RNA extractions and gene expression quantification

For the RT-qPCR experiments (reverse transcription and quantitative real time polymerase chain reaction), representative root samples from each root system were collected and immediately frozen in liquid nitrogen. Eventually, other tomato organs were also collected. Tissues were immediately frozen in liquid nitrogen and stored at −80�C until RNA extraction. Total RNA was isolated from 0.2 g samples using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions and treated with RNase-Free DNase. One microgram of DNase-treated RNA was reverse-transcribed into cDNA using the iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) following the supplier’s protocol. For the quantitative real time PCR (qPCR), a 20 μl PCR was prepared containing 1 μl of diluted cDNA (1:10), 10 μl 2� SYBR Green Supermix (Bio-Rad) and 200 nM of each primer using a 96-well plate. The PCR program consisted of a 3-min incubation period at 95�C, followed by 35 cycles of 30 s at 95�C, 30 s at 58–63�C and 30 s at 72�C. The specificity of the PCR amplification procedure was checked using a melting curve after the final PCR cycle (70 steps of 30 s, from 60 to 95�C, at a heating rate of 0.5�C). Experiments were carried out on five biological replicates, and the threshold cycle (Ct) was determined in triplicate. Relative transcription levels were calculated using the 2^−ΔΔCt method (Livak and Schmittgen 2001). The Ct values of all genes were normalized to the geometric mean of Ct values from the LeEF-1α (accession number {"type":"entrez-nucleotide","attrs":{"text":"X14449","term_id":"19272","term_text":"X14449"}}X14449) and actin ({"type":"entrez-nucleotide","attrs":{"text":"NM_001321306.1","term_id":"1008806505","term_text":"NM_001321306.1"}}NM_001321306.1) housekeeping genes.

The RT-qPCR data for each gene are shown as the relative expression with respect to the reference treatment to which an expression value of 1 was assigned. The reference treatment generally corresponded to the non-AM inoculated treatment or control plants transformed with the empty vector. All genes, whose transcript abundance was measured by RT-qPCR, and the corresponding primers used are listed in Supplementary Table S1.