Horseradish peroxidase

Leaf discs (4-mm diameter) were excised from leaves of 6-week-old plants and incubated in water overnight. Leaf discs (n = 6) were placed in a 96-well plate (1 disc/well, Greiner, Kremsmünster, Austria) containing 18 μg/mL luminol L-012 (Wako, Osaka, Japan) and 18 μM horseradish-peroxidase (AppliChem, Darmstadt, Germany). flg22 (Biomatik, Kitchener, Canada) was added to yield indicated concentrations. Luminescence was recorded over time using a TriStar² S LB 942 plate reader (Berthold Technologies, Bad Wildbad, Germany). flg22 peptide was kindly provided by Georg Felix. Peptide was dissolved in water and diluted in a solution containing 0.1% BSA and 0.1 M NaCl.

The ROS burst in leaves was determined as described previously (de Torres Zabala et al., 2015 (link); Chang et al., 2020 (link); Yan et al., 2021 (link)). In tomato, flg22 was used to instead of Xanthomonas to measure the ROS burst (Bhattarai et al., 2016 (link)). Similar methods were applied to study the cassava resistance to Xam, such as MeCAMTA3 (Chang et al., 2020 (link)), MeRAV5 (Yan et al., 2021 (link)). Herein, to measure the ROS burst, 48 leaf discs (5 mm in diameter) of cassava were placed in 48 single wells of 96-well black plates and placed in the dark for 12 h in 100 μL double-distilled water. After 12 h, the 48 leaf discs were divided into two groups. In one group, the water was replaced with 100 μL incubation solution containing 0.2 μmol/L luminol (AppliChem, Darmstadt, Germany) and 10 μg/mL horseradish peroxidase (AppliChem, Darmstadt, Germany). In the other group, the water was then replaced with 100 μL incubation solution containing 0.2 μmol/L luminol, 10 μg/mL horseradish peroxidase and 1 μmol/L flg22 (Phyto Technology Laboratories, Lenexa, KS, United States). Luminescence was measured immediately for 30 min using a GloMax 96 Microplate Luminometer (Promega, Madison, WI, United States). Luminescence readout is given in relative light emitting units (RLU).