Strains and culture
Dictyostelium discoideum wild type AX2 was cultivated at 22°C in HL5 medium. The 45 knockout mutants by BSR cassette containing bsr (blasticidin-resistant gene) (Sutoh 1993) insertion of D. discoideum cDNA clones expressed specifically in stalks were prepared and grown in HL medium containing 10 µg/mL of blasticidin S (Funakoshi, Tokyo, Japan) (Takamoto et al. 2001; Sakuragi et al. 2005; Kamei et al. 2013). These D. discoideum cDNA clones were provided from the “Dictyostelium cDNA project in Japan, https://nenkin.nbrp.jp/ clone/list” (Morio et al. 1998). Development was started by washing the cells in 17 mM phosphate buffer, pH 6.1. The cells were agitated on a rotary shaker at 150 rpm or spread on agar plates, and incubated at 22°C as reported previously (Yoshida et al. 1991). Lentinula edodes mycelia were grown on MYPG agar (0.25% malt extract, 0.1% yeast extract, 0.1% peptone, 0.5% glucose, and 0.5% low melting temperature agar) plates at 25°C. Mycelia agar plate discs were used for inoculation into sawdust pots and culture was continued at 22°C for 150 days. For single-strand conformation polymorphism (SSCP) analysis, parental dikaryon, MCR14, was generated from the monokaryon D703PP-9 (mating-type: A1B1) obtained from D703, a New Zealand wild type strain and the monokaryon G408PP-4 (A2B2) obtained from G408, a Japanese wild type strain. The 23 tetrads were analyzed in the present study (Miyazaki and Neda 2004; Miyazaki et al. 2014).
Preparation of a knockout construct and transformation of Dictyostelium discoideum
The knockout vector by BSR insertion into the 45 D. discoideum cDNA clones was constructed. A BSR cassette was inserted into the restriction enzyme site of each cDNA in pBluescript KS (-), followed by PCR-mediated amplification using rTaq DNA polymerase, T3, and T7 primers (Merck, Darmstadt, Germany). PCR products were purified using QIAGEN-tip 20 (QIAGEN, Duesseldorf, Germany). The knockout construct was transformed into AX2 cells by electroporation and transformants were independently isolated. Knockout mutants of three clones, SSJ301, SSJ337, and SSK864, formed aberrant fruiting bodies; SSJ301, a fruiting body with a tiny sorus; SSJ337, a fruiting body with an abnormal stalk; SSK864, a tiny fruiting body (Fig. 1).
Preparation of Lentinula edodes protoplast
Protoplast cells were prepared from L. edodes Mori 252 (M252) mycelia. Protoplast cells from M252 were transformed with an eleven-fold higher efficiency than those from L. edodes Hokken 600 (H600) (Fujimoto rt al. 2004). M252 mycelia was inoculated into MYPG agar and cultured in the dark at 25°C, and used as an inoculum. They were inoculated into 30 mL of two-fold concentrated MYPG liquid medium and cultured with shaking at 96 rpm at 25°C for 3 days. The mycelia cultured in 30 mL in three Erlenmeyer flasks were transferred to a homogenizer cup and crushed in a homogenizer at 9,000 rpm for 5 min on ice. Twenty mL of the suspension of crushed mycelial fragments was filtered through a 100-µm cell strainer (BD Biosciences, MA, USA), added to 20 mL of MYPG liquid medium, and statically cultured for 4 days. They were centrifuged at 12,000 rpm at 4°C for 20 min, and mycelia were washed with 10 mL of SM buffer (50 mM succinate, pH 5.6, and 0.6 M mannitol). To lyse mycelial cell walls for protoplast isolation, 1 g wet weight of mycelia was suspended in 10 mL of 2.5% cellulase Onozuka RS (Yakult Honsha, Tokyo, Japan) and 1.0% Lysing Enzyme (Merck)/SM buffer in a centrifuge tube. The tube was installed horizontally, and enzyme treatment was carried out for 4 h with shaking at 60 rpm at 28°C.
Cloning of Lentinula edodes genes
For the L. edodes primordia cDNA library, total RNA of L. edodes H600 primordia was extracted using TRIzol®Reagent (Invitrogen, Carlsbad, CA, USA). Two hundred µL of TRIzol was added to 0.1 g of primordia and pulverized in a mortar. Next, 200 µL of chloroform was added and the mixture was vigorously stirred, followed by centrifugation at 12,000 rpm at 4°C for 15 min. The supernatant was recovered, 500 µL of isopropanol was added, and the mixture was set at room temperature for 10 min and then centrifuged at 12,000 rpm at 4°C for 10 min. After removing the supernatant, it was washed with 70% ethanol, air-dried for 10 min, and suspended in DEPC-DW. Poly (A)+ RNA was purified from total RNA using a poly (A)+ RNA purification kit (GM Healthcare, Buckinghamshire, UK) according to the manufacturer's specifications. The cDNA was made from 5 µg of poly (A)+ RNA using a cDNA synthesis kit (GM Healthcare). It was blunted, ligated to EcoRI adaptors, phosphorylated, and size-fractionated by column chromatography. Fractions containing cDNA of longer than 500 base pairs were collected, precipitated by ethanol, and ligated into λZAP II arms (Agilent Technologies, CA, USA). The ligated DNA was packaged using a GIGA Pack III Gold packaging kit (Stratagene, La Jolla, CA). The cDNA library was screened using 32P-labeled SSJ301, SSJ337, and SSK864 cDNA, respectively, as a probe. Random labeling was performed with a Radprime DNA labeling system (Invitrogen) and 20 µCi [α-32P]dCTP (GM Healthcare) according to the manufacturer’s protocol. Hybridization was carried out at 42°C overnight in 6 x SSPE containing 0.05% nonfat dried milk and 50% formamide. Filters were first washed in 2 x SSC, 0.1% SDS at room temperature, and then in 1 x SSC, 0.1% SDS at 68°C. Positive clones were sequenced on an ABI Prism®3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using BigDye®Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The sequences were then compared with those registered in the GenBankTM data bank. Open reading frames were predicted using GENETXY-MAC 8.0. Homology searches of nucleotide and deduced amino acid sequences were carried out using BLASTX (DDBJ; http:// www.ddbj.nig.ac.jp/welcome-j.html) and Simple Modular Architecture Research Tool (SMART; http://smart.embl- heidelberg.de/smart/change_ mode. pl).
Transformation of Lentinula edodes
The L. edodes expression vector pLG-RasF1 was constructed using the promoter of the Le. Ras gene (Hori et al. 1991) and constitutively expressed in L. edodes cells (Fujimoto et al. 2004). Each gene, Le-Dd5, Le-Dd6, Le-Dd7, Le-Dd9, Le-Dd10, Le-Dd11, Le-Dd12, Le-Dd13, Le-Dd14, and Le-Dd18, was inserted into pLG-RasF1 digested with SmaI and dephosphorylated. Each cDNA expression vector was constructed (Hamada et al. 2008). For transformation of L. edodes M252 protoplasts, the REMI method (Sato et al. 1998) was used in which gene transfer into protoplasts was carried out by osmotic action. STC buffer (10 mM Tris-HCl, pH 7.5, 10 mM CaCl2, and 1.2 M sorbitol) was added to the protoplast suspension to be 0.5 to 1.0 × 107/240 µL, and the protoplast suspension was agitated and transferred to a new 50-mL Falcon tube. Ten µL of STC buffer containing 10 µg of the cDNA expression vector and 10 units of SphI (or DraI or BspT104I) was added to the tube. It was gently stirred and incubated on ice for 20 min. Next, 62.5 µL of a PEG solution (60% PEG 4000 [Nacalai tesque, Kyoto, Japan], 10 mM Tris-HCl, pH 7.5, and 10 mM CaCl2) was added and incubated on ice for 20 min. Thereafter, 3.125 mL of the PEG solution was further added, and the mixture was kept at 25°C for 20 min. Next, 10 mL of STC buffer was added. After centrifugation at 2,500 rpm at 4°C for 10 min, the precipitate suspended in 4 mL of MS medium (2.0% malt extract and 0.6 M sucrose) was transferred to a plastic dish (BD Biosciences). The transformed protoplasts were cultured in an incubator at 25°C in the dark for 3 d. They were applied on a minimal agar medium (Kües, 2000) (2.0 g of glucose, 0.2 g of ammonium tartrate, 0.05 g of MgSO4·7H2O, 0.1 g of KH2PO4, 0.112 g of Na 2CO3, 0.132 g of fumaric acid, 0.0125 mg of thiamine · HCl, 1.0 mg of FeSO4·7H2O, 0.88 mg of ZnSO4 · 7H2O, 0.72 mg of MnCl2· 4H2O, and 0.2 g of Sanpearl CP [Nippon Paper Chemicals, Tokyo, Japan], pH 4.5, 2.0 g of agar/100 mL) including 5 µg/mL of hygromycin (Nacalai tesque). They were cultured in an incubator at 25°C in the dark for 4 days. Thereafter, only the culture solution was removed, and 9 mL of MYPG agar medium (0.5% low melting temperature agarose) including 20 µg/mL of hygromycin was layered. Colonies that appeared after stratification were cultured in an incubator at 25°C in the dark. The most marginal part of the mycelial tuft was punched with a cork borer. It was inoculated into 30 mL of MYPG medium supplemented with 0.2% Sanpearl CP and cultured in an incubator for 14 days.
Culture of L. edodes on block medium (Castanopsis sawdust medium)
Castanopsis sawdust and wheat bran were sterilized in an autoclave at 121°C for 60 min and then mixed with the composition rate of 36% sawdust, 4% bran, and 60% DW. The mixture (90 g) was placed in a cultivation pot sterilized at 121°C for 10 min, and was again sterilized at 121°C for 60 min in an autoclave. The transformed mycelia that were cultured in MYPG agar medium for 14 days were punched out with a cork borer. Nine disks were inoculated into a cultivation pot. The cultivation period of L. edodes mycelia was divided into three categories of 90 days, 120 days, and 150 days, and cultivation was carried out under 90% humidity and light irradiation at 22°C, and the growth rate of mycelia was measured. When the period of each culture was reached, sterilized water was added to the cultivation pot in the clean bench until the medium was immersed with sterilized water. Low-temperature treatment was performed at 4°C for 17 h, and sterile water in the cultivation pot was removed in the clean bench. Culture was continued under 90% humidity and light irradiation at 15°C to promote fruiting body formation. The number of days required for fruiting body formation after low-temperature treatment was measured.
Northern blotting analyses using L. edodes cDNA
A cDNA probe was labeled with digoxigenin (DIG) by the random hexamer procedure using a DIG DNA labeling kit (Roche Diagnostics, Mannheim, Germany). Total RNA was extracted with ISOGEN (Nippon gene, Toyama, Japan) from L. edodes mycelia or transformants with the RNAi vector for Le-Dd10. Northern blotting analysis was conducted using each cDNA as a probe. Ten µg of total RNA was separated on formaldehyde-agarose gel. After agarose gel electrophoresis, agarose gels were transferred onto HybondTM N+nylon membrane (GM Healthcare) with VacuGeneXL (GM Healthcare). Hybridization was carried out at 50°C in DIG easy hyb (Roche Diagnostics).
Construction of RNAi for Le-Dd10
The RNAi vector for Le-Dd10 was constructed with a 146-nucleotide sequence comprising the 40-bp short homologous hairpin dsRNA sequence, a flanking sequence (6 nucleotide), and a 60-nucleotide spacer sequence, as described in a previous paper (Nakade et al. 2011). To construct the Le-Dd10 homologous inverted repeat sequence expression vector (pivrLe-Dd10), two 146-base oligonucleotide sequences LeDd10ivrF and LeDd10ivrR (Table 1) were synthesized by Eurofins genomics (Tokyo, Japan). These contained a 40-bp homologous inverted repeat sequence from Le-Dd10 exon connected to a 60-nucleotide linked loop sequence from intron 2 of Lcc1. These oligonucleotides were heated to 105°C for 10 min and allowed to spontaneously anneal for 30 min while cooling to room temperature. After annealing, the oligonucleotides were blunted and the insert was ligated into the dephosphorylated SmaI-digested pLG-RasF1 vector.
Construction of the GST:Le-Dd10 fusion protein expression vector and preparation of a polyclonal antibody against the Le-Dd10 products
To prepare GST:Le-Dd10, Le-Dd10 cDNA fragments were prepared with the primers GSTLeDd10F and GSTLeDd10R (Table 1), digested with BamHI and EcoRI, and inserted into the BamHI and EcoRI site of pGEX-2T, a GST fusion protein expression vector (GM Healthcare). After transforming these into Escherichia coli BL21-Gold (DE3) cells (Agilent Technologies, CA, USA), transformants were incubated with 0.5 mM IPTG at 25°C or 37°C for 5, 10, 16 h (Fig. 2). GST:Le-Dd10 fusion proteins were adequately expressed at 37°C for 16 h. The GST:Le-Dd10 was cut out from a 10% gel of sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). A 50-kDa target band was formed from the 26 kDa of GST and 24 kDa of Le-Dd10 protein. The gel slices from cells incubated at 37°C for 16 h were stirred in 50 mM Tris-HCl buffer (pH 8.1) containing 0.1% SDS and 1 mM EDTA overnight at room temperature. They were suspended in PBS, and subcutaneously and intraperitoneally injected into rabbits to prepare the polyclonal antibody as described in a previous paper (Yoshida 1987).
Western blotting analysis of the Le-Dd10 product
PCR amplification was used to detect polymorphisms by designing specific primers and amplifying fragments for SSCP analysis. Each reaction mixture consisted of 20 mM Tris-HCl, pH 8.5, 50 mM KCl, 2.5 mM MgCl2, 0.16 mM each dNTP, 0.08 µM each primer, 5 ng of genomic DNA, and 0.25 units of Platinum Taq DNA polymerase (Invitrogen). The total volume of each reaction mixture was 12.5 µL, which was overlaid with mineral oil. The thermal cycling conditions were as follows: 30 cycles at 95°C for 30 seconds, 55°C for 90 seconds, and 72°C for 120 seconds, followed by 10-min incubation at 72°C and subsequent cooling to 4°C (PE480 thermal cycler; Applied Biosystems, Foster, CA, USA).
Biotin-labeled PCR products were diluted 50- to 100-fold in 1 X TBE buffer (89 mM Tris-HCl, pH 8.0, 89 mM boric acid, and 2 mM EDTA), 6% (w/v) sucrose, and 0.33% tartrazine. Double-stranded DNA in a diluted solution was denatured and maintained at 96°C for 5 min, and then the mixture was cooled on ice. The mixture was subsequently loaded on a 15 X 40-cm vertical 5% Hydro-Link Long acrylamide gel (BMA, Maine, USA) in 1 X TBE buffer and subjected to electrophoresis at 14°C for 90 min at 30 W. DNA samples were transferred to a nylon transfer membrane (MSI, MA, USA) and visualized using a Phototope-Star Detection Kit (New England Biolabs, MA, USA).