made use of, see Supplemental Table S19). For heterologous expression in yeast, the resulting constructs had been transformed into the engineered S. cerevisiae strain WAT11 (Pompon et al., 1996) making use of the S.c. EasyComp Transformation Kit (Invitrogen) in accordance with the manufacturer’s directions. Subsequently, 30- mL Sc-Leu minimal medium (6.7 g/L yeast N2 base without the need of amino acids, but with ammonium sulfate; 100 mg/L of each l-adenine, l-arginine, l-cysteine, l-lysine, l-threonine, ltryptophan, and uracil; 50 mg/L of every single l-aspartic acid, l-histidine, l-isoleucine, l-methionine, l-phenylalanine, l-proline, lserine, l-tyrosine, and l-valine; 20 g/L d-glucose) was inoculated with single yeast colonies and grown Caspase Inhibitor medchemexpress overnight at 28 C and 180 rpm. For key cultures, 100 mL YPGA (Glc) complete medium (ten g/L yeast extract, 20 g/L bactopeptone, 74 mg/L adenine hemisulfate, 20 g/L d-glucose) was inoculated with 1 unit OD600 with the overnight cultures and incubated beneath precisely the same circumstances for 305 h. Right after centrifugation (5,000 g, 16 C, five min), the expression was induced by resuspension of the cells in 100 mL YPGA (Gal)Cloning and heterologous expression of OMT genes in E. coliThe full open reading frames of FOMT2 (W22) and FOMT4 (W22) were amplified from cDNA obtained from B. maydis-infected W22 leaves with all the primer pairs listed in Supplemental Table S20. FOMT3 and FOMT5 were amplified from plasmids containing the synthesized codon-optimized open reading frames (see paragraph beneath). The PCR solutions had been cloned in to the expression vector pET100/DTOPO (Invitrogen, Waltham, MA, USA) or pASK-IBA37plus (IBA Lifesciences, Gottingen, Germany) and totally sequenced. BX10 (B73), BX11 (B73), BX12 (CML322), and BX14 (B73) have been offered as pASK-IBA37plus constructs by Vinzenz HSP90 Antagonist medchemexpress Handrick (Meihls et al., 2013; Handrick et al., 2016). For heterologous expression, the expression constructs were transferred in E. coli strain BL21 (DE3; Invitrogen). Liquid cultures had been grown in lysogeny broth at 37 C and 220 rpm until an optical density at 600 nm (OD600) of 0.8, induced with a final concentration of 1 mM IPTG or 200 mg/L anhydrotetracycline, and subsequently incubated at 18 C and 220 rpm for 15 h. The cells had been harvested by centrifugation at 5,000 g and four C for 10 min, resuspended in refrigerated extraction buffer (50 mM Tris Cl pH 8, 500 mM NaCl, 20 mM imidazole, 10 (v/v) glycerol, 1 (v/v) Tween20, and 25 U/mL Benzonase Nuclease (Merck, Kenilworth, NJ, USA;Formation of O-methylflavonoids in maizePLANT PHYSIOLOGY 2022: 188; 167|medium (see above, but which includes 20 g/L galactose rather of d-glucose) and grown for a further 158 h at 25 C and 160 rpm. The cells were harvested by centrifugation (7,500 g, ten min, 4 C), resuspended in 30 mL TEK buffer (50 mM Tris Cl pH 7.5, 1 mM EDTA, 100 mM KCl) and centrifuged again. Then, the cells have been meticulously resuspended in two mL TES buffer (50 mM Tris Cl pH 7.five, 1 mM EDTA, 600 mM sorbitol; freshly added: ten g/L bovine serum fraction V protein and 1.five mM b-mercaptoethanol) and glass beads (0.450.50 mm diameter; Sigma-Aldrich) had been added till they reached the upper level of the cell suspension. For cell disruption, the suspensions had been shaken by hand 5 instances for 1 min, with cooling on ice for 1 min in involving. The crude extracts have been recovered by washing the glass beads four times with five mL TES. The combined washes had been centrifuged (7,500 g, 10 min, four C), along with the supernatant containing the microsomes was transferred into an ultracentrifuge tu