Glucanase

To identify the targeted binding components, B. bassiana spores were treated in the following ways, and then used for binding assays. To remove spore surface proteins, spores were suspended and incubated in the binding buffer containing 0.5 µg/µL Protease K (Roche) at room temperature for 12 h. High concentration of HCl can hydrolyzate proteins (22 (link)). In order to remove all proteins, spores were also incubated in 37% HCl at room temperature for 12 h. To remove chitin component, spores were suspended and incubated in the binding buffer (200 µL) containing 1 µg/µL chitinase (Roche) and 1-mM phenylmethylsulfonyl fluoride (PMSF) (to inhibit protease contamination in this product according to our assay). To remove β-1,3-glucan component, spores were suspended and incubated in 200 µL of 0.2 M of citric acid–sodium citrate buffer (pH 5) containing 10 µg/µL glucanase (Sigma) and 1 mM PMSF at 37°C for 2 h. After these separate treatments (37% HCl, chitinase, and glucanase), spore samples were separated from chemical and washed in binding buffer for three times. Subsequently, the samples were incubated with rPPO1 or rPPO1-GFP as described above. In order to examine the enzyme hydrolysis effect, the dye Calcofluor White (Sigma) was applied for chitin staining (18 (link)), and β-1,3-glucan was detected by using Aniline Blue Fluorochrome (Biosupplies) (23 (link)).

RNA interference was used for knockdown of genes. The two complementary cDNA fragments from GSNOR (300 bp), IoTrx1 (200 bp), IoTrx (150 bp) and IoTrx3 (200 bp) were amplified, respectively, using the primers listed in Supplemental Table 1, and inserted to vector pCIT that flanked to the intron (Supplemental Fig. 1A,B) to form silencing construct (Supplemental Fig. 1C). The silencing construct including PtrpC and TtrpC was released by Xho1 and Sac I and subcloned to the sites of Xho1 and Sac I in vector pCH containing hygromycin resistant gene to form pCH-silence plasmids (Supplemental Fig. 1D). For preparing the competent cells, the I. obliquus mycelia were incubated by rotation in 10 ml osmotic medium36 (link) containing lysing enzyme 45 mg (Sigma), yatalase 30 mg (Takara), glucuronidase 50 μl (Sigma), glucanase 20 mg (Sigma) and BSA 50 mg (MP Biomedicals, USA) at 37 °C for 18 h followed by protoplast capture according to the protocols described elsewhere36 (link). Transformation of the plasmids were conducted on an osmotic PDA medium containing 0.1 μg/ml hygromycin B (Sigma) as the procedure previously described36 (link). The positive mutants were further screened using hygromycin B containing PDA medium and used for further experiments.

Fixation in formaldehyde was adapted from the protocols described by Bertaux and Wang (Bertaux et al., 2003 (link); Wang et al., 2017 (link)). Fungal cultures grown under varied conditions were fixed in a 4% solution of formaldehyde (w/v, Thermo Fisher Scientific) in PBS overnight at 4°C. Fungal biomass was washed 3× with PBS and then treated with cocktail combinations of cell wall digestive enzymes: 1 mg•ml⁻¹ solution of lysozyme, or 0.5 mg•ml⁻¹ solution of chitinase, and/or 5 mg•ml⁻¹ solution of glucanase (reagents purchased from Sigma-Aldrich) for 1 h at 37°C and washed 3× with PBS. The cultures were then dehydrated with a step-wise series of ethanol treatments: 50%, 75%, 100%, 75%, 50%, and finally replaced with PBS for 3 min between each step.