P-355 - Characterising hydrolase-inhibitor interactions in the apoplast

The apoplast represents the first line of defence against many colonising pathogens. In response to pathogen perception, plants secrete hundreds of hydrolases into the apoplast that can harm invading microbes. Successful microbes deploy counter-defence mechanisms, including the secretion of small secreted proteins (SSPs) that inhibit, and thus suppress, the activity of apoplastic hydrolases (Sueldo et al., 2023). Activity-based proteomics has determined that at least 60 of the detectable hydrolases in N. benthamiana during infection are suppressed by the bacterial plant pathogen Pseudomonas syringae. How the activity of these hydrolases are suppressed, and by what, has so far remained elusive. Using Alphafold Multimer (AFM), we conducted a large screen of interactions between 211 predicted P. syringae SSPs and 48 activity-suppressed hydrolases in the apoplast. Top SSPs were screened for their potential to block the active site of apoplastic hydrolases and tested in vitro. Here we report the results of a transient overexpression screen of suppressed hydrolases and their effect on virulence of P. syringae DC3000 (ΔhQ) in planta. We also present the characterisation of one AFM-identified complex between an abundant hydrolase (Pathogenesis Related 3; PR3) and a P. syringae SSP, demonstrating the power of AI-based predictions of protein-protein interactions in the apoplast and its potential to accelerate the engineering of resistant crops to pathogens.