P3IPs activate autophagy by disrupting the GAPC2-ATG3 interaction and target TuMV 6K2 for degradation.

Our previous work identified p3-interacting protein (P3IP) as a novel plant factor that interacts with rice stripe virus p3 protein and activates autophagy to mediate its degradation, thereby restricting infection. However, the mechanism of P3IP-mediated autophagy and the evolutionary conservation of its antiviral function remain unknown. This study demonstrates that two Arabidopsis thaliana homologs, AtP3IP and AtP3IPH (Arabidopsis P3IP homologs, AtP3IPs), similarly activate autophagy and confer resistance to turnip mosaic virus (TuMV). We show that AtP3IPs activate autophagy by competitively disrupting the interaction between glyceraldehyde-3-phosphate dehydrogenases (GAPCs), known negative regulators of autophagy, and the autophagy-related protein ATG3. This represents the first identification of an endogenous host factor that modulates autophagy through targeting the GAPC-ATG3 regulatory axis. Functional analyses revealed that AtP3IP-overexpressing plants exhibit enhanced TuMV resistance, whereas loss-of-function mutants are more susceptible. Notably, AtP3IPs directly interacted with TuMV 6K2 protein and facilitated its autophagic degradation. Collectively, our findings demonstrate the conserved role of P3IPs in autophagy activation and reveal a novel mechanism through which P3IPs stimulate autophagy by disrupting the inhibitory GAPC-ATG3 regulatory module. Additionally, TuMV 6K2 is identified as a new target of host autophagy. These expand our understanding of plant antiviral defenses and provide potential targets for engineering broad-spectrum resistance against viral pathogens.