Trospray ionization (ESI) mass spectra in positive and unfavorable ion modes in the selection of 100000 mz, respectively. Total ICN, 4OH-ICN, and camalexin amounts had been quantified applying regular curves of requirements ready in cyp79B2 cyp79B3 seedling extract and integrated places inside the UV chromatographs at 260 nm for 4M-I (retention time [RT] = 7.7 min); 340 nm for ICN (RT = 11.5 min); 280 nm for ICN degradation item L-Prolylglycine site ICA-ME (RT = 9.five min), and co-eluting 4OH-ICN degradation items 4OH-ICA and 4OH-ICA-ME (RT = ten.1 min); and 320 nm for camalexin (RT = 12.1 min). For Fig. 1b, total camalexin amounts were quantified using integrated places inside the FLD chromatograph. Forsome experiments, 2.five L 200 M indole butrytic acid (IBA; RT = ten.1 min) was added per mg sample dry weight alternatively of 4M-I. Relative amounts of ICN, 4OHICN, and amounts have been quantified by dividing the peak locations at mz 169 [M-H]- (ICN), 174 [M-H]- (ICA-ME), 176 [M-H]- (4OH-ICA), 190 [M-H]- (4OHICME), and 201 [M + H]+ (camalexin), by the peak region at mz 202 [M-H]- (IBA).Glucosinolate extraction and LC-DAD-FLD-MS. For glucosinolate extraction, a 96-well 0.45 m polyvinylidene fluoride (PVDF) filter plate (EMD Millipore, Billerica, MA) was charged with 45 mg DEAE Sephadex A25 (GE Heathcare) and 300 L of water per effectively, and equilibrated at space temp for two h. Prior to sample homogenization, the plate was centrifuged at 400 g for 1 min to remove the water. The homogenate was extracted with 500 L 70 (vv) aqueous methanol at 67.5 for ten min and centrifuged at 16,000 g for 2 min. Added to the supernatant was 3 L of IS (1.25 mM sinigrin (Sigma-Aldrich) in 80 (vv) ethanol) per mg sample dry weight. Extract was applied to and incubated around the ion exchanger for ten min. The sephadex resin was washed three instances with 70 (vv) methanol, three times with distilled deionized water (ddH2O), and two instances with 20 mM sodium acetate (pH 5). Twenty microliters of 25 mgmL aryl sulfatase (Sort H1 from Helix pomatia, Sigma-Aldrich) was applied to and incubated around the sephadex resin at RT overnight. The plate was centrifuged at 400 g for 1 min and desulfoglucosinolates had been eluted from the sephadex resin by two one hundred L washes with 60 (vv) methanol and two one hundred L washes with ddH2O. Eluate volume was decreased to 25050 L employing an evaporator. Samples were separated on an Ultimate 3000 HPLC system, employing a three.5 m, three 150 mm Zorbax SB-Aq column together with the gradient shown in Supplementary Table 7. A coupled DAD-3000RS diode array detector, FLD-311 fluorescence detector, and MSQPlus mass spectrometer collected UV absorption spectra at 229 nm, fluorescence spectra at 275350 nm (exem), and ESI mass spectra in optimistic negative ion modes at 100000 mz, respectively. Glucosinolates had been quantified applying integrated locations of desulfoglucosinolates within the UV chromatographs at 229 nm and published response factors73.ChIP and PCR. For ChIP experiments on wrky33DEX:WRKY33-flag nuclear extracts, roughly two hundred and ten 9-day-old seedlings have been inoculated with Psta to OD600 of 0.013 and co-treated with mock remedy of dimethyl sulfoxide (M) or 20 M dex (D) for 9 h. Total protein was extracted in 25 mL of Extraction buffer 1 (0.four M sucrose, ten mM Tris-Cl [pH 8], ten mM MgCl2, five mM 2mercaptoethanol, 0.1 mM AEBSF, Comprehensive EDTA-free protease inhibitor cocktail [Roche]. After a 10 min fixing step with 1 (vv) formaldehyde Fenamic acid supplier resolution in addition to a five min quenching step with 2 M glycine, seedlings had been washed three instances with deionized wa.
