Peroxidase

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.

Secondary antibodies for immunofluorescence microscopy: Chicken anti‐mouse immunoglobulins conjugated to Alexa488 (Invitrogen, Carlsbad, CA, USA, #A21200). Donkey anti‐mouse immunoglobulins conjugated to Alexa568 (Invitrogen, #A10037). Donkey anti‐rabbit immunoglobulins conjugated to Alexa568 (Invitrogen, #A10042). Chicken anti‐rabbit immunoglobulins conjugated to Alexa488 (Invitrogen, #A21441). Goat anti‐mouse immunoglobulins conjugated to Alexa647 (Invitrogen, #A21236). Monovalent Fab fragments rabbit anti‐mouse [unconjugated; Fab rabbit anti‐mouse IgG (H&L); Rockland Immunochemicals, #810‐4102 via Biomol GmbH, Hamburg, Germany], used for immunofluorescence microscopy in a 1:50 dilution. Chicken anti‐goat immunoglobulins conjugated to Alexa594 (Invitrogen, #A21468). Secondary antibodies for Western blot analyses: goat anti‐rabbit immunoglobulins conjugated to peroxidase (Sigma, #A6154), used in a 1:5,000 dilution for Western blot analyses; goat anti‐mouse immunoglobulins conjugated to peroxidase (Sigma, #A3673), used in a 1:5,000 dilution for Western blot analyses.

Flg22-triggered ROS generation was monitored as described earlier (Felix et al., 1999 (link)). ROS production was induced with 200 nM flg22 and measured with help of a luminol/peroxidase solution [250 μM luminol (Sigma), 10 μg/ml peroxidase (Sigma)]. Apoplastic fluid derived from N. benthamiana plants that produced dspFo3, spFoa3 or spGFP was infiltrated into leaves of 4–5-week-old N. benthamiana plants and 3 h later leaf discs were excised and kept overnight on MQ. Flg22-triggered ROS production was measured the next morning.
Flg22-triggered MAPK activation was performed as described previously, with a few modifications (Flury et al., 2013 (link)). Three days after Agro-infiltration, N. benthamiana leaves were infiltrated with 0.5 μM flg22 or MQ and 10 min later leaf discs corresponding to 15 mg leaf material were collected and immediately frozen in liquid nitrogen. Total protein was isolated as described earlier and MAPK activation was monitored by Western blot using an anti-p44/p42 MAPK antibody (D13.14.4E; Cell Signaling Technology). Apoplastic fluid derived from N. benthamiana plants that produced dspFo3, spFoa3 or spGFP was infiltrated into leaves of 4–5-week-old N. benthamiana plants and 3 h later 1 μM flg22 or MQ was infiltrated. Ten minutes after flg22 treatment, leaf discs were collected, frozen in liquid nitrogen, and processed as described above.