Chitin

Binding capacity of C. glabrata cells to fungal cell wall components and extracellular matrix (ECM) collagen was also evaluated by flow cytometry following a similar procedure as described above for 4 h adhesion to polystyrene, using the same amount of cells. However, before incubation with C. glabrata, the microtiter plates were coated with chitin (Sigma; 250 μg/mL in 1% acetic acid), laminarin (β-1,3-glucan, Thermo Fisher; 500 μg/mL in milli-Q water), pustulan (β-1,6-glucan, Calbiochem; 500 μg/mL in 50 mM potassium acetate buffer) or bovine collagen (Sigma; 250 μg/mL in 0.2 M bicarbonate buffer, pH 9.6) by adding 500 μL of the respective solutions, and allowing for passive adsorption (1 h at 30°C followed by overnight incubation at 4°C), in the case of chitin and collagen, or evaporation (overnight at 37°C), in the case of laminarin and pustulan.

Isolation and purification of PGN were carried out from the Xoo strain PXO99^A as described previously (Barnard and Holt, 1985 (link)). Briefly, the bacterial cells were boiled in 100 volumes of 5% SDS for 30 min. After cooling down to room temperature, the SDS-insoluble material was collected by centrifuging at 30,000 g at 4°C for 30 min. The pellet was treated with trypsin digestion to remove protein contamination and dialyzed extensively against distilled water.
Arabidopsis and rice protoplasts were treated with flg22 (Sigma-Aldrich) at a final concentration of 1 μM. Chitin (Sigma-Aldrich) was applied to rice protoplasts at a final concentration of 10 μM. Before flg22 or Chitin treatment, rice and Arabidopsis protoplasts were isolated and incubated for about 12 h in W5 solution under dark, then incubated under light for 2 h in fresh W5 solution. After treatment (e.g., 10 min), protoplasts were harvested for immunoblot analysis. For PGN treatment, protoplasts were collected after 12 h post-transfection of effector gene constructs and resuspended in 100 μl of W5 solution. After recovered under light for 2 h in the 30°C growth chamber, protoplasts were treated with 5 mg/ml PGN for 15–60 min and then harvested for immunoblot analysis.

5-6-week-old A. thaliana, 4-5-week-old N. benthamiana plants were used for the assay. N. benthamiana was infiltrated with A. tumefaciens and incubated for 48h. Leaf disks (4mm) were floated in water o.n. Water was removed, and elicitors were added. flg22 elicitation solution was: Horseradish peroxidase (HRP 10 μg/ml, Sigma-Aldrich cat# P6782), L-012 (34ug/ml Fujifilm WAKO cat# 120–04891) and flg22 (100nM) in H₂O. Chitin elicitation solution was prepared as follows: 50mg of Chitin (Sigma-Aldrich cat# C9752) were ground with mortar and pestle in 5 ml of H₂O for 5 min, transferred to a falcon tube, microwaved for 40 s, sonicated for 5 min, centrifuged at 1800 g for 5 min, supernatant was transferred to a new tube, vortexed for 15 min and stored at 4°C. Before use, the suspension was diluted 1:1 in H₂O and supplemented with HRP and luminol (34 μg/ml Sigma-Aldrich cat# 123072). ROS production was monitored by luminescence over 30–40 minutes in a microplate reader (Synergy H1, BioTek). At least three plants per construct/genotype were used in each experiment. Experiments were performed at least three times.

Differences in the binding capacity to cell wall components or to the mammalian extracellular matrix (ECM) molecules collagen and galactose was evaluated by flow cytometry. Twelve-wells microtiter plates were coated with pustulan (β-1,6-glucan, Calbiochem; 250 μg/mL in 50 mM potassium acetate buffer), laminarin (β-1,3-glucan, Thermo Fisher; 250 μg/ mL in milli-Q water), chitin (Sigma; 250 μg/mL in 1% acetic acid), mannan (mannan from S. cerevisiae, Sigma; 250 μg/ml in milli-Q water), mannose (D-(+)-mannose, Sigma; 250 μg/ml in milli-Q water), galactose (D-(+)-galactose LS, Panreac; 250 μg/ml in milli-Q water) or bovine collagen (Sigma; 250 μg/mL in 0.2 M bicarbonate buffer, pH 9.6) by adding 500 μL of the respective solutions, and allowing for evaporation (overnight at 37°C), in the case of laminarin, pustulan, mannan, mannose, and galactose, or passive adsorption (1 h at 30°C followed by overnight incubation at 4°C), in the case of chitin and collagen. The plates were then washed with PBS. Overnight cultures of strains of interest were adjusted to 10⁶ cells/mL in PBS, and coated wells were filled with 500 μL of the cell suspension. After 4 h of incubation at 37°C, unbound cells were aspirated and further removed by two washes with PBS. Adhered cells were loosened with a 2.5% trypsin solution, resuspended in 500 μL PBS, and measured using a MacsQuant flow cytometer.