Activity-based protein profiling in plants

摘要: Activity-based probes (ABPs) are reporter-tagged inhibitors that label enzymes in an activity-dependent manner. Using ABPs, activity-based protein profiling (ABPP) portrays active complex proteomes. A collection of ABPs was screened and characterized for labeling Arabidopsis leaf extracts tomato apoplastic fluids (AFs). We focused on four ABPs: epoxide probe DCG-04; fluorophosphonate FP; vinyl sulfone MV151; β-lactone IS4. First, we optimized the conditions identified targets. Second, performed comparative ABPP detected proteins whose activities differential during benzothiadiazole (BTH)-induced plant defenses pathogen infections. Third, competitive targets pathogen-derived chemically-synthesized inhibitors. The major findings as follows: (i) DCG-04, labeled seven papain-like cysteine proteases (PLCPs) AFs, found activity PLCP PIP1 is induced upon BTH treatment inhibited by Cladosporium fulvum effector AVR2. also C14 activated 0.03% SDS native AFs Phytophthora infestans EPIC1/2B. (ii) FP, showed diversity serine hydrolase unchallenged Botrytis cinerea-infected plants. differentials treatment. (iii) MV151, three catalytic β subunits proteasome, selective inhibition aldehyde-based discovered a posttranslational, NPR1-dependent upregulation proteasome Arabidopsis. (iv) While characterizing IS4 extracts, occurs at N-terminus chloroplast PsbP through peptide bond requires RD21. This finding eventually led us to discovery RD21 acts ligase vitro. In conclusion, demonstrated powerful tool dynamically track plants, which facilitates functional analysis enzymes.