Estimation of the relative amount of the virus
To estimate the relative amount of the virus in apricot scions grafted
onto the four transgenic plum rootstocks, RNA extraction was performed
from plants, which were revealed to be RT-PCR positive.
Leaves harvested were snap-frozen in liquid nitrogen and stored at −
80°C until use. Plant RNA extraction was performed using the commercial
”RNeasy Plant Mini Kit” (Qiagen), following manufacturer instructions.
RNAs were digested with DNase I using the DNA-free Kit (Ambion, Austin,
TX, USA) and quantified using a spectrophotometer Nanodrop ND-1000
(Nanodrop Technologies, Wilmington, USA). cDNA was synthesized using the
RETROscript cDNA Synthesis Kit (Promega, Madison, WT, USA) following
manufacturer instructions. PPV concentration was established by
real-time RT-PCR using the GeneAmp 7500 sequence detection system
(Applied Biosystems, Foster City, CA, USA). The EF1-a gene was used for
the normalization. The cDNA was synthesized as described above, and the
qPCR was carried out using the SYBR Green Master Kit (Applied
Biosystems).
Primers used in this study were PPV-U (TGAAGGCAGCAGCATTGAGA) and PPV-RR
(CTCTTCTTGTGTTCCGACGTTTC) to amplify PPV, designed by Varga and James,
(2005), and q35s5F (CTCATTCACTTGCCACCTCG) and q35s5R
(ATGCACGTTACTGACTTGGC) for the transgene, specifically designed for this
study. For the housekeeping EF1-α gene amplification primers TEF2-f
(GGTGTGACGATGAAGAGTGATG) and TEF2-r (TGAAGGAGAGGGAAG GTGAAAG) were used.
Each set of primers was mixed at a final concentration of 300 nM with 2
µl cDNA and 1 × SYBR Green. After denaturation at 95 °C for 10 min, a
two-step procedure of 15 s denaturation and 1 min of annealing and
extension at 60 °C for 40 cycles was adopted. These conditions were used
for target and reference genes, and the absence of primer dimers was
checked in controls lacking templates. Each biological replicate was a
pool of three different plants, and three biological replicates were
used. For each biological replicate, six technical replicates were run.
For the calculation of relative virus content, the
2-ΔΔCt method was used (Livak and Schmittgen, 2001).
The Ct value was adjusted automatically, and the threshold cycle value
difference (ΔCt) between the Ct of the target gene (virus or transgene)
and Ct of EF1α (internal control) was used to normalize the amount of
the virus gene. As long as the target gene and the internal control have
similar amplification efficiencies, Ct values were normalized using the
difference (ΔCt) between the internal control and target gene. This
value is calculated for each sample to be quantified. Finally, the
relative quantification of the virus gene in each sample was calculated
according to the formula where the reference sample was the susceptible
line St5’-7. Relative quantification = 2−ΔΔCt, where
ΔΔCt = ΔCt (unknown sample) - ΔCt (reference sample).