Quantitative RT-PCR analysis
The expression of marker genes for the different defence related
pathways was analysed by real-time quantitative polymerase chain
reaction (qRT-PCR) using the gene specific primers shown in table S1.
Total RNA from leaves and roots was extracted using Tri-Sure (Bioline,
London, UK) according to the manufacturer’s instructions. The RNA was
treated with NZY DNase I (NZYtech, Portugal), purified through a silica
column using the RNA clean and concentratorTM (Zymo Research, USA) and
stored at −80 °C until use. The first-strand cDNA was synthesized with 1
μg of purified total RNA using the PrimescriptTM RT master mix (Takara,
Japan) according to the manufacturer’s instructions. The qRT-PCR was
conducted using the StepOnePlus™ (Applied Biosystem). Six independent
biological replicates were analysed per treatment. Expression values
were normalized using the housekeeping gene Actin (Solyc03g078400) and
relative quantification of specific mRNA levels was performed using the
comparative 2–Δ(ΔCt) method (Livak and Schmittgen, 2001).