RNA extraction and libraries construction

Total RNA for both mRNA-Seq and RT-PCR was extracted from ~80 mg of frozen root apices sampled 10 days after imposition of the stress using the “Spectrum™Plant total RNA Kit” according to manufacturer's instructions (Sigma®). RNA quality and concentration were determined by both UV spectrophotometry (Nanodrop™, Thermo Scientific™) and gel electrophoresis analyses. NGS analysis was performed in 101.14 plants on two biological replicates per each condition. mRNA was purified from the total RNA using the Dynabeads mRNA Direct kit (Invitrogen pn 610.12). A variable quantity of mRNA ranging from 0.4 to 1.6% with respect to the amount total of RNA was obtained. Samples for ligation sequencing were prepared according to the “SOLiD Whole transcriptome library preparation” protocol (pn 4452437 Rev. B). Before RNase III digestion samples were purified with Purelink RNA micro kit columns (Invitrogen™), digested from 3′ to 10′ depending on the starting amount of mRNA, retro-transcribed, size-selected using Agencourt AMPure XP beads (Beckman Coulter pn A63881) and barcoded during the final amplification. Obtained libraries (2.5 samples per lane) were sequenced using Applied Biosystems, SOLiD 5500 XL, producing paired-end reads of 75 and 35 nucleotides for the forward and reverse sequences, respectively. Reads were aligned to the v1 prediction of grapevine PN40024 reference genome (http://genomes.cribi.unipd.it/grape) using PASS aligner (Campagna et al., 2009). The percentage identity was set to 90% and one gap was allowed whereas the quality filtering parameters were set automatically by PASS. A minimum reads length cut-off of 50 and 30 nt was set for the forward sequences and reverse reads, respectively. The PASS-pair tool of the PASS package was used to perform the pairing between the forward and the reverse reads and to selected only those sequences that were uniquely aligned. Finally htseq-counts program (http://www.huber.embl.de/users/anders/HTSeq/doc/count.html) was used to quantify gene abundance.