Glucanex

For immunoblotting, cells were grown to mid-log phase and cells were collected and resuspended in 5 ml of buffer E (50 mM sodium citrate, 100 mM sodium phosphate, pH 6.0, and 0.8 M sorbitol). Protoplasts were generated by incubation with Glucanex (1 mg/ml; Sigma-Aldrich) and Zymolyase-100 T (3 mg/ml; ICN Biomedicals) for 1 h. Then, the protoplasts were resuspended in lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100, 1 mM DTT, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin). Protein concentrations were determined using the BioRad Protein Assay kit (BioRad). 50 μg of protein extracts were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) on 10% gels and probed with anti-GFP (JL-8 Living Colors, Clontech), polyclonal DsRed (Living Colors, Clontech), anti-HA Rat monoclonal antibody (3F10, Roche) or anti-tubulin (Sigma 75168). Protein transfer, blotting, and chemiluminescence detection were performed using standard procedures ^{49 (link)}.

Fungal mycelium was grown on cellophane-covered PDA plates and 10 g of fungal mass was used for TkTV1 particles purification and transmission electron microscopic (TEM) visualization as previously described by Boine et al. (2012) (link). TkTV1-free protoplasts were prepared from TkTV1-free isolates of T. koningiopsis (isolate TkF) and C. rosea (isolate CrF) using Glucanex and β-Glucuronidase as cell wall lytic enzymes (Sigma-Aldrich) as described by Shi-Wang et al. (2007) (link) and Sun et al. (2017) (link), respectively. For protoplast transfection, purified particles were passed through 0.45-μm syringe filters and used in PEG-mediated transfection tests of the previously prepared TkTV1-free protoplasts as described by Hillman et al. (2004) (link). Transfected protoplasts were regenerated on regeneration medium for 4–5 days and 25 colonies for each fungus were randomly picked and transferred to PDA plates. DsRNA and total RNA were extracted as above and TkTV1 transmission was verified by RT-PCR amplification of an 879 bp-long fragment using specific primer pair TVF5 (5′-GTAAAGTAGGAGCCGTCC-3′) and TVR6 (5′-CTTCCAATTCGAGTGTTTCC-3′).

T. thlaspeos cultures were grown in YEPS light medium to an OD₆₀₀ of 0.5–0.8 for 3 to 4 days. Fungal mycelium was collected using cell strainers with 40 µm mesh size (VWR TM Darmstadt, Germany) and washed with protoplasting buffer (0.1 M sodium citrate, 0.01 M EDTA 1.2 M MgSO₄, and pH 5.8) to remove residual culture medium. T. thlaspeos tissue was resuspended in 9 mL protoplasting buffer, supplemented with 10 mg/mL Yatalase (Takara Bio, Kusatsu, Japan) and 20 mg/mL Glucanex (Sigma-Aldrich, St. Luis, MI, USA) per 100 mL cell culture, and incubated for 30–60 min at room temperature. Protoplast formation was controlled microscopically. When protoplasting was finished, protoplasting buffer was added to a total volume of 24 mL. Aliquots of 6 mL crude protoplast solution were overlayed with 5 mL trapping buffer (0.6 M sorbitol, 0.1 M Tris/HCl pH 7.0) and centrifuged at 4863× g (5000 rpm) in a swing out rotor at 4 °C for 15 min. The interphase was collected from all tubes and diluted with 2 volumes of ice-cold STC buffer (0.01 M Tris/HCl pH 7.5, 0.1 M CaCl₂, and 1.0 M sorbitol). Protoplasts were pelleted at 4863× g (5000 rpm) in a swing out rotor at 4 °C for 10 min and resuspended in 500 µL ice-cold STC buffer. 100 µL protoplast aliquots were used for transformation immediately. A bullet point version of the protocol is available with the supplementary files (Protocol S1).

About 10–20 mg of spores were enzymatically treated for 2 hours at 37°C in 150 μl solution of 10 mg·ml^-1 Lysing Enzymes from Trichoderma harzianum (syn. Glucanex^®, Sigma-Aldrich, USA), 1% Triton X-100 (Sigma-Aldrich, USA), 4% 2-mercaptoethanol (Sigma-Aldrich, USA), and 50 mmol·l^-1 EDTA (Sigma-Aldrich, USA), pH 5.6. Samples were stirred several times during the treatment. Subsequently, 100 μl of lysis solution consisting of 500 mmol·l^-1 NaCl, 100 mmol l^-1 Tris-HCl, 50 mmol·l^-1 EDTA (pH 8.0), 0.02% Sodium Dodecyl Sulphate, (Serva, Germany), 0.5% w/v L-ascorbic acid (Sigma-Aldrich, USA), 0.03% w/v proteinase K (Roche Diagnostics, Switzerland) and 4% 2-mercaptoethanol was added to the suspension. The lysis was carried out by incubation 45 min at 65°C. Isolated DNA was then purified by phenol-chloroform extraction. Finally, the DNA was precipitated with isopropanol and dissolved in sterile deionized water. RNA was eliminated from the samples by incubation 20 min at 37°C with 10 mg·l^-1 ribonuclease A (Sigma-Aldrich, USA).

To confirm HCT, CHEF electrophoresis was performed. Preparation of protoplasts and pulsed‐field gel electrophoresis were performed as described previously (Ma et al., 2010). Fusarium strains were cultured in 100 ml NO₃ medium (0.17% yeast nitrogen base, 100 mM KNO₃ and 3% sucrose) for 5 days at 25°C. Then, conidia were collected by filtering through a double‐layer of miracloth and the concentration of spores were measured. 5 × 10⁸ conidia were transferred to 40 ml PDB (BD biosciences). After approximately 16 h of growth at 25°C, germinated spores were re‐suspended in 10 ml MgSO₄ solution (1.2 M MgSO₄, 50 mM sodium citrate, pH 5.8) supplemented with 50 mg ml⁻¹ Glucanex (Sigma) + 5 mg ml⁻¹ driselase (Sigma, D9515) and incubated for approximately 17 h at 30°C in a shaking water bath (65 r.p.m.). The protoplasts were filtered through a double layer of miracloth, collected by centrifugation and casted in InCert agarose (Lonza) at a concentration of 2 × 10⁸ protoplasts ml⁻¹. Plugs were treated with 2 mg ml⁻¹ pronase E at 50°C. Chromosomes were separated by pulsed‐field electrophoresis for 260 h in 1% Seakem Gold agarose (Lonza) at 1.5 V cm⁻¹ in a CHEF‐DRII system (Bio‐Rad) in 0.5 × TBE at 4°C, with switch times between 1200 and 4800 s. The gels were stained with 1 μg ml⁻¹ ethidium bromide in 0.5 × TBE.

To test the effects of the cell wall-perturbing agents on Guy11 and the mutants, comparisons of the growth phenotypes of the wild-type strain Guy11 and the null mutants were performed on CM supplemented with various agents [200 μg/ml Congo red, 0.01% SDS and 100 μg/ml CFW (calcofluor white)]. The plates were incubated at 28 °C for 6 days in dark. The growth-inhibition rate was calculated as follows: growth inhibition rate = [(Diameter on CM−Diameter on CM with stress)/Diameter on CM] × 100. Each test was repeated three times, with three replicates each time.
To test the sensitivity of Guy11 and the mutants to the cell wall digesting enzyme, cultures were incubated in CM liquid medium at 25 °C and 150 rpm for 48 h, and 0.5 g samples of mycelia were harvested by filtration and digested in 2 ml of 0.7 M NaCl buffer with 7.5 mg/ml Glucanex (Sigma, USA). The protoplasts release and cell wall degradation were examined after a 30 °C incubation on a shaker at 80 rpm for 30 min.

Protein-rich enzymatic hydrolysates from chicken and turkey cut-offs were provided by BIOCO AS (Hærland, Norway) and were kept at 4 °C until further use. The poultry hydrolysates contained 50.37 ± 0.03% dry matter out of which 88% was protein, according to product specifications. Glucose was purchased from VWR chemicals (Radnor, United States), and xylose, mannose, lactic acid, acetic acid, ethanol, sulfuric acid, sodium hydroxide, sodium acetate, potassium phosphate, 37% formaldehyde, biotin, glucosamine and Glucanex were purchased from Sigma-Aldrich (Missouri, USA). Urea was kindly provided by Yara International ASA (Oslo, Norway). Kjeltabs for Kjeldahl analysis were purchased from Thomson and Capper Ltd. (Cheshire, UK). Enzymatic hydrolysates of BALI™ pretreated spruce were kindly provided by Borregaard AS (Sarpsborg, Norway). The BALI™ pretreatment [25 (link)] involves sulfite pulping of chipped spruce wood (Picea abies), with chip size up to 4.5 × 4.5 × 0.8 cm. The carbohydrate composition of the spruce hydrolysate is shown in Supplementary Table S1.