Phosphorylation profiling of systemin versus MAMP signaling reveals signaling specificity in plant immunity

Plasma membrane-localized pattern recognition receptors (PRRs) detect both microbe-associated molecular patterns (MAMPs) such as flg22 and chitin, and host-derived signals like systemin. While early signaling events (phosphorylation, ROS burst, MAPK activation) are largely similar, they lead to different biological outcomes: • MAMPs activate innate immunity, • Systemin induces wound and herbivore defense responses. To investigate how specificity arises, phospho-proteomics was applied in tomato cells (wild type vs. systemin receptor-deficient). In total, 4701 phospho-sites on 2119 proteins were identified. Using imputation and clustering, seven temporal phosphorylation patterns were observed: • Systemin uniquely caused dephosphorylation at 1–15 min, • flg22 triggered an early rapid phosphorylation response, • Chitin mainly induced late phosphorylation events. Specificity was illustrated by three protein groups: 1. RLCK/PBL kinases → different family members respond in an elicitor-specific way. 2. S-type anion channel SLAC1 → carries a single regulatory site, differentially phosphorylated depending on the elicitor. 3. Phosphatase PLL1a → has multiple elicitor-specific phospho-sites (flg22: Ser50–59, chitin: Ser86–89, systemin: Ser163–181). Network analysis suggested that PLL1a regulates RBOHs, which was functionally validated using phospho-mimic/dead PLL1 mutants in Nicotiana.