Glucanex

DNA was prepared from A. nidulans as described by Specht et al. [41 (link)], and hybond-N membranes (Amersham/GE Healthcare) were used for Southern blots [42 ]. Southern hybridizations were done by DIG DNA Labeling and Detection Kit (Roche) according to the manufacturer's instructions. Transformations of A. nidulans protoplasts were performed as described by Antal et al. [43 (link)]. The protoplasts were prepared from mycelia grown over cellophane [44 (link), 45 ] using a 1% solution of Glucanex (Novozymes, Switzerland) in 0.7 M KCl. Transformation of 5x10⁷ protoplasts was carried out with 100–500 ng of fusion PCR products or plasmid vectors.

Conidia were stained with Calcofluor White (CFW, 5 µg/ml) and observed under fluorescent microscope (EVOS FL Cell Imaging System, Life Technologies, Thermo Fisher Scientific). For Concanavalin-A (ConA) and wheat germ agglutinin (WGA) staining, conidia were washed extensively with PBS containing 1% BSA (PBS-BSA) and incubated for 30 min in dark with ConA/WGA conjugated with fluorescein isothiocyanate (FITC) at a concentration of 5 µg/ml constituted in PBS-BSA. Following, conidia were washed three times with PBS and observed under fluorescent microscopy (EVOS FL Cell Imaging System) (Alsteens et al., 2013 (link)). β-1,3-Glucan and α-1,3-glucan labeling for dormant conidia, and GAG labeling for germinating conidia were performed following the protocols described earlier (Aimanianda et al., 2009 (link); Fontaine et al., 2011 (link); Valsecchi et al., 2020 (link)). Permeabilization of the germinating conidia was performed similar to dormant conidia (Mouyna et al., 2016 (link)) but prolonging the incubation with Glucanex (Novozymes) for 60 min. All the fluorescent labeling was performed with PFA-fixed dormant or germinating conidia.

Protoplast preparation and subsequent transformation was performed as previously described [21 (link)]. Conidia from a four day PDA culture were harvested and filtered through miracloth. 10⁵ conidia were used for the inoculation of 10 mL liquid MM, incubated at 26°C and 150 rpm overnight and collected with centrifugation at 3000 × g for 5 min and diluted in 10 mL of 1.2 M MgSO₄, pH 5.8. Glucanex (Novozymes – 0.2 g) was added and the mixture was incubated for 1.5 h at 100 rpm and 30°C. Ten mL of Sorbitol 0.6 M, MOPS 10 mM, pH 6.3, were added gently and the solution was centrifuged at 1000 × g. Protoplasts were then collected from the interface, washed in a solution of Sorbitol 1 M, MOPS 10 mM, pH 6.3, and suspended, prior to transformation, to Sorbitol 1 M, MOPS 10 mM, CaCl₂ 40 mM.
Plasmids were diluted in 10 mM Tris buffer, pH 7.5, 1 mM EDTA and 40 mM CaCl₂. Five μg of each plasmid solution were mixed with 100 μL protoplast solution. The mixture was kept on ice for 20 min and then 160 μL PEG4000 60% (w/v in 1 M Sorbitol, 10 mM MOPS, pH 6.3) were added. Following incubation at room temperature for 15 min, it was diluted and washed in 1 M Sorbitol, 10 mM MOPS and 40 mM CaCl₂ solution. It was then mixed with SMM with 4 g^.L⁻¹ agar and poured on Petri dishes with SMM with 15 g^.L⁻¹ agar and 50 μg^.mL⁻¹ hygromycin. Transformants were isolated after 7 days incubation at 26°C.

Total DNA was prepared from A. nidulans as described by Specht et al.^{37 (link)}. Transformations of A. nidulans protoplasts were performed as described by Antal et al.^{38 (link)}. The protoplasts were prepared from mycelia grown over cellophane^{35 (link),36 (link)} by using a 4% solution of Glucanex (Novozymes, Switzerland) in 0.7 M KCl solution. Transformation of 5 × 10⁷ protoplasts was carried out with 100–1 µg of fusion PCR products or with 1–5 µg of plasmids. Transformation of S. cerevisiae was carried out with the lithium acetate method using single-stranded carrier DNA^{39 (link)}. Genomic DNA of S. cerevisiae was prepared as described by Bodi et al.^{40 (link)}. For cloning procedures, E. coli JM109^{41 (link)} was used and transformation of E. coli was performed according to Hanahan^{42 (link)}. Plasmid extraction from E. coli and other DNA manipulations were done as described by Sambrook et al.⁴³ .