Activity-Profiling of Vacuolar Processing Enzymes and the Proteasome during Plant-Pathogen Interactions

Programmed cell death (PCD) is an interesting natural phenomenon, common to many organisms. PCD has been extensively studied during animal apoptosis and several regulators have been identified. Cysteine proteases called caspases, and the proteasome were found to be main players in PCD in animals. In plants, PCD is regulated by vacuolar processing enzymes (VPEs) and the proteasome. Both proteolytic machineries exhibit caspase-like activities. In this work, the activity of VPEs and the proteasome were characterized using activity-based protein profiling (ABPP). ABPP involves fluorescent or biotinylated probes that react with the catalytic residue of proteases in an activity-dependent manner. Specific probes that target γVPE, the most abundant VPE in vegetative tissue, were selected from screen with legumain probes. Further characterization of γVPE activity revealed an unexpected, post-transcriptional up-regulation of γVPE activity during compatible, but not during incompatible interactions of Arabidopsis with Hyaloperonospora arabidopsidis (Hpa). Sporulation of Hpa was reduced in the absence of VPEs indicating that VPEs promote pathogen fitness. These findings introduce a new tool to study VPEs and reveal a new role of VPEs during compatible interactions. New, selective probes that target the plant proteasome are also introduced in this thesis. The proteasome is a multi-subunit proteolytic complex containing three subunits with different catalytic activities: β1, β2 and β5. ABPP was applied to further characterize the inhibition of the plant proteasome by Syringolin A (SylA), a non-ribosomal cyclic peptide produced by the bacterial pathogen Pseudomonas syringae pv. syringae. This work shows that SylA preferentially targets β2 and β5 of the plant proteasome. Structure-activity analysis revealed that dipeptide tail of SylA contributes to β2 specificity and identified a nonreactive SylA derivative. The selectivity of SylA for the catalytic subunits is discussed and the subunit selectivity is explained by crystallographic data. Importantly, it was discovered that SylA production promotes colonization of distant tissue by Pseudomonas syringae pv. syringae. SylA was found to suppress both effector-triggered immunity and salicylic acid-dependent acquired resistance. Distant colonization is a new phenomenon, common to other P. syringae strains, and undetected by classical pathogen assays