Qiaamp column

Total RNA was extracted from Las infected and uninfected Citrus paradise (Grapefruit cultivar Duncan) and Citrus sinensis (Sweet orange cultivar Valencia) leaves using TRIzol reagent according to the manufacturer's protocol. DNA contaminations were removed by treating RNA extraction with RNase-free DNase (QIAGEN, West Sussex, UK) and subsequently purified with QIAamp columns (QIAGEN). First-strand cDNA was synthesized at 42°C from total RNA using M-MLV (Invitrogen) reverse transcriptase according to the manufacturer's instructions with the individual PCR reactions containing 1 μl of cDNA, 0.5 μl of each specific primer (10 pmol/μl) (Table S1), 10 μl of 2X Green GoTaq® Reaction Buffer (Promega) in a final volume of 20 μl. The following standard thermal profile was used for all amplifications: 95°C for 3 min followed by 30 cycles of 95°C for 30 s, 58°C for 30 s, and 70°C for 30 s.

Total RNA was isolated using PeqGOLD TriFast reagent (PeqLab, Germany) according to manufacturer’s recommendation. QIAamp columns (Qiagen, Germany) were used for DNA isolations. Nucleic acids were quantified with a Nanodrop 100 spectrophotometer (Peqlab, Germany) and RNA quality checked with an Agilent 2100 bioanalyzer on RNA 6000 nanochips.

Total RNA was extracted from GPA exposed to test plants using TRIzol reagent (Life Technologies, Paisley, UK). DNA was removed by treating RNA extractions with RNase-free DNase (QIAGEN, West Sussex, UK) then purified with QIAamp columns (QIAGEN). GPA mRNA was also obtained using a Dynabeads mRNA DIRECT kit (Life Technologies) according to the manufacturer’s instructions. First-strand cDNA was synthesized at 37 °C from RNA isolations using M-MLV (Invitrogen) reverse transcriptase according to the manufacturer’s instructions. Quantitaive real-time PCRs (qRT-PCRs) were laid out in 96-well plates (Thermo Scientific), with each sample represented by the gene of interest and two reference genes [L27 and glyceraldehyde phosphate dehydrogenase (GAPDH)] as determined by GeNORM (see below). Two or three technical replicates were included for each cDNA–primer combination. Individual reactions contained 3 μl of cDNA, 0.5 μl of specific primers (forward and reverse primer at 10 pmol ml^–1), and 10 μl of 2× SYBR Green (Sigma-Aldrich) in a final volume of 20 μl. Plates were sealed using adhesive PCR Film (Thermo Scientific). Plates were run in a CFX connect™ machine (Bio-Rad) at 90 °C for 3min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and finally 10min at 72 °C. The SYBR-specific fluorophore was quantified during the reaction by the instrument.