P-370 - Investigating the P69 Proteases of Tomato: Evidence for an Evolutionary Arms Race with Pathogen Effectors?

The apoplast is a crucial battleground in plant immunity, as many pathogens attempt to invade and colonize this extracellular space. To defend against them, plants pack this compartment with various defence-related proteins, including hydrolases of different classes. The P69 family of subtilisin-like serine proteases is a key group of these hydrolases, present in Solanum plants. In tomato (Solanum lycopersicum), the P69 family consists of 10 genes, all clustered within a single locus as adjacent, intron-less genes. These proteases are abundant in the apoplast and become transcriptionally activated upon infection by diverse pathogens, including Pseudomonas syringae, Cladosporium fulvum, and Phytophthora infestans. A recent AlphaFold Multimer screen identified P69B as a potential target of at least four distinct pathogen-secreted inhibitors. Bioinformatics analysis and structural modelling shows that despite high sequence similarity of P69s, there is high variability in residues surrounding the substrate binding groove, suggesting that these residues might be under evolutionary pressure to diversify. To investigate this further, we expressed and purified tomato P69s and assessed their interactions with the identified pathogen inhibitors in vitro. The uncovered differential binding abilities support the concept that the P69 family may be engaged in an evolutionary arms race with pathogen-secreted proteins in tomato and likely other Solanum species.