## 4 Materials and Methods
### Isolation of Total RNA and Expression of O. vulgare Genes in Response to S. littoralis Herbivory  
To evaluate the effect of _S. littoralis_ herbivory on _O. vulgare_ terpenoid metabolism, we analyzed the expression level of terpene synthases and cytochrome P450s previously known to be involved in the production of the main _O. vulgare_ terpenoids [(8; 10; 35)]. Total RNA was extracted from 50 mg of _O. vulgare_ leaves after 6 h _S. littoralis_ feeding and control unwounded leaves using the Agilent Plant RNA Isolation Mini Kit (Agilent Technologies, Santa Clara, CA, USA). To remove residual genomic DNA, RNA was treated with RNAse-free DNAse I set (Qiagen, Venio, The Netherlands). RNA quality was checked using the Agilent 2100 Bioanalyzer with RNA 6000 Nano LabChip. Quantitative analysis was performed using the Nano Drop ND-1000 micro-scale spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). For cDNA synthesis, high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used according to manufacturer's instructions. Reactions were prepared by adding 1.5 ug total RNA, 2 uL of 10\(\times\) RT Buffer, 0.8 uL of 25\(\times\) dNTPs mix (100 mM), 2 uL 10\(\times\) RT primer, 1 uL of Multiscriber\({}^{\text{TM}}\) Reverse Transcriptase and nuclease-free sterile water up to 20 uL. Reaction mixtures were incubated at 25 \({}^{\circ}\)C for 10 min, 37 \({}^{\circ}\)C for 2 h, and 85 \({}^{\circ}\)C for 5 s. Samples were stored at \(-20\)\({}^{\circ}\)C for further analyses. Primer pairs for the selected genes were designed using BeaconDesigner (version 5.0; PremierBiosoft, Palo Alto, CA, USA). Primers were designed for regions with the largest possible difference between the P450 sequences whilst primer binding sites were chosen for identical regions of all known alleles for the respective genes from _O. vulgare_ and _Thymus vulgaris_. Each primer pair was tested for potential cross-hybridisation with the other P450s as templates; none was observed at a 60 \({}^{\circ}\)C annealing temperature except that the CYP71D179 primers cross-reacted to some extent with the CYP79D178 and D182 sequences due to their close sequence similarities. Primers are listed in Supplementary Table S1. Primer efficiencies for all pairs were calculated using the standard curve method [71]. qPCR analysis was on a Stratagene Mx3000P Real-Time System (La Jolla, CA, USA) using SYBR green I with ROX as internal loading standard, using 10 \(\mu\)L of mixture consisting of 5 \(\mu\)L 2X MaximaTM SYBR Green qPCR Master Mix (Fermentas, Waltham, MA, USA), 0.6 \(\mu\)L cDNA and 300 nM primers (Integrated DNA Technologies, Skokie, IL, USA). Controls included non-RT controls (using total RNA without reverse transcription to monitor genomic DNA contamination) and non-template controls (water template). PCR conditions were: 10 min at 95 \({}^{\circ}\)C, 40 cycles of 15 s at 95 \({}^{\circ}\)C, 20 s at 60 \({}^{\circ}\)C, 30 s at 72 \({}^{\circ}\)C. Fluorescence was read after each annealing and extension phase. All runs were followed by a melting curve analysis from 55 to 95 \({}^{\circ}\)C. The linear range of template concentration to threshold cycle value (C\({}_{\text{t}}\) value) was determined by performing a dilution series using cDNA from three independent RNA extractions analyzed in three technical replicates. qPCR reactions were run using specific primers [52]. Three distinct reference genes: Elongation factor 1 alpha, actin and 185 rRNA were used to normalize the results of the real-time PCR. The most stable gene, selected using Normfinder software [72], was the elongation factor 1 alpha. All amplification plots were analyzed with MX3000P(tm) software (Agilent Technologies, Santa Clara, CA, USA) to obtain _Ct_ values. Relative RNA levels were calibrated and normalized with the level of the elongation factor 1 alpha mRNA. Isolation of Total RNA and Expression of S. littoralis Savenging and Detoxifing Genes in Response to O. vulgare Terpenoids  
Larvae were fed with ACD, ACD + Tween 20, ACD + _O.