Identifying apoplastic plant proteins involved in plant-pathogen interactions on the basis of their evolutionary profile

To sustainably feed a growing global population, yield can be increased by reducing crop losses to plant pathogens. During infection of plants, several pathogens attempt to colonise the intercellular space, the apoplast. Apoplastic defence against attempted invasions includes the secretion of defence-related proteins, turning the apoplast into a molecular battlefield. While few apoplastic hydrolases have been characterised in plant immunity, many secreted proteins remain uncharacterised. Thus, in order to improve our understanding of apoplastic plant defence responses, we aimed to identify novel apoplastic proteins involved in immunity in this thesis. We combined comparative proteomics with molecular evolution and bioluminescence-based high-throughput disease assays. First, to identify core extracellular tomato proteins in immune responses, we studied apoplastic fluids (AF) of different biotic stress-induced tomato species S.lycopersicum, S.peruvianum and S.pennellii by mass spectrometry (MS). We identified that P69 subtilases were the most abundant proteins in tomato AFs during defence. Molecular evolution studies further implicated P69 subtilases in plant immunity and implicated another 20 biotic stress-induced tomato proteins under positive selection in plant immunity. Second, we compared apoplastic proteomic landscapes between tomato species and N.benthamiana, G.max and A.thaliana which marked distinct differences in profiles of accumulating defence proteins, implicating subtilases from G.max and chitinases from tomato species in plant immunity. Third, we established a bioluminescence-based, high-throughput disease assay for the N.benthamiana-Pseudomonas syringae pv. tomato (Pto) pathosystem, but failed to identify increased immunity in N.benthamiana against Pto for three proteins implicated in plant immunity, including P69B. Last, we used AlphaFold2 multimer as a high-throughput in silico screening tool to identify inhibitor-enzyme complexes from the plant-pathogen interface and identified 13 novel, putative hydrolase inhibitors, including six putative P69B inhibitors. This thesis has contributed to understanding apoplastic defence responses by identifying proteins implicated in plant immunity.