CDNA synthesis and qRT-PCR validation of microarray

One microgram of RNA from each of the five samples from each treatment were reverse transcribed by M-MuLV Reverse Transcriptase (New England Biolabs, Ipswich, MA, USA) with oligo-(dT)₁₅ primers to obtain cDNA. To create highly specific primers for PR gene family members, nucleotide sequences of for the PR-1, PR-3, PR-4, and PR-10 families were aligned using MUSCLE [72] in Geneious [73]. qRT-PCR primers were designed to target bases that differentiate family members. Primer sequences are listed in Additional file 18: Table S15. qRT-PCR was performed in a total reaction volume of 10 μL containing 4 μL of diluted cDNA (1:8), 5 μL of SYBR Green PCR Master Mix (TaKaRa, Mountain View, CA, USA), 0.2 μL of Rox and 0.4 μL of each 5 μM primer. Each reaction was performed on each of the five samples per treatment in technical duplicate using the Applied Biosystem Step One Plus Realtime PCR System (Nutley, NJ, USA) with the following program: 15 min at 94 °C, 40 cycles of 15 s at 94 °C, 20 s at 60 °C, and 40 s at 72 °C. The specificity of the primer pair was verified by dissociation curve.

Data normalization, a statistical randomization test, and relative pathogen-treated vs. water-treated expression ratios were computed using REST [64]. Fold changes with p-values less than 0.05 were considered significant.