GC-TOF-MS system was composed of a CTC CombiPAL autosampler, an Agilent 6890N GC and a LECO Pegasus III time-of-flight mass spectrometer running in EI+mode. Metabolites were identified by comparison to database entries of authentic standards and relative quantification was performed as described in [74]. For secondary metabolites extraction for the LC-MS injection were done as follows: 50 mg of frozen plant tissues were homogenized in 10 mL of extraction solvent (80% methanol with 10 mg/ml of isovitexin, EXTRASYNTHASE, Genay Cedex, France) per mg of fresh weight of tissue in a mixer mill MM300 (Retsch, Haan, Germany) for 3 min at 30 Hz. After centrifuga- tion, 400 ml were transferred to new tube for evaporation. Dried pellets were resolved by 400 ml of 80% Methanol. After filtration by centrifugal device NanoSep MF GHP 45 mm filter (PALL, NY, USA) with centrifugation at 3,000 g. The filtrates were immedi- ately used for injection. HPLC-MS analysis was performed on HPLC system Surveyor (Thermo Finnigan, USA) coupled to Finnigan LCQ-Deca system (Thermo Finnigan, USA). Five ml of the plant extract was injected onto the column (Luna 3 m C18(2), 100A, Phenomenex, Torrance, CA, USA) with elution buffer A (water with 0.1% of formic acid) and buffer B (acetonitrile with 0.1% formic acid). Flow rate was 0.2 ml/min. Separation conditions were as follows: 0–2 min, isocratic elution with 100% A; 2–4 min, linear gradient from 0% to 15% B; 4–14 min, linear gradient from 15% to 32% B; 14–19 min, linear gradient from 32% to 50% B; 19–21 min, isocratic elution with 100% B. The mass analyzer was used for the detection in a negative/positive ion scanning mode with the following setting: capillary temperature, 350uC; source voltage, 5.00 kV and capillary voltage, 60.0 V in positive ion detection and 210.0 V in negative ion detection. Peak areas were integrated using Xcalibur software 2.1 (Thermo Finnigan, USA). Rutin and chlorogenic acid were used for the identification of the peaks in the plant extracts based on co-elution profile for retention times and mass fragmentation by tandem MS analysis. Other peaks which are not available of standard compounds were annotated by comparing their m/z values and MS2 fragmentation patterns with reference compounds, the reported data [75,76,77] and metabolite databases [78,79]. Tagfinder software for GC-MS results and Xcalibur software for LC-MS was used to assign the peaks identity [80]. Metabolites abundance was calculated based in the Ribitol or Isoxitexin intensity and tissue weight. Averages, standard errors and Student’s t-tests calculated. VANTED software was used to show and analyze metabolites abundance and statistical changes in a network, manually draw based on published databases and papers [81]. Correlation analysis Non-parametric Spearman correlation analyses were calculated using Infostat software (InfoStat software [InfoStat version 2008. Grupo InfoStat, FCA, Universidad Nacional de Co´ rdoba, Argentina]), filtered at p = 0.001 for metabolites and p = 0.01 for miRNA data. Significant correlations were drawn using netdraw (http://www.analytictech.com/). RNA extraction and quantitative real-time polymerase chain reaction For relative quantification of miRNAs and target mRNAs accumulation levels, experiments were carried out using five biological replicates. The RT-qPCR performed complies with the MIQE requirements, for the detailed data on each experiment see Supplemental Figure S7. Total RNA was isolated from leaves using TRIZOLH Reagent (Invitrogen), quantified using spectro- Metabolic and miR Profiling of TMV Infected Plants photometer (NanoDropTechnologies), and treated with DNase I (Invitrogen). First-strand cDNA was synthesized either using Superscript III (Invitrogen) and stem-loop specific primers (for miRNAs accumulation analysis) or MMLVI and oligo d(T)20 primers (for targets mRNA accumulation analysis). Real-time qPCR to detect miRNAs was performed as described by Chen et al [82]. The oligonucleotide primer sets for mRNA quantifi- cation were designed using Primer Express 2.0 software (Applied Biosystems) to amplify a fragment containing the miRNA target recognition site. All qPCR reactions were performed using PlatinumH Taq DNA Polymerase (Invitrogen) and SYBRHGreen stain (Roche). qPCR data analysis and primer efficiencies were obtained using LinRegPCR Software [83]. To select the reference genes a stability test was performed using GeNorm, NormFinder and Bestkeeper algorithms on three candidate genes (Supplemen- tal Figure S8). The candidates genes were Tabacco elongation factor-1a (EF-1a, SGN-U446573), tobacco Actin gene (SGN- U431117) and Ubiquitin 3 gene (Ubi-3, GB: X58253). Based on the result observed EF-1a was selected and used as internal control given that it was the most consistent along the three algorithms used and it was in agreement to the reference genes proposed by Lilly et at [84] for virus infections even thought the three selected genes show similar stability. Relative expression ratios and statistical analysis were performed by using FgStatistics interface (Di Rienzo J.A, (2009). Statistical software for the analysis of experiments of functional genomics. http://sites.google.com/site/ fgStatistics/). This software makes use of Pfaffl algorithm [85] to calculate the expression ratio of a given gene in a improved multivariate user friendly interface and calculate the statistic significance using a permutation test (See Supplemental Figure S7). The cut-off for statistically significant differences was set as * means p-value,0.1; ** means p-value,0.05 and *** means p- value,0.01. All primer sets are listed in Supplemental Figure S9 [85]. sRNAs extraction and MicroArray Assay High-quality sRNAs were extracted from tobacco leaves using miRNAVana kit (Ambion, USA) following manufacturer’s indi- cations and then quantified using a spectrophotometer (Nano- DropTechnologies). Combimatrix CustomArrayTM 4X2K miRNA array was used, each slide has four identical array sectors that can be hybridized with different microRNA samples simultaneously, each array sector carry anti-sense oligonucleotide probes with a median length of 22 nucleotides and an average Tm of 55 to 60uC. The 2000 probes on each array/sector contain the complete set of plants mature miRNAs available on MirBase 9.0 in triplicates along the array (Arabidosis thaliana miRNAs probes are repeated four times). The negative controls for RNA degradation and positive controls (U6 and tRNAs) were used to accept array data. Preparation and labeling of miRNA samples for hybridiza- tion were performed following manufacturer’s protocols (Combi- matrix). Images were collected and digitalized using a laser scanner (arrayWoRxe Standard Biochip Reader; Applied Preci- sion, WA, USA). The Microarray Imager software from Combimatrix was used in order to extract raw data from images. A total of n = 6 plants were separately tested for each time point (5, 15 and 22 dpi) of each treatment (mock- and virus-inoculation). Samples were randomly assigned in blocks from the same dpi (mock/virus inoculations) into the three chips used (each with four independent chambers or arrays), which were subsequently stripped and reused twice following manufacturer’s protocol. Therefore a total of 36 arrays, each one belonging to one biological replicate were performed. Raw fluorescence values were normalized (As suggested by Combimatrix), background-sub- PLoS ONE | www.plosone.org 11 December 2011 | Volume 6 | Issue 12 | e28466