Oligo dt 18 primer

Total RNA was extracted using TRI Reagent (Sigma #T9424) or by Renozol (Genoplast #BMGPB1100–2) followed by Total RNA Mini column purification kit (A&A Biotechnology #031–100). 2 μg of RNA was subjected to reverse transcription using M-MLV enzyme (Promega #M1705), dNTP mix 100 mM each (BLIRT #RP65) and oligo (dT)₁₈ primers (Bioline #BIO-38029). The RT-qPCR reaction was performed using SensiFAST SYBR Hi-ROX Kit (Bioline #BIO-92020) on the StepOnePlus™ platform (Thermo Fisher Scientific) according to MIQE guideline. Primers sequences are listed in the Supplementary Information. The comparative 2^-ΔΔCt method was used to determine the relative mRNA level using StepOnePlus software. 18SrRNA was used as a reference control. Data are presented as mean values ± SD; n = 3–5. Statistical significance was assessed using unpaired Student’s t-test with Welch’s correction and p ≤ 0,05 was estimated as significant (*p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.001; ****p ≤ 0.0005).

Total RNA was extracted using Tri-Reagent RNA/DNA/Protein Isolation Reagent (Molecular Research Center, Cincinnati, OH, USA) and then treated with RNase-free DNase (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was purified using an RNA Clean & Concentrator column (Zymo Research, Irvine, CA, USA). Two micrograms of DNase-treated total RNA was used to prepare cDNA by reverse transcription with M-MLV reverse transcriptase (New England Biolabs Inc., Ipswich, MA, USA) and oligo(dT)18 primers (Bioline, London, UK), according to the manufacturer’s protocol. Gene expression was determined by quantitative RT-PCR (MyiQ real-time PCR detection system, Bio-Rad Laboratories Inc., Hercules, CA, USA) using gene-specific primers (Table S1) and SensiMix SYBR & Fluorescein Kit (Bioline, London, UK), following the manufacturer’s instructions.
The Arabidopsis TON1A amplicon (At3g55000) was used as an internal control to normalize all data. The efficiency of the quantitative RT-PCR reactions was higher than 95%.
Quantitative RT-PCR reactions were carried out with cDNA synthesized from three pools of 36–60 roots, depending on the treatment used, harvested randomly.

Total RNA from powdered leave material was extracted with the Plant/Fungi Total RNA Purification Kit (Norgen Biotek Corp., Thorold, Canada) and treated with the Turbo DNA-free^™ (Thermo Fisher Scientific, Waltham, MA, USA). Prior to and after the DNase treatment, the concentration and quality of total RNA were assessed by a NanoDrop^™ 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), while the integrity was checked by the 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA).
Single-strand cDNA was synthesized from DNase-treated RNA using SuperScript^® IV (Thermo Fisher Scientific, Waltham, MA, USA) reverse transcriptase with oligo(dT)₁₈ primer (Meridian Bioscience, Cincinnati, OH, USA). The assessment of cDNA quantity and quality was performed both spectrophotometrically by the NanoDrop ^™ 2000c and in real-time PCR.