Nzy dnase 1

Total RNA from Arabidopsis leaves was extracted using Tri-sure (Bioline) and subsequently purified using the RNA Clean and Concentrator-5 kit (Zymo Research). RNA samples were treated with NZY DNase I (NZYTech). First-strand cDNA was synthesized from 1 μg of purified total RNA by using the PrimeScript RT Master Mix (Takara). Real-time quantitative PCR (RT–qPCR) reactions were performed using SYBR®Premix Ex Taq^TM (Takara) and an iCycler 5 (Bio-Rad). All kits were used according to the manufacturers’ instructions. Relative quantification of specific mRNA levels was performed using the comparative 2^–ΔΔCt method (Livak and Schmittgen, 2001 (link)) by using the gene-specific primers described in Supplementary Table S1. Expression values were normalized using the Arabidopsis housekeeping genes TUBULIN-4 (At5g44340) or ACTIN7 (At5g09810). Fungal infection was measured by analysing the B. cinerea β-TUBULIN gene (XM_001560987.1) relative to the Arabidopsis TUBULIN-4 gene.

For RNA purification, 1 µg RNA was incubated with 1 µL NZY DNase I (NZYtech, Lisbon, Portugal) at 37 °C for 20 min. Then, 200 µL absolute ethanol (Merck, Darmstadt, Germany) was added, and samples were stored at −20 °C overnight. On the following day, a centrifugation at 15,000× g for 15 min at 4 °C was performed, precipitated RNA was suspended in 100 µL of 75% (v/v) ethanol, and further centrifuged at 15,000× g for 15 min at 4 °C. The pellet was air dried and afterwards was hydrated with DEPC-treated water and incubated in a heat block at 60 °C for 10 min. The quality and concentration of isolated RNA was assessed using a NanoDrop^® ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and purified RNA was stored at −80 °C.

The expression of marker genes for the different defence related pathways was analysed by 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 concentrator™ (Zymo Research, Irvine, CA) and stored at −80°C until use. The first‐strand cDNA was synthesized with 1 μg of purified total RNA using the Primescript™ 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. We measured the expression of three different housekeeping genes, actin (Solyc03g078400), elongation factor 1‐α (Solyc06g005060) and β‐tubulin (Solyc04g081490) and, to find the optimal normalization gene among these three, we used the Normfinder software (https://moma.dk/normfinder-software). According to the results, expression values were normalized using the housekeeping gene elongation factor 1‐α (EF‐1α), and relative quantification of specific mRNA levels was performed using the comparative 2–Δ(ΔCt) method (Livak and Schmittgen, 2001).