Flg22

N. benthamiana leaves were agroinfiltrated with desired constructs for transient protein expression. 36 hours after infiltration, either water control or 100nM flg22 (PhytoTechnology Laboratories, Shawnee Mission, USA) were injected to induce NbAcer31 and NbWRKY22 expression. Total RNA from agroinfiltrated leaf discs treated with flg22 for 1 hour was isolated with TRIzol reagent (Invitrogen, Carlsbad, USA). One microgram of total RNA was treated with DNase I (Invitrogen, Carlsbad, USA), followed by reverse transcription using a Super Script II reverse transcriptase (Invitrogen, Carlsbad, USA). qRT-PCR analysis was performed on an ABI Prism 7100 sequence detection system using Power SYBR Green reagents (Life Technologies, Carlsbad, USA). The N. benthamiana EF1 gene was used as an internal control for normalization [43 (link)]. Relative expression ratios were determined based on the comparative CT method (ΔΔCT) using the StepOne Software. Primers used in qRT-PCR are listed in S1 Table.

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).