Targeting ALA3 with propiconazole regulates plant growth and enables discovery of promising inhibitor leads

Propiconazole (PCZ) is widely misused growth regulator in leafy Brassica vegetables. Developing green strategies for managing plant architecture has become an urgent agricultural priority. Here, we identified from a membrane-protein-defective yeast library a P4-ATP phospholipid flippase, aminophospholipid ATPase 3 (ALA3), as a target sensitive to PCZ. ALA3 exhibits high binding affinity for PCZ, which inhibits its ATPase activity. Knockdown of ALA3 rendered yeast, Arabidopsis , and Brassica rapa less sensitive to PCZ and conferred a growth-inhibited phenotype. This dwarfing phenotype is mediated through the interaction between ALA3 and CYP51G1 that jointly acts within the brassinosteroid regulatory pathway. Furthermore, we identified lead compounds A01 and A15 as ALA3-targeting agents, and compared to PCZ, they display superior binding affinity and reduced toxicity. Our work establishes ALA3 as a key mediator of PCZ-induced dwarfism and provides dual strategies—creating promising varieties through gene editing and developing targeted green pesticides—to reduce PCZ use.