Biostack ready

The MdWRKY87 ORF sequence without stop codon was cloned into the pBD-VP16 vector (Han et al., 2016 (link)). The reporter vector contained a GAL4-luciferase (LUC) containing five copies of the GAL4-binding element and a minimal CaMV35S promoter at the 5’ end of the LUC gene (Han et al., 2016 (link)).The effector vectors or reporter vectors were introduced into A. tumefaciens strain GV3101. The A. tumefaciens cells was re-suspended to an OD₆₀₀ of 1.0 using the buffer (10 mM MgCl₂, 10 mM MES-KOH, pH 5.6; 200 μM acetosyringone). A. tumefaciens cells harboring effector vector and reporter vector were mixed 1:1, then injected into the tobacco (N. benthamiana) leaves by using a 1 mL needleless syringe. After spraying 1 mM luciferin onto the leaves, luciferase imaging was performed using NEWTON 7.0 (VILBER LOURMAT, Paris, France). An assessment of LUC and REN activities was conducted using the Duo-Lite Luciferase Assay System (DD1205–01, Vazyme, Nanjing, China) and BioStack Ready (BioTek Instruments Inc., Winooski, Vermont, USA). LUC/REN ratio was used to calculate the results. The primers used for construction are listed in Supplemental Table S1.

The stock solutions of peptides (2 mg/mL) were prepared in DMSO. Specific amounts of peptide from stock solution were added to a 96-well microtiter plate containing 100 μL of growth media and then serially diluted two-fold. A further 100 μL of growth media containing microbial cells were added to each well and incubated at 30–37 °C for 24–48 h. The optical density (OD) was measured at 600 nm using a BioStack Ready (BioTek Instruments, Winooski, VT, USA) microplate reader to analyze microbial growth inhibition, and IC₅₀ and MIC values were calculated. To visualize the growth and inhibition pattern, a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in water was added to the plate and incubated for 2–3 h. MTT reacts with the mitochondrial dehydrogenases, which are present only in a viable cell, to give blue colored formazan crystals. Therefore, a darker color indicates a high number of viable cells; however, non-viable cells appear pale or colorless.