Strain
The Oidiodendron maius (MUT1381/ATCC MYA-4765) isolate used here had been isolated from roots of Vaccinium myrtillus growing in zinc-contaminated experimental plots in the Niepolomice Forest, Poland according to Pearson and Read (58; 59). The isolate is capable of forming typical ericoid mycorrhizae with axenic Calluna vulgaris (60) and Vaccinium myrtillus (40) seedlings. The genome of O. maius as well as transcriptomes for both the free-living mycelium and the fungus in symbiosis with Vaccinium myrtillus, have been sequenced (44).
In-silico analysis of RNA-seq data
Five RNA-seq datasets from O. maius Zn were analyzed in the current study: three unpublished data sets (E. Feldman, unpublished) and two from Kohler et al. (44; the complete data sets have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE63947). In all cases, fungal cultures were grown for 45 days on Modified Melin Norkrans (MMN) plates, overlaid with sterile cellulose membranes (autoclaved in ddH20 twice, with 24 hours in between), with a range of amendments (Table 3). The MMN medium contained: 0.075 g L− 1 (NH4)2HPO4 (filter sterilized, 0.2 µm, added after autoclaving), 1 g L− 1 glucose, 0.5 g L− 1 KH2PO4, 0.066 g L− 1 CaCl2.2H2O, 0.025 g L− 1 NaCl, 0.15 g L− 1 MgSO4.7H2O, 0.1 g L− 1 thiamine HCl (filter sterilized, 0.2 µm, added after autoclaving), 1 mg L− 1 FeCl3.6H2O and 10 g L− 1 agar; pH was adjusted to 4.7.
Table 3
Amendments made to Modified Melin Norkrans media to support growth of Oidiodendron maius.
Treatment Name
|
Study
|
Glucose (1 g L− 1)
|
(NH4)2HPO4
|
BSA
(0.1 g L− 1)
|
Peat1
(~ 10 g dry weight)
|
Notes
|
FLM/NH4
|
present work
|
+
|
+
|
-
|
-
|
|
FLM/Peat
|
present work
|
-
|
-
|
-
|
+
|
|
MYC/BSA
|
Kohler et al. 2015
|
-
|
-
|
+
|
-
|
O. maius colonizing Vaccinium myrtillus roots
|
1 Sunshine brand, Sun Gro Horticulture Canada Ltd.; sterilized via electron beam radiation (Iotron Industries Canada Inc.; 35 kGy) |
Three criteria were applied to these five transcriptomes to generate a short list of potential candidate reference genes: (1) the fold change (based on Reads Per Kilobase Million; RPKM) was equal to approximately one (between 0.8 and 1.2) among all treatments (uniform expression despite treatment difference, as suggested by Manoli et al. (61)), (2) annotation was available (including a valid annotation from InterPro (excluding “protein of unknown function”) and a valid Gene Ontology name), and (3) homologous genes had been used previously in other fungi as internal reference genes for qPCR. The online tool Heatmapper (62) was utilized to construct the heatmap from RPKM Means; no clustering method was used.
Cultivation of Oidiodendron maius for use in ddPCR
Because we planned to assess expression of carbohydrate-active genes in future research, we cultivated O. maius on a greater variety of carbon sources than those previously examined by RNA-SEq. O. maius cultures were maintained on Czapek-glucose agar (3 g L− 1 NaNO3; 1 g L− 1 K2HPO4; 0.5 g L− 1 MgSO4.7H2O; 0.01 g L− 1 FeSO4.7H2O; 0.5 g L− 1 KCl; 20 g L− 1 glucose; 10 g L− 1 agar; pH adjusted to 6) for 45 days in the dark at 25 °C. Fungal plugs from these plates were transferred to glass mesh filters (Whatman Grade GF/F Glass Microfiber Filters, Binder Free, GE Healthcare, 0.6–0.8 µm particle retention) overlaid on MMN 0.1% glucose plates supplemented with one of eleven additional carbon sources (Table 4) for a further 45 days in the dark at 25 °C (plates contained (NH4)2HPO4 as the sole nitrogen source, unless otherwise stated, to provide a nitrogen source where it may be limiting). The experimental ddPCR assays were performed on cDNA samples from these eleven experimental treatments using at least three biological replicates.
Table 4
Treatment groups for growth of Oidiodendron maius for use in ddPCR*
Carbon Source
|
Source C Added (g L− 1)
|
N source (g L− 1)
|
glucose (0.1%)
|
|
(NH4)2HPO4 (0.075)
|
glucose (0.5% total)
|
0.4
|
(NH4)2HPO4 (0.075)
|
peat
|
0.1
|
(NH4)2HPO4 (0.075) + peat
|
peat dissolved organic matter (DOM)
|
200 mL L− 1
|
(NH4)2HPO4 (0.075) + peat DOM
|
field soil
|
0.1
|
(NH4)2HPO4 (0.075) + field soil
|
field soil organic matter (SOM)
|
200 mL L− 1
|
(NH4)2HPO4 (0.075) + field SOM
|
Bovine Serum Albumin (BSA)
|
0.1
|
(NH4)2HPO4 (0.075) + BSA
|
cellulose
|
0.113
|
(NH4)2HPO4 (0.075)
|
chitin
|
0.113
|
(NH4)2HPO4 (0.075)
|
pectin
|
0.119
|
(NH4)2HPO4 (0.075)
|
lignin
|
0.08
|
(NH4)2HPO4 (0.075)
|
*Cultures were grown on solid Modified Melin Norkrans media in preparation for RNA extraction and subsequent transcript abundance studies. Added source C is in addition to the 0.1 g L− 1 glucose contained in the base media. |
RNA Extraction and cDNA synthesis
Fungal tissue was removed from glass mesh filters with a sterile scalpel, placed in pre-weighed RNase-free 1.5 mL Eppendorf tubes and flash-frozen in liquid nitrogen. Up to 100 mg of flash-frozen fungal tissue was mechanically disrupted by grinding in liquid nitrogen in a sterile mortar and pestle and placed in RNase-free 1.5 mL Eppendorf tubes on ice. To each tube, 700 µL extraction buffer (100 mM Tris-HCl pH 8, 100 mM NaCl, 20 mM Na-EDTA, 0.1% PVP, 1% sodium-lauryl sarcosine, prepared in diethylpyrocarbonate (DEPC) water) and 700 µL acid phenol were added prior to gently inverting the tubes. Tubes were centrifuged for 5 minutes at 14000 rpm (4 °C); all subsequent centrifugation was also performed at 14000 rpm (4 °C). The uppermost phase of the supernatants was transferred to new Eppendorf tubes on ice, to which an equal volume of acid phenol-chloroform-isoamyl alcohol (25:24:1) was added. Tubes were gently inverted several times, then centrifuged for 5 minutes. Supernatants were transferred to new tubes containing an equal volume of chloroform, tubes were gently inverted several times and then centrifuged for 5 minutes. This chloroform extraction and centrifugation was repeated once. Total nucleic acids were precipitated by addition of an equal volume of isopropyl alcohol to the supernatant and gentle inversion. Tubes were incubated for 30 minutes at -80 °C, then centrifuged 30 minutes. Supernatant was discarded and the pellet was resuspended in 500 µL DEPC water. To each tube 500 µL 6 M LiCl was added, then the tubes were gently inverted and kept overnight in ice at 4 °C. Tubes were centrifuged 30 minutes, then the supernatant was discarded and the remaining pellet was washed with 150 µL 70% ethanol (prepared with DEPC water). The tubes were centrifuged for an additional 5 minutes, the supernatant discarded and the pellet was dried on ice in the fume hood. Once completely dry, the pellet was resuspended in 25 µL DEPC water, then the concentration and quality were determined using a NanoDrop ND-1000 UV-visible light spectrophotometer (NanoDrop, Wilmington, DE, USA). Only RNA samples with 260/280 nm wavelength ratio of approximately 2 and 260/230 nm wavelength ratio of approximately 2 were retained. RNA solutions were treated with PerfeCTa® DNase I according to manufacturer’s instructions (Quanta Biosciences™, Beverly, MA, USA). DNase-treated RNA was converted to cDNA using the BioRad iScript RT Supermix for RT-qPCR according to manufacturer’s protocol (Bio-Rad Laboratories, Inc., Hercules, California, USA) and stored at − 20 °C.
Primer Design and Validation by Conventional PCR Detection
Primers were designed for the three candidate reference genes using the IDT PrimerQuest tool which incorporates Primer3 software (version 2.2.3; Integrated DNA technologies, Skokie, Illinois) using the qPCR Intercalating Dyes parameters. Additional parameters included a product size of 75–200 bp (optimum = 125 bp), melting temperature of 50─65 °C (optimum = 59 °C), GC content of 50─60% (optimum = 55%), GC clamps on both ends (3’ GC clamp = 2 nt), 50 mM salt concentration, 300 nM oligonucleotide concentration and minimum overlap of 4 nt at either end. Three primer pairs were chosen for each transcript based on forward and reverse primers having similar GC content and melting temperature, where the target sequence had a single hit when BLASTed against the O. maius model filtered transcript dataset (40).
cDNA from the MMN + Peat treatment was used for all primer validation by conventional PCR. PCR reaction conditions were 1x GoTaq buffer, 200 µM dNTPs, 1 U GoTaq, 0.1 µM F primer, 0.1 µM R primer, 100 ng cDNA, 5% DMSO. Touchdown PCR was run: 3 min. @ 94 °C + 10(1 min. @ 94 °C + 1 min. @ 65Δ − 1 °C + 1 min. @ 72 °C) + 30(1 min. @ 94 °C + 1 min. @ 60 °C + 1 min. @ 72 °C) + 9 min. @ 72 °C + ∞@ 4 °C.
All PCR products were run for 60 minutes at 90 V on a 1% agarose gel containing Invitrogen™
SYBR™ Safe. Gels were photographed under UV light and bands containing the correctly sized amplicons were excised. These excised gel fragments were cleaned using the QIAGEN QIAquick Gel Extraction kit as per manufacturer’s instructions. The resulting extractions were sequenced on an Applied Biosystems 3130xl DNA sequencer in the Fragment Analysis and DNA Sequencing Services lab at the University of British Columbia Okanagan campus. Sequenced amplicons were reBLASTed against the O. maius model filtered transcript dataset (40) to ensure a single hit with the intended target.
ddPCR Assay with EvaGreen
Validated primers were used in a ddPCR Assay using a QX200™ ddPCR™ system (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's standard EvaGreen® protocol. Briefly, each reaction contained 2 µL of cDNA, 100 nM of each forward and reverse primer, 1X ddPCR EvaGreen Supermix, 5% DMSO and molecular-grade water to 20 µl. Reactions were loaded into the sample wells of a DG8 droplet generation cartridge (Bio-Rad). Seventy µl of Droplet Generation Oil for EvaGreen (Bio-Rad) were loaded into the oil wells, and the cartridge was placed in the QX200Droplet Generator (Bio-Rad). The resulting droplets were transferred to a 96-well Bio-Rad PCR plate. The PCR plate was then heat-sealed with a foil seal and placed in the thermocycler. Reaction conditions consisted of initial enzyme activation period at 95 °C for 5 min; followed by 40 cycles of denaturing at 95 °C for 30 s and annealing/extension for 1 min; then dye stabilization at 4 °C for 5 min and 90 °C for 5 min; the ramp rate was 2.5 °C/sec. Optimal annealing temperature was determined by running a temperature gradient for each primer pair ranging from 54 °C to 60 °C.
After the amplification, plates were loaded into the Bio-Rad QX200 DropletReader for enumeration of the number of positive and negative droplets based on fluorescence. The number of template molecules per microliter of starting material was estimated by the QuantaSoft®AP software (version 1.6.6.0320, Bio-Rad) using an internal Poisson algorithm to analyze clusters; only droplets above a minimum amplitude threshold were counted as positive. For each primer pair, the PCR reaction mixture without matrix was used as negative control (no template control, NTC). Three biological replicates were run for each carbon source, unless otherwise specified.
ddPCR Stability Analysis
The stability of the putative reference genes was assessed using the geNormv3 (18) and the NormFinder (54) add-ins for Microsoft Excel. The geNorm add-in allows the calculation, for each reference gene, of the gene expression stability value M, which is the average pairwise variation of a particular gene with all other genes. The most stable genes present the lowest M values; genes with M value ≤ 1.5 are considered highly stable across analyzed samples. Normfinder uses a model-based approach that provides an estimate of both intra- and intergroup expression variation, and calculates a gene stability value; the smaller the stability value, the more appropriate the use as a reference gene.