Peroxidase pod assay kit

R. solani AG1IA cultures were treated using the methods described in Section 2.9. A Superoxide Dismutase (SOD) Assay Kit (A001-3, Nanjing Jiancheng Bioengineering Institute) was utilized to test SOD activities. A Peroxidase (POD) Assay Kit (Cat#BC0090, Beijing Solarbio Science & Technology Co., Ltd.) was utilized to test POD activities. A Cytochrome P450 Enzyme Assay Kit (A114608, Shanghai Fusheng Industry Co., Ltd.) was utilized to test P450 function.

The CDSs of Sspep1 and pod-1a were amplified by PCR using the corresponding primer pairs pGEX-pep1-BamHI-F/pGEX-pep1-BamHI-R and pET30a-pod-BamHI-F/pET30a-pod-BamHI-R (Table S1) with cDNA generated from the whit part of a whip of infected sugarcane plants serving as a template. The fragments were cloned into the prokaryotic expression vectors pGEX4-1 and pET30a to yield the constructs pGEX4-Sspep1 and pET-Pod-1a. The plasmids were transformed into BL21 Competent Cells (Vazyme, Nanjing, China), and the expressed recombinant proteins were purified using Ni–NTA Agarose beads or GST-tag Purification Resin (both from Beyotime, Shanghai, China). In vitro horseradish peroxidase (HRP) activity was assayed by the Peroxidase (POD) Assay Kit (Solarbio, Beijing, China).

Hyphae (0.1 g) were soaked in 20 mL of 400 μg/mL Ag-NP solution for different time intervals (2 h, 4 h, 6 h, 8 h, and 10 h). The hyphae were rinsed with distilled water for 3 times and dried with adsorbent paper. The fresh hyphae were ground in liquid nitrogen and homogenized with buffers in an ice bath to extract superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) enzymes. Then, the homogenate was placed in a centrifuge machine at 4 °C and 8000× g r/min for 10 min. SOD was detected using a superoxide dismutase (SOD) assay kit (Code No. BC0170; Solarbio, Beijing, China); POD was detected using a peroxidase (POD) assay kit (Code No. BC0090; Solarbio, Beijing, China); and CAT was detected using a catalase (CAT) assay kit (Code No. BC0200; Solarbio, Beijing, China) according to the manufacturer’s guidelines.