Activity-based proteomics uncovers suppressed hydrolases and a neo-functionalised antibacterial enzyme at the plant-pathogen interface

center dot The extracellular space of plant tissues contains hundreds of hydrolases that might harm colonising microbes. Successful pathogens may suppress these hydrolases to enable disease. Here, we report the dynamics of extracellular hydrolases in Nicotiana benthamiana upon infection with Pseudomonas syringae. center dot Using activity-based proteomics with a cocktail of biotinylated probes, we simultaneously monitored 171 active hydrolases, including 109 serine hydrolases (SHs), 49 glycosidases (GHs) and 13 cysteine proteases (CPs). center dot The activity of 82 of these hydrolases (mostly SHs) increases during infection, while the activity of 60 hydrolases (mostly GHs and CPs) is suppressed during infection. Active beta-galactosidase-1 (BGAL1) is amongst the suppressed hydrolases, consistent with production of the BGAL1 inhibitor by P. syringae. One of the other suppressed hydrolases, the pathogenesis-related NbPR3, decreases bacterial growth when transiently overexpressed. This is dependent on its active site, revealing a role for NbPR3 activity in antibacterial immunity. Despite being annotated as a chitinase, NbPR3 does not possess chitinase activity and contains an E112Q active site substitution that is essential for antibacterial activity and is present only in Nicotiana species. center dot This study introduces a powerful approach to reveal novel components of extracellular immunity, exemplified by the discovery of the suppression of neo-functionalised Nicotiana-specific antibacterial NbPR3.