Luminol

To examine ROS burst elicited by VmNIS1 recombinant protein, Pep13 (peptide derived from VmNIS1) or flg22 PAMP, leaf disks (diameter 0.5 cm) were collected from healthy N. benthamiana leaves using a cork-borer set. To examine suppression of flg22 PAMP-induced ROS burst, leaf disks were collected from N. benthamiana leaves transiently expressing VmNIS1, VmNIS2 or GFP. The disks were put in a 96-well plate and floated in ultra-pure distilled water overnight. Prior to luminescence measurement with a Varioskan LUX multimode microplate reader (Thermo Scientific), water in each well of the plate was replaced with 100 μL reaction solution containing 100 μM luminol (Solarbio, Beijing, China), 20 μg mL⁻¹peroxidase (Solarbio, Beijing, China), and 1 μM VmNIS1 purified protein or flg22 peptide. Eight biological replicates were used each time. The experiments were repeated three times with similar results. Pep13 peptide was synthesized by Sangon Biotech (Shanghai, China), and flg22 peptide was purchased from Genscript Biotech (Nanjing, China).

N, N-dimethylformamide (DMF) was purchased from Aladdin Chemical Co. Ltd. (Shanghai, China). Hemin, luminol, 20× phosphate-buffered saline (pH 7.4), 1M Tris-HCL (pH 7.4) and 1M Tris-HCL (pH 9.0) were purchased from Solarbio Life Sciences (Beijing, China). The 1 mM Hemin and 30 mM luminol were prepared in DMF and stocked in the dark at 4 °C. H₂O₂ (30%), methanol (HPLC grade), magnesium chloride hexahydrate and potassium chloride were obtained from Beijing Jingri Jindian Technology Co., Ltd. (Beijing, China). Zearalenone (ZEN), aflatoxin B₁ (AFB₁), deoxynivalenol (DON) and ochratoxin A (OTA) were ordered from Meizheng Bio-Tech (Beijing, China). Costar 96-well white flat-bottom microplates were purchased from Corning (Beijing, China). All the oligonucleotides were synthesized by Tsingke Biotechnology (Beijing, China) and prepared as stocks of 10 μM in nuclease-free water.

Isothiocyanobenzyl-EDTA (ITCBE) was purchased from Dojindo (Kyushu, Japan). N, N’-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), dimethyl formamide (DMF), 3, 3’, 5, 5’-tetramethylbenzidine (TMB) and luminol were purchased from Solarbio (Beijing, China). HAT medium, keyhole hemocyanin (KLH) and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). Goat anti-mouse IgG-HRP was purchased from Thermo (Waltham, MA, USA). Pb(NO₃)₂, HgSO₄, 3CdSO₄·8H₂O, Cr₂(SO₄)₃·6H₂O, CuSO₄, CoCl₂·6(H₂O), NiSO₄.6H₂O, ZnSO₄·7H₂O and FeSO₄·7H₂O were purchased from Sinopharma chemical reagent (Shanghai, China). OriginPro 8.1 (OriginLab, Northampton, MA, USA) was used for processing the analytical data.