Flg22

Rice protoplasts preparation and transformation were conducted according to the method of Zhang et al., 2011 (link). The sheaths and stems of 30–40 seedlings were cut into 0.5 mm strips and incubated immediately in 10 mL enzyme solution for 4–5 h in the dark at 25 °C with gentle shaking (60 rpm). The protoplasts were purified and resuspended in 1–2 mL MMG solution at a concentration of 5 × 10⁶ cells mL⁻¹. Plasmids (5–10 μg) prepared by an EndoFree Plasmid Midi Kit (CWBIO, Beijing, China) were used for transfection. For the protein degradation assay, DMSO (mock) or 20 μM MG132 (Millipore), H₂O (0 μM), 1, 5, and 25 μM E‐64 (Sigma‐Aldrich) or Leupeptin (Sigma‐Aldrich) were added to the protoplasts 12 or 4 h after transfection and treated for 4 or 12 h. For the time course treatment, 20 μM MG132 and/or 50 μM cycloheximide (CHX, Sigma‐Aldrich) were added to the protoplasts 12 h after transfection and treated for 2 h, 4 h, and 6 h. For pathogen mimic treatment, H₂O (0 μM), 0.5, 1, 2, and 5 μM Flg22 (Sangon Biotech) were added to the protoplasts 16 h after transfection and treated for 2 h. Transfection experiments were repeated at least three times.

Reactive oxygen species (ROS) production was monitored with an L-012/peroxidase-based assay on leaf discs collected from N. benthamiana, soybean, tomato, and Arabidopsis plants. The leaf discs were floated overnight on 200 μL of ddH₂O in a 96-well plate. The ddH₂O was replaced with a working solution [20 μM L-012 (Waco), 20 μg/mL peroxidase (Sigma-Aldrich), 1 μM flg22 (Sangon), or 1 μM purified protein reaction solution]. After the addition of the working solution, the plate was immediately moved to a GLOMAX96 microplate luminometer (Promega, Madison, WI, USA) for measurement of luminescence.
ROS detection in soybean leaves in response to zoospores of different P. sojae strains was monitored with an L-012 /peroxidase-based assays as previous reported with some minor modifications^{63 (link)}. Briefly, Leaf discs was collected from 2-week-old soybean plants and then floated overnight on 200 μL of ddH₂O in a 96-well plate. The ddH₂O was replaced with a working solution [20 μM L-012 (Waco), 20 μg/mL peroxidase (Sigma-Aldrich), zoospores (10000 zoospores per mL) of WT, PsRLK6-knockout (ΔPsRLK6) or PsRLK6-GFP overexpression transformants (OT-24). After the addition of the working solution, the plate was immediately moved to a GLOMAX96 microplate luminometer (Promega, Madison, WI, USA) for measurement of luminescence.

The indicated constructs were transiently expressed in N. benthamiana leaves by agroinfiltration for 48 h. The leaf disks were collected and incubated overnight with 200 µL ultra-pure distilled water in a 96-well white plate to eliminate the wounding effect. Water was replaced by 100 µL of a reaction solution containing 100 µg/mL luminol (Sigma) and 1 mg/mL horseradish peroxidase (Sigma) supplemented with 1µM flg22 (Sangon). Luminescence was measured with a microplate luminometer (TECAN, Männedorf, Switzerland) for a period of 40 min. ROS production was indicated as means of relative light units (RLU).