Strains, plasmids and media
The dikaryotic P. ostreatus strain CCMSSC00389 from the Center for Mushroom Spawn Standards and Control of China was used in this study as a parent strain for OE and RNAi experiments. The wild-type (WT), OE and RNAi strains were maintained on potato dextrose agar (PDA) at 4 °C. For the selection and maintenance of transformants, complete media (CM) was supplemented with 90 μg/mL hygromycin (hyg) (Invitrogen, U.S.A.). Agrobacterium tumefaciens (A. tumefaciens) GV3101 (IMCAS, Beijing, China) was grown in Luria-Bertani (LB) medium (Oxoid, England) containing 100 μg/mL kanamycin (kan) (VWR Life Science, U.S.A.) and 50 μg/mL rifampicin (rif) (MP Biomedicals, France) and used to transform P. ostreatus. Escherichia coli (E. coli) DH5α and BL21 (DE3) (Tiangen, Beijing, China) were used for plasmid construction, and grown in LB broth containing kan (50 µg/mL). Restriction endonucleases were purchased from New England Biolabs (NEB), and DNA polymerase, a reverse-transcription kit, and a DNA Gel Extraction kit were purchased from Vazyme (Nanjing, China). Primer synthesis and DNA sequencing were performed by Sangon Biotech (Shanghai, China). The plasmid pCAMIBA 1300 was purchased from YRGen Biotech Company (Changsha, China).
Identification, cloning and sequence analysis of pal genes
The two pal genes were identified by keyword search in the annotated genome database of P. ostreatus strain PC15 , via the Joint Genome Institute website (https://genome.jgi.doe.gov/PleosPC15_2/PleosPC15_2.home.html), and two protein sequences with the following protein IDs were obtained: 1112899 and 173727. Subsequently, the two sequences were used to BLAST against the CCMSSC00389 genome database to identify homologs. The nucleotide sequences were used to design primers (pal1 and pal2, listed in Table 1) to amplify full-length sequences from CCMSSC00389 complementary DNA (cDNA). Total RNA and DNA were extracted using TRIzol (Omega Bio-Tek, U.S.A.) and cetyltrimethylammonium bromide (CTAB), respectively. The first-strand cDNA was synthesized using a PrimeScript™ RT-PCR kit (Vazyme). The amplified products were purified and cloned into the vector pGEM-T (Promega, Madison, WI, U.S.A.) for sequencing. All primers used in the experiment are shown in Table 1.
Bioinformatics analysis of the pal genes
DNAMAN software was used for multiple sequence alignment. The molecular weight, distribution of amino acids, isoelectric point, and signal peptide of pal were predicted using the online ProtParam (http://web.expasy.org/protparam/). The structural domains of the PAL proteins were analyzed online (http://smart.embl-heidelberg.de/) . A phylogenetic tree was constructed using the neighbor joining method in MEGA 5.0 based on the PAL nucleotide sequences obtained from GenBank and the maximum composite likelihood model. The three-dimensional (3D) structure of the PAL proteins were predicted using Modeller.
Expression and purification of pal1 and pal2 in E. coli
Expression and purification of pal1 and pal2 in E. coli were performed as previously described with slight modifications . The PCR products were digested with two restriction enzymes (EcoRI-HF and NotI-HF) and ligated into the vector pET28a (Novagen, Inc., Madison, WI, U.S.A.) that had been digested with the same enzymes. The recombinant plasmids that were confirmed by DNA sequencing were named pET28a-pal1 and pET28a-pal2 and were subsequently transformed into E. coli BL21 (DE3) cells for protein expression. The transformed strains were inoculated into LB medium containing 50 µg/mL kan and incubated at 37 °C with shaking at 180 rpm until reaching an OD600 nm of 0.6-0.8. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to reach a final concentration of 1 mM to induce protein expression, and the culture was incubated overnight at 16 °C with shaking at 180 rpm.
The cultured cells were centrifuged at 4 °C and 5000 rpm for 5 min, washed with PBS buffer, and then suspended in lysis buffer. After the cells were lysed by ultrasonication, the enzymes were retained in the supernatant after centrifugation. The supernatant was loaded onto an Ni-NTA column (Qiagen, Duesseldorf, Germany) that had been preequilibrated with binding buffer. Subsequently, the column was eluted with binding buffer, washing I buffer, washing II buffer, elution I buffer and elution II buffer. Finally, the fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The PAL activities of the purified protein were determined using a Phenylalanine Ammonia-Lyase Assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.
OE and RNAi vector construction
The original pCAMBIA1300 vector was modified to harbor the hyg phosphotransferase gene (hyp), which was expressed under the control of the upstream lac promoter [28, 43]. The pal gene OE cassettes were constructed as follows. The P. ostreatus gpd promoter was PCR amplified, after which the pal1 and pal2 cDNA was obtained. The two cassettes were individually cloned into vector to generate the pal gene expression cassette driven by the P. ostreatus gpd promoter (Fig. 1A, B, C). Finally, the vector was introduced into A. tumefaciens GV3101. RNAi-F and RNAi-R fragments were obtained by PCR, after which the two amplicons were individually inserted into the vector to construct the interference vectors (Fig. 1D, E, F). Finally, the interference vectors were transferred into P. ostreatus by A. tumefaciens GV3101. The primers used to construct the vectors are shown in Table 1.
P. ostreatus mycelia were inoculated onto PDA plates and cultured at 28 °C until the colony diameter was 5.5-6 cm. Mycelial pellets were cut from the edge of the colony using a cork borer with a 5-mm diameter. Subsequently, 200 pellets were placed into 100 mL of CM liquid medium at 28 °C for 2 days without shaking. A. tumefaciens GV3101 containing the OE-pal or RNAi-pal plasmid was cultivated at 28 °C with shaking at 180 rpm in LB medium with the selective antibiotics (100 μg/mL kan and 50 μg/mL rif) for at least 16 h. A. tumefaciens cells were collected in sterile tubes (50 mL capacity) by centrifugation at 4500 rpm and 4 °C for 10 min. The bacterial cells were suspended in induction medium (IM, ; supplemented with 200 μM acetosyringone) and incubated for 5 h (90 rpm, 28 °C) to preinduce A. tumefaciens GV3101 virulence. Then, the A. tumefaciens GV3101 and the mycelia pellets were cocultured at 28 °C for 5 h without shaking. After incubation, the mycelial pellets were dried with filter paper and placed onto IM solid medium at 28 °C for 3 days and then transplanted onto CM medium with selective antibiotics (90 μg/mL hyg and 50 μg/mL cef). Transformants were obtained after 25 days of culturing and were subsequently selected twice for hyg resistance. PCR analysis of the hyg and pal genes was performed using the primers listed in Table 1 .
Mushroom production experiment and sample collection
The strains were cultured on PDA plate for 7 days. Samples mycelia were collected, frozen with liquid nitrogen and stored at - 80 °C. The remaining mycelia were inoculated on cotton seed hull culture medium and cultured at 25 °C in dark. When the mycelia were full, they were transferred to a mushroom room for mushroom production. The primordia, fruiting bodies and spores were collected and stored at - 80 °C after quick freezing with liquid nitrogen.
Western blot analysis
Western blot analysis was performed according to a previous study. Briefly, equal amounts of total protein (20 µg) were loaded into the protein lane and separated in a 12 % (w/v) SDS-PAGE. After electrophoresis, proteins were transferred onto a polyvinylidene fluoride membrane. Western blot analysis was performed using antibodies against PAL1 and PAL2, which were synthesized by Shanghai genScript Company. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, PC15_1090663 (jgi)) was used as control .
Heat stress treatment
WT, pal-overexpressing (OE-pal1 7.11-9, OE-pal1 7.11-11, OE-pal2 7.11-7 and OE-pal2 7.12-11) and RNAi transformant strains (RNAi-pal1 8.1-26, RNAi-pal1 8.1-38, RNAi-pal2 7.18-1 and RNAi-pal2 7.18-19) were used in this study. The WT strain was cultured on PDA plates incubated at 28 °C for 5 days and then subjected to different temperatures to induce heat stress for 2 days. To assess the function of pal in the mycelial response to heat stress, the WT, OE-pal and RNAi-pal strains were cultured on PDA plates at different temperatures (28, 32, and 40 °C) for 6 days [19, 44].
Growth susceptibility assay
To assess the susceptibility of the WT, OE-pal and RNAi-pal strains to oxidative stress, mycelial tip pellets with 5-mm diameter were inoculated onto PDA plates supplemented with 5, 10 or 15 mM H2O2. The control groups were subjected to the nonexogenous addition of H2O2. The diameters of the strains were measured after incubation at 28 °C for 7 days .
Quantitative real-time PCR (qPCR)
To analyze the expression of pal at different developmental stages, samples were collected at the mycelia, primordia, fruiting body and spore stages. The levels of gene-specific mRNA expressed by the WT, OE-pal and RNAi-pal strains were analyzed using qPCR according to our previous study , with the β-actin gene used as a reference. The qPCR amplification procedure was as follows: 95 °C for 3 min, 40 cycles of 95 °C for 3 s and 60 °C for 32 s, and a final extension at 72 °C for 30 s. The relative gene expression was analyzed according to the 2−△△CT method.
Enzymatic activity assay
The WT, OE-pal and RNAi-pal strains were cultured on PDA medium incubated at 28 °C for 5 days. Subsequently, the mycelia were quickly scraped, mixed, and frozen in liquid nitrogen for further use. The activity of PAL was determined using a Phenylalanine Ammonia-Lyase Assay kit according to the manufacturer’s instructions.
Determination of malondialdehyde (MDA) and H2O2 contents
Intracellular MDA and H2O2 contents were determined using a Malondialdehyde and Hydrogen Peroxide Assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.
Determination of relative ion leakage and total respiratory rate
Ten pellet pieces (5 mm) were inoculated into 100 mL of potato dextrose broth medium for 5 days at 28 °C with shaking at 180 rpm. Heat stress was then applied for different durations at 40 °C (0, 3, 6, 12, 24, and 48 h). The conductivity of mycelial pellets (C1) was measured by washing off the electrolytes attached to the surface with deionized water and then placing the pellets them into 20 mL of deionized water at 28 °C for 2 h. Then, the sample was autoclaved for 30 min to determine the total conductivity (C2). The relative ion leakage rate (%) = C1/C2﹡100 . The respiration rate was determined by measuring the production of carbon dioxide with a carbon dioxide meter (MultiRAE IR PGM-54) in sealed containers. The total respiratory rate was measured according to previous studies .
GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA, U.S.A.) was used for statistical analysis. The values are reported as the means ± SE and were analyzed by one-way ANOVA, with a P value of <0.05 considered significant.