Deposition of cell wall-reinforcing papillae is an integral component of the plant immune response.The Arabidopsis PENETRATION 3 (PEN3) ATP binding cassette (ABC) transporter plays a role in defense against numerous pathogens and is recruited to sites of pathogen detection where it accumulates within papillae.However,the trafficking pathways and regulatory mechanisms contributing to recruitment of PEN3 and other defenses to the host-pathogen interface are poorly understood.Here,we report a confocal microscopy-based screen to identify mutants with altered localization of PEN3-GFP after inoculation with powdery mildew fungi.We identified a mutant,aberrant localization of PEN3 3 (alp3),displaying accumulation of the normally plasma membrane (PM)-Iocalized PEN3-GFP in endomembrane compartments.The mutant was found to be disrupted in the P4-ATPase AMINOPHOSPHOLIPID ATPASE 3 (ALA3),a lipid flippase that plays a critical role in vesicle formation.We provide evidence that PEN3 undergoes continuous endocytic cycling from the PM to the trans-Golgi network (TGN).In alp3,PEN3 accumulates in the TGN,causing delays in recruitment to the host-pathogen interface.Our results indicate that PEN3 and other defense proteins continuously cycle through the TGN and that timely exit of these proteins from the TGN is critical for effective pre-invasive immune responses against powdery mildews.