Xanthine

For most routine extractions of fungal tissue and our early extractions of ectomycorrhizal root tips, DNA was extracted using a CTAB protocol with 0.8% mercaptoethanol and incubating at 65°C for 1 hour [34 (link)]. An alternate extraction method modified from Ross [35 (link)] based on the action of xanthogenate [36 ] was later employed for rapid survey analyses of large numbers of ectomycorrhizal root tips. Single ectomycorrhizal root tips (approximately 1 mg tissue dry weight) were placed in each 1.5 mL tube with 600 μL Xanthine/Tween Buffer (100 mM Tris-HCl pH 7.5, 12.5 mM potassium ethyl xanthogenate (Fluka, Buchs, Switzerland, cat. no. 60040), 10 mM EDTA pH 8.0, 10% Tween 20, 500 mM NaCl). These were sonicated for 15 seconds and incubated 90–120 min at 60°C on a shaker. Tubes were then centrifuged 5 min at 10,000 × g to pellet undissolved tissue. Supernatant was removed to a new tube containing 1/5 volume of PEG/NaCl (20% PEG-8000/2.5 M NaCl [37 (link)]) and 8 μL 1.25 mg/mL Linear Acrylamide (Ambion, Austin, Texas, cat. no. 9520). This was mixed by gently tipping the tube, and the mixture was incubated at 30°C for 15 minutes. DNA was precipitated by centrifuging 5 minutes (room temp.) at 10,000 × g, and then recovered by removing the PEG solution. The final DNA pellet was obtained by rinsing twice with 200 μL 80% ice-cold ethanol while mixing by gently tipping the tubes, followed by centrifuging 5 minutes at 10,000 × g, and lastly, by drying the pellet under vacuum for 30–45 minutes. In both protocols DNA was resuspended in a final volume of 100 μL TE buffer (pH 8, 10 mM Tris: 1 mM EDTA) after extraction by heating to 37°C for 10 minutes.

For enzyme extracts and assays, fresh roots (0.1 g) were ground in liquid nitrogen, and then suspended in 0.9 mL solution containing 10 mM phosphate buffer (pH 7.4). The homogenate was centrifuged at 4°C, 2500 rpm for 10 min and the resulting supernatant was collected for determination of the activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) and glutathione peroxidase (GSH-Px, EC 1.11.1.9) using commercial assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All enzymes above were detected using a microplate reader (SpectraMax M5, USA), and 5 to 10 seedlings were used to provide enough amounts of root tissues in each experimental replicate (n = 3).
The activity of SOD was determined by measuring the inhibiting rate of the enzyme to O₂⁻· produced by the xanthine morpholine with xanthine oxidase using the SOD assay kit. Each endpoint assay was detected the red substances of the reaction system by absorbance at 550 nm after 40 min of reaction time at 37°C. And one unit SOD activity (U) was defined as the quantity of SOD required to produce 50% inhibition of reduction of nitrite in 1 mL reaction solution by measuring the change of absorbance at 550 nm.
The CAT activity was measured based on the hydrolysis reaction of hydrogen peroxide (H₂O₂) with CAT, which could be terminated by molybdenum acid (MA) to produce yellow MA-H₂O₂ complex. CAT activity was calculated by the decrease in absorbance at 405 nm due to the degradation of H₂O₂, and one unit is defined as the amount of enzyme that will cause the decompose of 1 µmol hydrogen peroxide (H₂O₂) per second at 37°C in 1.0 g fresh tissue according to CAT assay kit.
The POD activity was measured based on the change of absorbance at 420 nm by catalyzing H₂O₂. One unit was defined as the amount of enzyme which was catalyzed and generated 1 µg substrate by 1.0 g fresh tissues in the reaction system at 37°C. POD activity was calculated as the formula according to POD assay kit.
The GSH-Px activity was also measured using the assay kit based on the principle that oxidation of glutathione (GSH) and hydrogen peroxide (H₂O₂) could be catalyzed by GSH-Px to produce oxidized glutathione (GSSG) and H₂O. In addition GSH reacts with 5, 5′-dithiobis (2-nitrobenzoic acid) (DTNB) to produce stable yellow substances and the decrease of GSH at 412 nm during the reaction is indicative of GSH-Px activity in tissues. One GSH-Px unit of GSH-Px activity (U) was calculated as the amounts of enzyme that will oxidize 1 µmol/L GSH in reaction system at 37°C per minute in 1.0 g fresh tissue according to the assay kit. All of the enzymes were expressed as in U/g FW.

To determine SOD activity, the Fluka 19160 test kit (Germany) was used, which is based on the indirect method of nitrotetrazolium blue chloride (NBT). This assay uses xanthine and xanthine oxidase to generate superoxide radicals, which react with 2-(4-)-3-(4-nitrophenol)-5-phenyl tetrazolium chloride to produce a compound, which absorbs light at 450 nm. The inhibition of chromogen production is proportional to the SOD activity present in the sample. The reading was carried out using a spectrophotometer and the results are reported in U/L.

RHΔhxgprtΔku80 strain parasites expressing firefly luciferase were filtered, washed, and resuspended in Cytomix (10 mM KPO₄, 120 mM KCl, 0.15 mM CaCl₂, 5 mM MgCl₂, 25 mM HEPES, 2 mM EDTA). Parasites were mixed with 50 μg of each gRNA1 and gRNA2 CRISPR plasmid for each gene, along with the PCR-amplified targeting fragment for each gene, and supplemented with 2 mM ATP and 5 mM glutathione (GSH). Parasites were electroporated by Gene Pulser II (Bio-Rad Laboratories). Selection by growth for 14 days in 25 μg/mL mycophenolic acid (Sigma) and 50 μg/mL xanthine (Wako) were used to obtain stably resistant clones. Next, parasites were plated in limiting dilution in 96-well plates to isolate single clones. To confirm the disruption of the gene, we analyzed mRNA of IWS1, SUB2, RAMP4 or DRL1 from WT and each KO (knockout) parasite by quantitative RT-PCR using the primers listed in Table S3.

The IWS1-deficient parasites lacking HXGPRT were filtered, washed, and resuspended in Cytomix. Parasites were mixed with 50 μg of the ROP18 expression vector containing ROP18 cDNA (18 (link)), in which 1 kb promoter of the SAG1 gene was fused to the coding sequence of ROP18 cDNA, followed by the poly A sequence and hxgprt expression cassette in pBlueScript plasmid vector (Fig. S2B), and supplemented with 2 mM ATP and 5 mM GSH. Parasites were electroporated using a Gene Pulser II. Selection by growth for 14 days in 25 μg/mL mycophenolic acid (Sigma) and 50 μg/mL xanthine (Wako) was used to obtain stably resistant clones. Then, parasites were plated in limiting dilution in 96-well plates to isolate single clones. To confirm expression of rop18, we analyzed ROP18 mRNA from the rescued parasites by quantitative RT-PCR using the primers listed in Table S3.