DCG-04 is a biotinylated derivative of cysteine protease inhibitor E-64, which irreversibly reacts with papain-like cysteine proteases (PLCPs) when these proteases are active. Using DCG-04, seven active proteases are labelled in Arabidopsis leaf extracts. Of these, RD21 (responsive to desiccation-21) was found to have increased activity during the infection with avirulent Pseudomonas syringe in Arabidopsis cell cultures. Infection with a virulent strain caused post-translational suppression of RD21 activity. These data suggest that RD21A plays a role in defence. We therefore challenged single, double and triple knock-out lines of RD21-like proteases with several pathogens and detected an altered susceptibility for Botrytis cinerea, but not the other pathogens tested. Presumably because adapted pathogens might use inhibitors that make them insensitive for RD21A. As an alternative reverse genetic approach, we silenced the RD21 orthologs of Nicotiana benthamiana using virus-induced gene silencing with Tobacco Rattle Virus (TRV)-based silencing vectors. NbRd21 silencing resulted in retarded growth and spreading cell death, most likely triggered by a combination of NbRd21 silencing and TRV presence. Interestingly, silencing of autophagy-related genes, ATG3 and ATG6, pheno-copied NbRD21 silencing. Furthermore, DCG-04 activity profiling assay showed the suppression of NbRD21 activity and up-regulation of NbRd21 transcript in ATG3 (and ATG6) silenced plants, which implies a connection between RD21, cell death and autophagy. To identify other defence-related PLCPs, we applied benzothiadiazole (BTH) to trigger the salicylic acid�regulated defence pathway in tomato. Of the seven PLCPs tested, transcription of only PIP1 and RCR3 were induced. Sequencing of PLCP alleles of tomato relatives revealed that same proteases, PIP1 and RCR3, are under diversifying selection, resulting in variant residues around the substrate binding groove. Taken together these data indicate that some PLCPs are involved in plant-pathogen interactions
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