Glutathione transferase photoaffinity labeling demonstrates GST activation by safeners and NPR1-independent activation by BTH

Abstract

ABSTRACTGlutathione transferases (GSTs) represent a large and diverse enzyme family involved in detoxification of small molecules by glutathione conjugation in crops, weeds and model plants. Here, we introduce an easy and quick assay for photoaffinity labeling of GSTs to study global GST activation in various plant species. The small molecule probe contains glutathione, a photoreactive group, and a minitag for coupling to reporter tags via click chemistry. Under UV irradiation, this probe quickly and robustly labels GSTs in crude protein extracts. Enrichment and MS analysis of labeled proteins from Arabidopsis identified ten GSTs from the Phi(F) and Tau(U) classes. Photoaffinity labeling of GSTs demonstrated GST activation in wheat seedlings upon treatment with safeners, and in Arabidopsis leaves upon infection with avirulent bacteria. Photoaffinity labeling and proteomics identified six Phi- and Tau-class GSTs that are induced upon treatment with salicylic acid (SA) analog benzothiadiazole (BTH) and these were tested for enhancing immunity in disease assays. Our data confirm that BTH-induced GST activation is independent of NPR1, the master regulator of SA signaling.