Tri reagent rna isolation system

Total RNA was isolated (TRI Reagent RNA Isolation system, Sigma #T9424) and DNase I treated (DNAfree TM , Ambion) before reverse transcription (Superscript III First-Strand Synthesis System, Invitrogen) according to the manufacturer's protocol as previously described [24] , except 0.5µl Superscript III was used for each reverse transcription reaction instead of 1µl.
PCR was performed as previously described [25] using GoTaq Green Master Mix (Promega) according to the manufacturer's instructions. Briefly, 1µg cDNA was combined with 2x Master Mix and 10µM primers (sequences shown in Table 1) and performed on a Veriti Thermal Cycler (Applied Biosystems). PCR products were visualized with GelRed Nucleic Acid Stain (Biotium) on a 1.6% agarose gel. qPCR was performed as previously described [10] . Briefly, qPCR analyses were performed on the ABI 7500HT fast block real time PCR system (Applied Biosystems, Foster City, CA, USA) in triplicate in 384-well Micro Optical plates (Applied Biosystems) with the Power SYBR green master mix (Applied Biosystems ) and 200 nM primers (sequences shown in Table 1).
The PCR and qPCR protocol was 95°C for 10 min and 40 cycles of 95°C for 15 s followed by 60°C for 1 min. qPCR relative expression levels were calculated by the comparative cycle threshold method (ΔΔCt), with 18S ribosomal RNA serving as the endogenous control for normalization.

The in-situ accumulation of hydrogen peroxide (H 2 O 2 ) was determined by histochemical analysis according to the 3,399-diaminobenzidine (DAB)-uptake method (Thordal-Christensen et al. 1997) . Cotyledon disks of 1 cm in diameter were incubated in 1 mg of DAB per milliliter (pH 3.8) (Sigma-Aldrich, St. Louis) overnight, in the dark, at room temperature. After incubation, the leaf disks were treated with boiling ethanol to stop the reaction and clear the disks. Finally, the leaf disks were analyzed under a bright field microscope for brownish-red precipitates corresponding to H 2 O 2 accumulation. In the same disks the progress of the P. xanthii infection was microscopically assessed. The haustoria were visualized as dark brown spots beneath the P. xanthii hyphae inside epidermal cells.
RNA extraction and cDNA synthesis.
Total RNA was isolated from samples, using a TRI Reagent RNA isolation system (Sigma-Aldrich) according to the manufacturer's instructions. The RNA concentration was estimated using the NanoDrop spectrophotometer ND-1000 (Thermo Scientific, Waltham, MA, U.S.A.). For the elimination of contaminating DNA, the TURBO DNA-free kit (Invitrogen) was used. cDNAs were synthetized using Superscript III Reverse transcription (Invitrogen) and the Oligo dT(20) primer (Invitrogen) according to the manufacturer's recommendations.