Combination of PAMP‐induced peptide signaling and its regulator SpWRKY65 boosts tomato resistance to Phytophthora infestans

SUMMARY Late blight, caused by Phytophthora infestans ( P. infestans ), seriously compromises tomato growth and yield. PAMP‐induced peptides (PIPs) are secreted peptides that act as endogenous elicitors, triggering plant immune responses. Our previous research indicated that the exogenous application of PIP1 from Solanum pimpinelifolium L3708, named SpPIP1, enhances tomato resistance to P. infestans . However, little is known about the roles of additional family members in tomato resistance to P. infestans . In addition, there remains a significant gap in understanding the receptors of SpPIPs and the transcription factors (TFs) that regulate SpPIPs signaling in tomato defense, and the combination of SpPIPs signaling and TFs in defending against pathogens is rarely studied. This study demonstrates that the exogenous application of SpPIP‐LIKE1 (SpPIPL1) also strengthens tomato resistance by affecting the phenylpropanoid biosynthesis pathway. Both SpPIP1 and SpPIPL1 trigger plant defense responses in a manner dependent on RLK7L. Tomato plants overexpressing the precursors of SpPIP1 and SpPIPL1 (SpprePIP1 and SpprePIPL1) exhibited enhanced expression of pathogenesis‐related genes, elevated H 2 O 2 and ABA levels, and increased lignin accumulation. Notably, SpWRKY65 was identified as a transcriptional activator of SpprePIP1 and SpprePIPL1 . Disease resistance assays and gene expression analyses revealed that overexpression of SpWRKY65 (OEWRKY65) confers tomato resistance to P. infestans , while wrky65 knockout led to the opposite effect. Intriguingly, transgenic tomato studies showed that either spraying OEWRKY65 with SpPIPs or co‐overexpressing SpprePIP1 and SpWRKY65 further augmented tomato resistance, underscoring the potential of gene stacking in enhancing disease resistance. In summary, this study offers new perspectives on controlling late blight and developing tomato varieties with improved resistance. The results emphasize the potential of exogenous SpPIPs application as an eco‐friendly strategy for crop protection, laying a theoretical foundation for advancing crop breeding.