All quantitative Real time PCR primers were designed using AlleleID® (Primear Biosoft) using default parameters for SYBR green except the primer annealing temperature was changed to 60-62°C.
2.5.2 PCR Cycling conditions
Relative quantification or transgene copy number prediction was performed using a LightCycler 480 SYBR Green 1 DNA Master kit (Roche, Penzberg Germany). 2 μL of 10-fold serially diluted cDNA were added to 8 μL PCR mix and subjected to 45 cycles of PCR (10 sec at 95°C, then 30 sec at each of 60°C and 72°C) with an acquisition temperature of 72°C. For melting curve analysis PCR products were heated to 95°C and then rapidly cooled to 65°C for 10 sec and then gradually increased to 97°C at the rate of 0.2°C/sec with continuous measurement of fluorescence at 520 nm. The Ct value is the cycle number at which the fluorescence emission of the PCR amplicon could be distinguished from the background. For all the expression analysis Ct = 35 cycles, were
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considered for analysis. Real time PCR was carried out using a LightCycler® 2.0 and all
the data were analysed using RealQuant® version 1.1.1 (Roche, Basal, Switzerland)
2.5.3 Standard curves and gene expression analyses
For Real time PCR, a standard curve was prepared for each of the primer pairs when required, total RNA was extracted and cDNA synthesized as mentioned in section 2.7.1. A standard curve was constructed by plotting the logarithm of six 10-fold serial dilutions of D. septosporum cDNA or genomic DNA against the cycle threshold (Ct) values with three replications for each dilution. All data were processed using Real Quant 1.1.1 (Roche) and the standard curve was saved as an external standard in the database.
Expression analysis of all the dothistromin biosynthesis genes in various mutants and wild type strain of D. septosporum was done relative to the 18s rRNA gene
or β-tubulin. Three technical replicates of each of three biological replicates were used for calibrator normalized relative quantification analysis, and the values were normalized by comparing to expression levels in the NZE10 wild type.
The results were expressed as a normalized ratios.
Example: To study AflR gene expression in LaeA knockout strain: Target gene is AflR;
Reference gene is βtubulin or 18s rRNA gene (stable constitutive expression). Control is Wild type (WT) strain
(E
target)
ΔCt target (control – sample)(
E
ref)
ΔCt ref (control – sample)Ratio =
50 Sample is Δ LaeA strain
Etarget is the PCR efficiency of target gene primer ; Eref is the PCR efficiency of reference
gene primer.
For target gene; ΔCt target = Ct of AflR expression in WT – Ct of AflR expression in Δ
LaeA strain.
For reference gene; ΔCt ref = Ct of βtubulin or 18s rRNA expression in WT – Ct of
βtubulin or 18s rRNA expression in Δ LaeA strain. (Pfaffl, 2001).
The expression studies were done using β-tubulin or 18s rRNA as reference genes and the results obtained were consistent but only the β-tubulin data are presented.
2.5.4 Quantification of fungal biomass in infected pine needle
To estimate the biomass of D. septosporum in the infected pine needles, DNA was extracted from lesions of infected needles for each strain as mentioned above in 2.3.1 and vacuum-dried if necessary to yield a minimum concentration of 500 ng/L. Primer and TaqMan probe sets for real-time PCR assays (Table 2.3) were designed to detect D. septosporum polyketide synthase PksA (as target gene) and Pinus radiata cinnamyl alcohol dehydrogenase CAD (as reference gene). The PksA probe was labelled with 6- carboxyfluorescein (FAM) at the 5’ end and quencher dye BHQ-1 at the 3’ end. The CAD probe was labelled with HEX (5’) and BHQ-1 (3’). qPCR was carried out as mentioned in 2.5.2 with slight modification, 0.2 μM of each FAM-labeled and HEX- labeled probe and 0.4 μM forward and reverse primer for each target and reference gene, in a reaction volume of 10 μL was used. To estimate fungal biomass, standard curves were prepared as mentioned above in 2.5.3 with serial dilutions of pure genomic DNA of D. septosporum and P. radiata in distilled water (1000, 200, 40, 8, 1.6, 0.32, 0.064 ng for pine and pg for D. septosporum). The relative amounts of fungal biomass
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in the samples were calculated as fungal target/plant reference and normalised by dry weight of the samples.
2.5.5 Predicting the copy number of transgene
Transgene copy number prediction was done following the strategy outlined by (Solomon, et al., 2010). To verify the copy number of the AflJ complemented strains by qPCR firstly a single copy reference gene (DsAflR) gene was selected whose copy number had been verified previously by an in silico genome search and Southern hybridisation. The standard curves for each of the primer pairs were prepared as mentioned in section 2.5.3. Genomic DNA from each of the AflJ complemented strains
was extracted (2.3.2) and diluted to 10 ng/μl as this concentration was found to give
optimal efficiency. qPCR was performed as mentioned above, and all concentrations were normalised to the single copy DsAflR. D. septosporum wild type strain DNA was taken as a positive control to cross check the copy number of DsAflJ and to validate the use of qPCR to determine gene copy number. All analysis was done using RealQuant 1.1.1 (Roche, Basel, Switzerland) software. Subsequent melting curve analysis was done to confirm the absence of any non-specific amplification.