Quantitative RT–PCR

Arabidopsis plants were grown and infected with nematodes as described above. Root segments containing the infection zone were cut, and total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s instructions. Contaminating DNA was digested with DNase1 using a DNA-free™ DNA Removal Kit (Ambion) and the RNA was used to synthesize cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosynthesis, Darmstadt, Germany) following the manufacturer’s instructions. Quantitative reverse transcription–PCR (qRT–PCR) was performed with the StepOne Plus Real-Time PCR System (Applied Biosystems) using the primers given in Supplementary Table S1. Each sample contained 10 μl of Fast SYBR Green qPCR Master Mix (Invitrogen), 2 mM MgCl₂, 0.5 μl each of forward and reverse primers (10 μM), 2 μl of cDNA, and water in a 20 μl total reaction volume. UBQ5 and β-tubulin was used as an endogenous control except for assays involving nematode feeding sites (galls and syncytia). For galls and syncytia, 18S and UBP22 were used as housekeeping genes as recommended previously (Hofmann and Grundler, 2007). cDNA was diluted 1:100 for 18S amplification. Data were analysed using Pfaffl’s method (Pfaffl, 2001). Data shown are an average of three independent experiments. Each experiment consisted of three technical replicates. Primer sequences used for qRT–PCR analysis along with their respective efficiencies are listed in Supplementary Table S1.