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

Abstract

ABSTRACTThe extracellular space of plant tissues contains hundreds of hydrolases that might harm colonizing microbes. Successful pathogens may suppress these hydrolases to enable disease. Here, we report the dynamics of extracellular hydrolases in leaves upon infection withPseudomonas syringae.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). The activity of 82 of these hydrolases (mostly SHs) increases during infection, whilst the activity of 60 hydrolases (mostly GHs and CPs) is suppressed during infection. Active β-galactosidase-1 (BGAL1) is amongst the suppressed hydrolases, consistent with production of the BGAL1 inhibitor byP. syringae. One of the other suppressed hydrolases, the pathogenesis-relatedNbPR3, decreases bacterial growth when transiently overexpressed. This is dependent on its active site, revealing a role forNbPR3 activity in antibacterial immunity. Despite being annotated as a chitinase,NbPR3 does not possess chitinase activity, and contains a E112Q active site substitution that is essential for antibacterial activity and is conserved only inNicotianaspecies.This study introduces a powerful approach to reveal novel components of extracellular immunity, exemplified by the discovery of the suppression of neo-functionalisedNicotiana-specificantibacterialNbPR3.