The interaction between the fungal pathogen Cladosporium fulvum and its host tomato (Solanum lycopersicum) is an ideal model to study suppression of extracellular host defenses by pathogens. Secretion of protease inhibitor AVR2 by C. fulvum during infection suggests that tomato papain-like cysteine proteases (PLCPs) are part of the tomato defense response. We show that the tomato apoplast contains a remarkable diversity of PLCP activities with seven PLCPs that fall into four different subfamilies. Of these PLCPs, transcription of only PIP1 and RCR3 is induced by treatment with benzothiadiazole, which triggers the salicylic acid-regulated defense pathway. Sequencing of PLCP alleles of tomato relatives revealed that only PIP1 and RCR3 are under strong diversifying selection, resulting in variant residues around the substrate binding groove. The doubled number of variant residues in RCR3 suggests that RCR3 is under additional adaptive selection, probably to prevent autoimmune responses. AVR2 selectively inhibits only PIP1 and RCR3, and one of the naturally occurring variant residues in RCR3 affects AVR2 inhibition. The higher accumulation of PIP1 protein levels compared with RCR3 indicates that PIP1 might be the real virulence target of AVR2 and that RCR3 acts as a decoy for AVR2 perception in plants carrying the Cf-2 resistance gene.

Papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes associated with development, immunity, and senescence. Although many properties have been described for individual proteases, the distribution of these characteristics has not been studied collectively. Here, we analyzed 723 plant PLCPs and classify them into nine subfamilies that are present throughout the plant kingdom. Analysis of these subfamilies revealed previously unreported distinct subfamily-specific functional and structural characteristics. For example, the NPIR and KDEL localization signals are distinctive for subfamilies, and the carboxyl-terminal granulin domain occurs in two PLCP subfamilies, in which some individual members probably evolved by deletion of the granulin domains. We also discovered a conserved double cysteine in the catalytic site of SAG12-like proteases and two subfamily-specific disulfides in RD19A-like proteases. Protease activity profiling of representatives of the PLCP subfamilies using novel fluorescent probes revealed striking polymorphic labeling profiles and remarkably distinct pH dependency. Competition assays with peptide-epoxide scanning libraries revealed common and unique inhibitory fingerprints. Finally, we expand the detection of PLCPs by identifying common and organ-specific protease activities and identify previously undetected proteases upon labeling with cell-penetrating probes in vivo. This study provides the plant protease research community with tools for further functional annotation of plant PLCPs.

Localized control of cell death is crucial for the resistance of plants to pathogens. Papain-like cysteine proteases (PLCPs) regulate plant defence to drive cell death and protection against biotrophic pathogens. In maize (Zea mays), PLCPs are crucial in the orchestration of salicylic acid (SA)-dependent defence signalling. Despite this central role in immunity, it remains unknown how PLCPs are activated, and which downstream signals they induce to trigger plant immunity. Here, we discover an immune signalling peptide, Z. mays immune signalling peptide 1 (Zip1), which is produced after salicylic acid (SA) treatment. In vitro studies demonstrate that PLCPs are required to release bioactive Zip1 from its propeptide precursor. Conversely, Zip1 treatment strongly elicits SA accumulation in leaves. Moreover, transcriptome analyses revealed that Zip1 and SA induce highly overlapping transcriptional changes. Consequently, Zip1 promotes the infection of the necrotrophic fungus Botrytis cinerea, while it reduces virulence of the biotrophic fungus Ustilago maydis. Thus, Zip1 represents the previously missing signal that is released by PLCPs to activate SA defence signalling.