Figure 7.

Pst_13661 accumulates in the apoplast and has affinity to chitin. (a) Transiently expressed Pst_13661‐mCherry localized in the apoplast of N. benthamiana. Plasmolysis was induced by 50 mm NaCl. Arrows represent plasmolysis regions. BF, bright field, Bar = 20 μm. (b) Pst_13661 has high affinity to chitin and cellulose. Pst_13661 or GFP protein was diluted and incubated with different concentrations of affinity materials at 4 °C for 4 h. Samples of the supernatant and affinity precipitate were detected with anti‐His by Western blot. GFP protein was a negative control. S, supernatant; P, affinity precipitation. Different concentrations of affinity materials are 200 mg, 40 mg, 10 mg and 4 mg. (c) Pst_13661 protein binds the fungal cell wall. Pst_13661, Pst_13662 or GFP proteins were diluted and incubated with germ tubes of Pst at 4°C for 4 h. The supernatant and affinity precipitate were detected with anti‐His by Western blot. GFP protein was a negative control. S, supernatant; P, affinity precipitation. (d) Deacetylase enzyme activity of Pst_13661 (100 µg/mL) was detected with the chitin deacetylase (CDA) ELISA kit. The left panel indicates the colour change in substrate solution in the presence of Pst_13661 protein and standard samples. GFP was a negative control. Standard samples were 0, 50, 100, 200, 400, and 800 U/L. The right panel shows the standard curve of deacetylase enzyme activity according to the dilution of standard samples. The blue circle represents Pst_13661.