44
Chapter 2: Matherial and Methods
2.1 Fruiting bodies
Fruiting bodies belonging to the various T. magnatum accessions were collected from natural ground in central, northern Italy (Tuscany, Piedmont, Marche and Umbria) in different years as reported in Table 3. At least three fruiting bodies were used for each biological replicate and their protein extracts were then mixed to increase the homogeneity of the sample.
Table 3: Sample under analysis Accessions
(Collection place)
Host Plant Region Collection period
San Miniato Wood Tuscany November 2008-2011 (4 years) Crete Senesi Wood Tuscany November 2008-2009 (2 years) Mugello Tilia sp. Tuscany November 2008-2009 (2 years) Montaione Wood Tuscany November 2008-2009 (2 years) Lucca (Populus tremula) Tuscany November 2008-2009 (2 years)
Casentino
(Populus tremula), (Quercus robur),
(Salix spp.)
Tuscany November 2010-2011 (2 years) (only for Casentino P) Umbria Wood Umbria November 2010-2011 (2 years)
Alba 1 (Populus tremula) Piedmont November 2008-2011 (4 years) Alba 2 (Quercus ruber) Piedmont November 2008-2011 (4 years) Msa Marche (Populus tremula) Marche November 2010-2011 (2 years) Mma Marche Wood Marche November 2010-2011 (2 years)
45
Table 3: Samples collected in two years in different Italian areas. In four cases it was not possible to trace back the specific host plant. Msa Marche: sample were obtained from Sant’Angelo in Vado; (PU), Mma Marche: sample were obtained from Mercatello sul Metauro (PU); Umbria: samples were obtained from Corciano (PG).Two accession from Casentino (host plant Quercus robur e Salix spp.), red marked, were not include in the first bioinformatic analysis (analysis of two years sample)
Fruiting bodies were thoroughly washed several times with distilled water and subsequently dipped in absolute ethyl alcohol to remove external contamination. Finally the thin external layer of the peridium was removed. Samples were frozen in liquid nitrogen and the tissue was ground in a mortar, and then stored at -80 °C before being used for protein analysis.
2.2 Microscope analysis
A thin slice of fresh sample, approximately 5-10 µm thick was cut, placed in a glass slide and observed with an optical microscope to verify the maturation degree of the fruiting bodies.
The degree of maturation of the fruiting bodies was assessed using the following categorised stages on the basis of the percentage of ascidia containing mature spores after microscope analysis, as described by (Zeppa
et al., 2002): stage 0, 0%; stage 1, 5%; stage 2, 6–25%; stage 3, 26–50%; stage 4, 51–75%; and stage 5, 76–100%. The maturation stage of the spores was defined morphologically: the mature spores were yellow-reddish brown in colour, with reticulate ornamentation.
46
2.3 Microbial analysis
Microbiological analysis, carried out to verify the presence of micro-organisms inside the gleba (inner tissue of the fruiting bodies), showed that after ethanol treatment the residual microbial contaminants were still present, but the number of CFU were reduced to such a low level that the detection of their proteins would not be possible (data not shown). Microbial isolates were used for the 16S rDNA analysis for their identification (Fig 23).
The inner gleba (0.3 g) was taken from three different carpophores (Molise, Alba and San Miniato), mixed and homogenated for 1 min with 3 ml of filter sterilized physiologic solution (0.9% NaCl). Further decimal diluitions were made and then 0.1 ml was pipetted onto the surface of each of the three different substrates used to isolate bacterial strains:
• TSA (Tryptic Soy Agar): (30g/l of Tryptic soy broth plus 20 g/l of Agar); Broad range media
• King’s B medium: Bacto peptone 20g, Glycerol 10 ml, Potassium Solphate (K2SO4 ) 10g, Magnesium Chloride 1,4g, Agar 15g. Final volume 1L, pH 7.2 (KOH added): General media suitable for the isolation of Pseudomonacee (Gram negative bacteria)
• Waksman’s Glucose Agar: Glucose 10g, Peptone 5g, Beef Extract 5g, NaCl 5g, Agar 12,5 g. Final Volume 1l , pH 7.4 .Selective media for Aerobic Gram positive species (e. g. Streptomyces) through the addition of two antibiotics: Cycloheximide (100 g/l), an inhibitor of
47 protein biosynthesis in eukaryotic organisms and Streptomycin(100 g/l) a prokaryotic protein synthesis inhibitor highly active against gram-negative aerobic
The number of CFU/ml was calculated as follow:
Cfu/ml = (no. of colonies x dilution factor) / volume of culture plate.
Three technical replicates (TSB substrate) were performed for each sample and a mean error with standard error were calculated.
Figure 23: Flow diagram for the 16S rDNA sequencing
Selection of isolates was based on picking all colonies growing on agar plates prepared from the appropriate serial dilution. After the first isolation, microbial colonies were transferred separately in their respective media.
48
Each microbial strain was isolated three times during the second isolation phase. The last step is represented by the colony transfer in a liquid media, 48h at 27º, a crucial step to obtain enough biomass for the DNA extraction. The bacterial colonies were then washed once in filtered sterile TES buffer (20 mM Tris, 50 mM EDTA, 150 mM NaCl, pH 7.9), centrifuged for 2 min at 13,000 rpm and resuspended in 1.2 ml of TES buffer (Barbieri et al., 2007). For DNA extraction, fruiting bodies (200 mg) were ground in liquid nitrogen and homogenized with 0.7 mL of CTAB extraction buffer (2% CTAB, 1,4M Nacl, 20 mM EDTA pH 8, 100 mM Tris (pH 8), 2% PVP-40, 1% 2-mercaptoethanol) according to (Petersen and Scheie 2000) with some modifications. The suspension was then placed in a 65ºC water bath for 30min. Following incubation, 0.7 mL of chloroform: isoamyl alcohol (24:1) was added to the samples. The samples were centrifuged and the aqueous phase was transferred to a new centrifuge tube. The DNA in the aqueous phase was precipitated with isopropanol, washed with 70% ethanol, and resuspended in distilled water. DNA concentration was measured by spectrophotometer at 260 nm. Purity of DNA was assessed using the ratio of OD260/280 with a ratio of 1.8–2.0 being of good purity (Ng et al., 2004). PCR product were obtained with the primer pairs (27f-1492r) (Lane 1991) using the following parameters: 95ºC for 3 min (initial denaturing period); 94ºC for 1.20 min (denaturing period), 58ºC for 1.00 min (annealing period), 72ºC for 1.30 min (extension period) for 35 cycles and 72ºC for 10 min (final extension period).The PCR product was of the predicted size for bacterial 16S rDNA (1500 bp), as determined by agarose gel electrophoresis (2 µl of PCR mixture, 2% agarose gel) ethidium bromide staining. The amplicons were sequenced (BMR Genomics, Padova Italy) and their relative
49 sequences were used for the microbial identification by blast analysis (blastn http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?PAGE=Nucleotides)
after having refined them to the same length. The resulting data were
subsequently used to perform a multiple sequence alignment using the ClustalW2 software (http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/) to obtain a phylogenetic tree of the identified species.
2.4 Protein extraction
Fruiting bodies (100 mg) were ground in liquid nitrogen and homogenized with 1.6 mL of extraction buffer (Urea 8 M, Tris-HCl 40 mM CHAPS 4%, DTT 60 mM) according to (Yang et al., 2007) with some modifications. The homogenates were centrifuged for 15 min at 13.000 rcf at 4°C in order to eliminate debris. Supernatants, containing extracted proteins, were precipitated using 13% TCA and 0.007% ß-mercaptoethanol in acetone, transferred to -20 °C for 2 hrs and finally kept at 4 °C for 2 hrs.
Samples were then centrifuged at 14000 rcfat4 ºC for 15 min and the pellet was washed twice with cold acetone (100%), re-centrifuged at the same speed, mixed with 50-500 µL extraction buffer, resuspended and centrifuged at 3,000 rcf at 4 °C for 25 min.
Protein quantification was done using 5 µL of solution composed by 2 µL of protein extract and to 18 µL of Milli-q water. This dilution was act to reduce the error due to manually operation. Bovine serum albumin (BSA) was using as standard at different concentration: 0; 2,5; 5; 7,5; 10; 15 µL and 20 µL. Bradford assay (BIO-RAD Hercules, CA) was used for protein quantification according to manufacturer’s instructions (Bradford 1976). Three charge forms of the Coomassie brilliant blue dye are present in
50 equilibrium at the usual acidic pH of the assay (Zor and Seliger 1996). The red, blue, and green forms have absorbance maxima at 470, 590 and 650 nm respectively (Chial et al., 1993). The blue is the form that binds the protein forming a complex that intensely absorbs light at 594 nm (Chial et al., 1993, Compton and Jones 1985). Development of color in Coomassie dye-based (Bradford) protein assays has been associated with the presence of certain basic amino acids (primarily arginine, lysine and histidine) in the proteins. Spectrophotometric analyses were carried out using a Cintral 101 spectrophotometer (GBC Scientific Equipment) at 595 nm in double beam mode.
2.5 Two-dimensional electrophoresis analysis
Two dimensional electrophoresis (2DE) analysis was performed combining IsoElectric Focusing (IEF) and Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS–PAGE) as describe in Corti et al., (2005) and Fanucchi
et al., (2012).
Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources (O’Farrell 1975).
Samples (1 mg) of protein were directly loaded by in-gel rehydration onto an IPG (Immobilized pH Gradient) gel strip for preparative analysis. IPG strips (18 cm, GE-Healthcare), with pH range 4-7, were rehydrated with 350 µL of IEF sample buffer (8 M urea, 2% w/v CHAPS, 40 mM DTT and 0.5% v/v IPG Buffer) containing the samples.
Strips were covered with mineral oil and focusing was carried out in a IPGphor apparatus (GE-Healthcare) applying the following conditions: 12
51 h of rehydration at 30V, 1 h at 300 V (in gradient), 1 h at 300 V (step and hold), 3 h at 3,500 V (in gradient), 3 h at 3,500 V (step and hold), 3 h at 8,000 V (in gradient) and a final step at 8,000 V (step and hold until reached a total of 50,000 Vhs). After focusing, the IPG strips were equilibrated, in two steps of 15 min (first step-equilibration buffer: 50 mM Tris-HCl, pH 8.8, 8 M urea, 30% v/v glycerol, 2% w/v SDS, 40 mM DTT; second step-equilibration buffer: in the same buffer in which DTT was replaced by 40 mM IAA).
IPGs are based on the bifunctional Immobilines reagents, which are chemically well-defined acrylamide derivatives with the general structure CH2=CH-CO-NH-R, where R contains either an amino or a carboxyl or
group, and form a series of acrylamido buffers with different pK values between pK 1.0 and 13. Since the reactive end is co-polymerized with the acrylamide matrix, extremely stable pH gradients are generated with increased reproducibility (Görg et al., 2009).
The equilibration fulfils two functions: the reduction and prevention of re-oxidation of disulfide bonds through a reducing agent (DTT or DTE) that cleave intra- and intermolecular disulfide bonds (for instance the thiol group of cystein) to achieve complete protein unfolding (Görg et al., 2004) and their alkylation to prevent their re-formation.
The second dimension, SDS-PAGE electrophoresis, was performed using BioRad Protean II XL (20×20 cm) vertical gel electrophoresis chambers, on 12% (%T) acrylamide gels (Sigma Aldrich Acrylamide/Bis-acrylamide, 30% solution: ratio 29:1) applying a current of 40 mA per gel. The size of the pores in a polyacrylamide gel is determined by two parameters: total solid
52 content (%T) and the ratio of cross linker to acrylammide monomer (%C) (Fig 24).
Figure 24: Total solid content (%T) and cross linker ratio equations (from Protein
Electrophoresis technical manual, Ge Healthcare)
The %T is the ratio of the sum of the weights of the acrylamide monomer and the cross-linker in the solution, expressed as % w/v. For example, a 20%T gel would contain 20% w/v of acrylamide plus bisacrylamide. As the %T increases, the pore size decreases. The second way to adjust pore size is to vary the amount of cross-linker. The %C is the weight/weight percentage of total linker weight in the sum of monomer and cross-linker weights. Thus, a 20%T 5%Cbis gel would have 20% w/v of acrylamide plus bis, and the bis would account for 5% of the total solids weight (acrylamide plus bis) (from Protein Electrophoresis technical manual, Ge Healthcare).
Molecular weight standards in a range from 10 to 150 kDa were from BioRad. Proteins resolved by 2DE, were visualized by colloidal Coomassie brilliant-blue G-250 staining for preparative analyses.
53 Coomassie Brilliant Blue G-250 differs from Coomassie Brilliant Blue R-250 by the addition of two methyl groups (Fig 25) that increase the sensitivity for the protein detection.
Figure 25: Chemical formula of Coomassie G-250 (on the left) and Coomassie R-250 (on the
right)
Coomassie Brilliant Blue forms strong, but non-covalent, complexes with proteins, most probably based on a combination of van der Waals forces and electrostatic interactions. Formation of the protein/dye complex stabilises the negatively charged anionic form of the dye producing the blue colour which may then be seen on the membrane or in the gel. The bound number of dye molecules is approx. proportional to the amount of protein present per spot.
For computer analysis, three stained gels per year were selected (Table 3) for each sample. The Brilliant Blue G-Colloidal Concentrate Coomassie
54 (Sigma) staining for preparative analysis was performed according to manufacturer’s instructions with some modifications. The protocol is described below:
- Fixing solution: (1h) 40% Ethanol and 10% Acetic Acid - Staining solution: (overnight) Coomassie Brilliant Blue
- Washing solution: (few minutes) 25% Ethanol 10% Acetic Acid - De-staining solution: (3h) 25 Ethanol
- Stop solution: (long period) 5% Acetic Acid
-2.6 Image analysis and Statistical analysis
High resolution (300 dpi) images were acquired using the ProXpress CCD camera system (Perkin Elmer). Computer-assisted 2D image analysis was done using Progenesis SameSpots vs 3.2.3 gel analysis software (NonLinear Dynamics) for three technical replicates for each biological condition (different years) from three independent extraction experiments procedures (Tab 3). This software offer several advantages, among which a complete statistical tool (for instance 1-way ANOVA) and an image detection tool able to analyze the quality of the scanned images and to detect saturation and grey depth problems that could decrease the analysis’ accuracy (Fig 26).
55 Figure 26: Samespots image check
Protein apparent relative molecular mass (Mr) was estimated by comparison with molecular weight (MW) reference markers (Precision, Bio-Rad, Hercules, CA) and pI values assigned to detected spots by calibration as described in the GE-Healthcare guide lines. The amount of protein was expressed as spot volume, which was defined as the sum of optical density of all the pixels that make up the spot as detected by the software. Protein
56 level increase/decrease was quantified comparing the spot volumes normalized as percentage of the total volume in all the spots present in the gel.
Spots were considered to represent differentially expressed proteins on the basis of their ANOVA values (q-value) and fold change as evaluated by the software. Image software automatically order spots on the base of these values. Post-test analysis (Tukey’s test) was performed on the basis of the ANOVA results, in order to identify specific correlations among the samples. The relevance of each spot in discriminating between samples from different places was evaluated by principal component analysis as software tool for different combinations of differentially expressed spots.
2.7 Protein identification by MALDI-TOF and
nLC-ESI-MS\MS
Protein spots of interest were excised from gels, reduced, alkylated, and digested overnight with bovine trypsin (Roche Diagnostics Corp.) as previously described by Shevchenko et al., 1996. Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins (Leiros et al., 2004, Rawlings and Barrett 1994). Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline. In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Aliquots of the supernatant (1 µL) were used for MS
analysis through MALDI-TOF (Matrix-Assisted Laser
Desorption/Ionization) mass spectrometer. MALDI-MS, first introduced in 1988 by Hillenkamp and Karas has become a widespread analytical tool for
57 peptides proteins, and most other biomolecules (oligonucleotides, carbohydrates, natural products, and lipids). MALDI is an ionization method that provides for the nondestructive vaporization and ionization of both large and small biomolecules (Fig 27). In MALDI analysis, the analyte is first co-crystallized with a large molar excess of a matrix compound, usually a UV-absorbing weak organic acid, after which laser radiation of this analyte–matrix mixture results in the vaporization of the matrix which carries the analyte with it. The matrix therefore plays a key role by strongly absorbing the laser light energy and causing, indirectly, the analyte to vaporize. The matrix also serves as a proton donor and receptor, acting to ionize the analyte in both positive and negative ionization modes, respectively (Hilenkamp et al., 1986).
Figure 27: MALDI ionization method (from Lewis et al., 2006)
Matrix assisted MS analysis was done using the dried-droplet technique, with α-cyano-4-hydroxycinnamic acid as a matrix. Mass spectra were obtained with a MALDI-TOF Voyager DE-STR from Applied
58 Biosystems/MDS Sciex. Ions were generated by irradiation with a pulsed nitrogen laser (337 nm UV, pulse duration 3 ns, pulse rate 3 Hz), and positive ions were accelerated and detected in the reflector mode. Instrument settings were: accelerating 20,000 V, grid 64%, guide wire 0%, delay time 200 ns, shots/spectrum 100, mass range 750-4000 Da and low mass gate 700 Da. Spectra were acquired via Voyager Control Panel 5.10 from Applied Biosystems. Once acquired, spectra were processed with Data Explorer 4.0 from Applied Biosystems and internally calibrated with trypsin autolysis products and matrix clusters. MALDI-TOF data led to extracted and manually curated peptide monoisotopic peak lists (deprived from trypsin and matrix clusters signals) that were searched, via in-house Mascot Server 2.2.07, against the target database as detailed below except for mass tolerance for monoisotopic data that was set to 50 ppm and significance threshold of p < 0.05 set for the probability based Mascot Mowse Score.
For Electrospray ionization (ESI) analysis, 5 µL of trypsin digested samples were injected in a capillary chromatographic system Agilent 1100 Series equipped with a Nano Pump, Iso Pump, Degaser and a 8 µL injection loop (Agilent). ESI is an ionization technique for small amounts of large and/or labile molecules such as peptides, proteins, organometallics, and polymers. The ESI source operates at atmospheric pressure. A sample solution is sprayed from a small tube into a strong electric field in the presence of a flow of warm nitrogen to assist desolvation (Fig 28). The droplets formed, evaporate in a region maintained at a vacuum of several torr causing the charge to increase on the droplets. The multiply charged ions then enter the analyzer. The most obvious feature of an ESI spectrum is that the ions carry
59 multiple charges, which reduces their mass-to-charge ratio compared to a singly charged species. This allows mass spectra to be obtained for large molecules. For example, apo-myoglobin with a molecular weight of 16,951.5 Da produces a series of ions with charge states from +8 to +27 with mass peaks from about 600 to 2000 Da.
Figure 28: Overview of an Electro Spray Ionization (LC-MS) Interface (from
http://people.whitman.edu/~dunnivfm/C_MS_Ebook/CH5/5_3_1.html)
Peptide separations occurred on a 10-15 cm fused silica emitter (75 µm i.d., 360 µm o.d.; Proxeon Biosystems) used as analytical RP (reverse phase) nano column. The emitter was packed in-house with a methanol slurry of reverse-phase, fully end-capped 3-µm ReproSil-Pur 120 C18-AQ resin (Dr. Maisch GmbH), using a pressurized “packing bomb” operated at 50-60 bars.
Mobile phases consisted of water with 2% acetonitrile, 0.1% formic acid (v/v; buffer A) and acetonitrile with 2% water, 0.1% formic acid (v/v; buffer B). A 55-min gradient from 8% to 80% buffer B at a constant flow rate of 200 nl/min was used for peptides separation.
60 Biosystems) and analyzed on an API QStar PULSAR (PE-Sciex) mass spectrometer. Analyses were performed in positive ion mode. The HV Potential was set up around 1.8-2.0 kV. Full scan mass spectra ranging from m/z 350 to 1600 Da were collected and for each MS spectrum the two most intense doubly and triply charged ions peaks were selected for fragmentation (MS/MS range from m/z 100 to 1600 Da).
MS/MS spectra data files from each chromatographic run were combined and converted to mgf files using Mascot.dll (version 1.6b27) through Analyst QS 1.1 (Applied Biosystems) and searched (via Mascot Daemon 2.2.2 and in-house Mascot Server 2.2.07), first against a custom contaminant database (trypsin and common keratins partly derived from the cRAP collection), unmatched signals were then searched against the UniProt_Complete Proteome_tuber 2012_07 (7679 sequences; 3339250 residues) database cause the lack of a specific T. magnatum protein dataset. Mass tolerance was set to 200 ppm and 0.3 Da for precursor and fragment ions respectively. Searches were performed with trypsin specificity, alkylation of cysteine by carbamidomethylation, and oxidation of methionine as fixed and variable modifications respectively; ion score cut-off set to 20; two missed cleavages were allowed for trypsin specificity; the quality of MS/MS identifications was manually checked.
Because the tandem mass spectra contain structural information related to the sequence of the peptide, rather than only its mass, these searches are generally more specific and discriminating (Mann et al., 2001), a main feature for the analysis of no sequenced organism.
Proteins obtained without functional identification were then used for Protein Blast Analysis (UniprotKb blast p) performed with default settings.
61
2.8 GO enrichment
Gene ontology (GO) term enrichment analysis to find statistically over- or under represented categories was performed with BiNGO 2.44 as a plugin for Cytoscape 2.8.0; the latest available ontology (obo 1.2 format) and Tuber spp. annotations files were downloaded respectively from the Gene Ontology and from the Gene Ontology Annotation (GOA) websites. Hypergeometric test, Benjamini & Hochberg false discovery rate correction and a significance level of 0.05 were chosen as parameters to visualize in Cytoscape the over-represented categories after correction. Due to the interdependency of functional categories in the GO hierarchy, it is very likely that not one category, but a whole branch of the GO hierarchy lights up as being significantly over-represented. In such cases the nodes which are furthest down the hierarchy were chosen to be reported.
2.9 SNPs analysis
The SNPs can be broadly defined as any single base substitution/indel in the genome of an individual (Brookes 1999). They are used as a powerful marker for mutational analysis in humans and are normally found in non-coding regions subject to less selection (Mello et al., 2005). Mello and co-workers discover in T. magnatum the presence of two distinct SNPs in one polymorphic SCAR-RAPD fragment (640 Bp), named as A21-inf. This sequence showed no significant similarity to known sequences in GenBank. These SNPs sequence is able to distinguish a wide range of T. magnatum samples in three different haplotype families (Fig 29).
62 Figure 29. Two SNPs, indicated by a star, in SCAR A21-inf originating the haplotypes I, II
and III. (from Mello et al., 2005)
DNA was extracted from fruiting bodies using CTAB extraction method (see above, Microbial analysis). Amplification was performed using the following protocol:
The following parameters for PCR amplification of the SCAR sequence (primer pair CL1/CL2, Table 4) were used: 94ºC for 5 min (initial denaturing period); 94ºC for 0.45 min (denaturing period), 55ºC for 0.45 min (annealing period), 72ºC for 1 min (extension period) for 35 cycles and 72ºC for 5 min (final extension period).The PCR product was of the predicted size SCAR A21-inf (572 bp), as determined by agarose gel electrophoresis (2 µl of PCR mixture, 2% agarose gel) ethidium bromide staining. The amplicons were sequenced (BMR Genomics, Padova Italy) and their relative sequences were used to perform a multiple sequence
63 (http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/) to obtain a phylogenetic tree.
Table 4: Primer pair designed on lucus A21-inf and size of the amplified product
2.10 Total RNA extraction and Real-Time PCR analysis
Total RNA was extracted from pulverized samples as described by (Chomczynski and Sacchi 2006).
This protocol was selected on the basis of its ability to remove contaminants from RNA extracted samples. Electrophoresis using 1% agarose gel was performed for all RNA samples to check for RNA integrity, followed by spectrophotometric quantification and quality control. RNA samples were then subjected to DNase treatment using a Turbo DNA-free kit (Ambion, USA) to remove possible DNA contamination. RNA was then reverse-trascribed using SuperScript® III Reverse Transcriptase kit (Life Technologies, UK) with random primers. Gene expression analysis was carried out using an ABI Prism 7300 sequence detection system (Applied Biosystems, USA) as described by (Licausi et al., 2010) Quantitative PCR was performed using 30 ng cDNA and iQ™ Sybr Green Supermix (BioRad laboratories), according to the manufacturer’s instructions. Two technical replicates were performed for each biological replicate (n=4).
Expression of T. magnatum (AF054901) 18S rRNA was used as a housekeeping gene. Relative expression levels were calculated using Genorm (http://medgen.ugent.be/genorm/). Primers were designed using
64 Primer 3. The following primer list is reported with its specific Tuber gene identification code (in brakets Uniprot code of the relative protein):
GSTUM_00009270001 (D5GJY5), gcactggcaccactcctacc-3’(forward), gaagaaggtgcccccaaaac-3’ (reverse); GSTUM_00007439001 (D5GGN7), 5’-gaccaggaataccgcaccaa-3’ (forward), 5’-tcctcctcagccttgtgagc-3’ (reverse); GSTUM_00008874001 (D5GJ78), gcgccatcaaggatattgga-3’ (forward), aacaccagtggcgatgtcct-3’ (reverse); GSTUM_00003332001 (D5G9M7), 5’-tcctctcgctcgcctatgag-3’ (forward), 5’-aacttcgacgaggtccacca-3’ (reverse); GSTUM_00005271001 (D5GAF9), gttttgacacccgccgataa-3’ (forward), aaggttcctgcacccacaga-3’ (reverse); GSTUM_00000555001 (D5GC43), 5’-gttgaaaacgcacgcctctc-3’ (forward), 5’-gccctcatcctcgacaacac-3’ (reverse); GSTUM_00005237001 (D5GAC6), acctgtgcgattctggtgct-3’ (forward), atccgtaggctcgccaaaat-3’ (reverse); GSTUM_00006427001 (D5GE86), 5’-gaagccaatctcggaggtga-3’ (forward), 5’-aaaacggcttccggtgtctt-3’ (reverse); GSTUM_00001447001 (D5G5R4), gagctcctcggaaagcatca-3’ (forward), 5’-ccaggaagaggggtttgtcc-3’ (reverse); DQ223686.1 (Q1ACW3), 5’-gaaggctctccgctacgaca-3’ (forward), 5’-accgcaagccttgactttga-3’ (reverse); AF054901, 5’-actagggatcgggcgatgtt-3’ (forward), 5’-cagccttgcgaccatactcc-3’
