affinity. This competitive binding module functions as a key molecular switch that mediates dynamic PSR modulation in tomato, thus yielding distinct functional outputs in response to varying intracellular Pi levels. Our findings uncover a previously uncharacterized regulatory layer in the PSR network, wherein a Mediator subunit and a Pi-sensing protein modulate PHR activity via competitive binding, thereby enhancing the mechanistic insight into Pi homeostasis regulation in plants.

Key words: phosphate starvation response, SlMED25‐SlPHR3‐SlSPX2 module, transcription regulation