The mycelia were homogenized in their medium, filtered through Miracloth (Calbiochem, Canada), rinsed with sterile 0.9% saline solution then resuspended in 50 mL of 2nd stage medium to induce mycotoxin production containing either 2 mM L-TRP, 0.2 mM TAM, 0.2 mM IAAld or no treatment (NT) and shaken at 200 rpm, at 28ºC in the dark. In control samples (no treatment), mycelia was transferred to 2nd stage medium without the addition of any intermediate.. Three biological replicates were grown for each treatment; mycelia were collected for 6 h after treatment, filtered and immediately submerged into liquid nitrogen.
Growth protocol
Mycelia from F. graminearum strain DAOM180378 was grown as follows: 1x106 macroconidia were inoculated into 50 mL of 1st stage medium (Taylor et al 2008. Proteomics. 8: 2256-2265) in 250 mL Erlenmeyer flasks, 200 rpm, 28 ºC, in the dark for 72 h. The mycelia were homogenized in their medium, filtered through Miracloth (Calbiochem, Canada), rinsed with sterile 0.9% saline solution then resuspended in 50 mL of 2nd stage medium (Taylor et al 2008) to induce mycotoxin production containing either 2 mM L-TRP, 0.2 mM TAM, 0.2 mM IAAld or no treatment (NT) and shaken at 200 rpm, at 28ºC in the dark. In control samples (no treatment), mycelia was transferred to 2nd stage medium without the addition of any intermediate.
Extracted molecule
total RNA
Extraction protocol
The frozen mycelia were ground using a mortar and pestle and stored at -80 ºC. RNA from the ground mycelia was extracted using TRIZOL reagent (Invitrogen, Life Technologies Co., Canada), following the manufacturer’s protocol, and then purified further using the RNeasy Mini Kit (Qiagen Science, Canada) and the RNA cleanup protocol which included a RNase-free DNase I treatment. The RNA quality and quantity were determined using the 2100 Bioanalyzer (Agilent Technologies Inc., Canada).
Label
Cy5
Label protocol
500 ng total RNA was labelled using the Two-Color Quick-Amp Labelling Kit (Agilent Technologies) using the manufacturer's instructions except using half the reagent volumes in Step 2 of the protocol.
The mycelia were homogenized in their medium, filtered through Miracloth (Calbiochem, Canada), rinsed with sterile 0.9% saline solution then resuspended in 50 mL of 2nd stage medium to induce mycotoxin production containing either 2 mM L-TRP, 0.2 mM TAM, 0.2 mM IAAld or no treatment (NT) and shaken at 200 rpm, at 28ºC in the dark. In control samples (no treatment), mycelia was transferred to 2nd stage medium without the addition of any intermediate.. Three biological replicates were grown for each treatment; mycelia were collected for 6 h after treatment, filtered and immediately submerged into liquid nitrogen.
Growth protocol
Mycelia from F. graminearum strain DAOM180378 was grown as follows: 1x106 macroconidia were inoculated into 50 mL of 1st stage medium (Taylor et al 2008. Proteomics. 8: 2256-2265) in 250 mL Erlenmeyer flasks, 200 rpm, 28 ºC, in the dark for 72 h. The mycelia were homogenized in their medium, filtered through Miracloth (Calbiochem, Canada), rinsed with sterile 0.9% saline solution then resuspended in 50 mL of 2nd stage medium (Taylor et al 2008) to induce mycotoxin production containing either 2 mM L-TRP, 0.2 mM TAM, 0.2 mM IAAld or no treatment (NT) and shaken at 200 rpm, at 28ºC in the dark. In control samples (no treatment), mycelia was transferred to 2nd stage medium without the addition of any intermediate.
Extracted molecule
total RNA
Extraction protocol
The frozen mycelia were ground using a mortar and pestle and stored at -80 ºC. RNA from the ground mycelia was extracted using TRIZOL reagent (Invitrogen, Life Technologies Co., Canada), following the manufacturer’s protocol, and then purified further using the RNeasy Mini Kit (Qiagen Science, Canada) and the RNA cleanup protocol which included a RNase-free DNase I treatment. The RNA quality and quantity were determined using the 2100 Bioanalyzer (Agilent Technologies Inc., Canada).
Label
Cy3
Label protocol
500 ng total RNA was labelled using the Two-Color Quick-Amp Labelling Kit (Agilent Technologies) using the manufacturer's instructions except using half the reagent volumes in Step 2 of the protocol.
Hybridization protocol
F. graminearum custom-designed 4x44K microarrays (Agilent Technologies, AMADID 020717) (NCBI, GEO record# GPL11046) were hybridized overnight at 65°C in the dark with 825 ng each cy3 and cy5 labelled probes in a Robbins Scientific Model 400 incubator and an Agilent Scientific G2530-60020 rotator. Hybridization was carried out as per manufacturer's protocol. Slides were washed as per protocol except omitting the 3rd wash (Stabilization and Drying Solution).
Scan protocol
Slides were scanned using a Genepix 4200a scanner (Molecular Devices) at 5µm resolution. Feature-finding analysis was performed in Genepix Pro 6.0 and then manually correct any misaligned features. Raw data was saved as .gpr files and imported in Acuity 4.0 (Molecular Devices) for analysis.
Data processing
Genepix Pro 6.0 .gpr files for each array were imported in Acuity 4.0. The data was normalized with the Normalization Wizard using Lowess M log ratio. A dataset was created and spike-in and other controls were removed. Features within the dataset that had Lowess A values above 7.5 in at least 2 of the 6 arrays were kept. Reverse-dye replicates were combined after cy3 test columns were swapped to cy5. The normalized data was analysed in Acuity 4.0 to identify candidate genes that were significantly differentially expressed, using the following cut-off parameters: t-test p-value < 0.05 and fold changes ≥ 4 in expression ratios (i.e. log2 ratio ≥ 2.0 or -2.0; L-TRP vs control).
