stracted and a mean value from technical replicates for each miRNA sub-type for each species-specific probe (e.g., ath|- miR156a) for every array was calculated. Afterwards a median value was obtained for every single miRNA type and species- specific probe (e.g., ath|miR156) at 5, 15 and 22 days post- inoculation (dpi) for all arrays and each treatment. Median values were only taken into account in the case of the existence of at least three independent arrays showing mean values above the background for each miRNA and probe. Later a log-2 quotient between infected and mock-inoculated was calculated for each miRNA type and species-specific probe fluorescence value. A median value was then calculated in order to obtain a single value for each miRNA for all probes spotted in the arrays. MiRNAs belonging to closely related families with similar mature sequences were put together for the calculations. The results were visualized as a log-2 false-color scale (MultiExperiment Viewer Version 4.2 August 1st, 2008) [86]. Supporting Information Figure S1 Virus severity comparison. Systemic symptoms on infected tobacco plants at 30 days post infection (TMV, and ShMV respectively). Percentage of plants showing systemic symptoms observed on inoculated plants with the selected viruses in two independent experiments at different days post infection, with a minimum of ten plants per assay. Similar virus concentrations were used (,2.5 mg/ml) as inocula. Number of plants with flowers and the average height of plants quantified 30 days post infection in two independent infection assays. (XLS) Figure S2 Excel table showing the data used to build Figure 2 and 3. A) Metabolite changes detected in systemic leaves from TMV and Mock-inoculated plants, data is presented normalized for each time point and as a ratio between the levels of each metabolite in infected versus the mock-inoculated plants in each time point, this ratios indicate the metabolic alteration produced by the virus infection at each time point. B) Metabolite changes detected in Mock-Inoculated plants along the time course of the experiment, data is showed as the ratio between data of each time point of mock-inoculated samples versus the first dpi of mock- inoculated. This ratio indicates the metabolic changes produced as a consequence of the sampled leaf development along the time of the experiment. In addition in both A and B tables information about the peak assignment is display. (XLS) Figure S3 CP-TMV detection by RT-PCR analysis in 5 and 22 dpi TMV and mock infected tobacco plants. It is observed that at 5 dpi there is no detectable virus in sampled tissue. For RT-PCR, first-strand cDNA was synthesized using M- MLV RT (Promega) and random primers according to manufac- turer’s instructions (Promega). The oligonucleotide primer set used for CP detection were designed using Vector NTI Software. The PCR was performed using Taq (Invitrogen) under the following program: 16 5 min 95uC, 306 15 sec 95uC, 30 sec 58uC, 30 sec 72uC. The product were analized in 1.2% Agarose gel. (TIF) Figure S4 Excel table showing the data used to build Figure 5. A) Fluorescence values extracted from microarray hybridizations using sRNA obtained fractions from systemic leaves of infected (TMV) or buffer-inoculated (MOCK) N. tabacum plants. Raw fluorescence values were normalized, background-sub- stracted and a mean value from technical replicates for each miRNA sub-type for each species-specific probe (e.g., ath| Metabolic and miR Profiling of TMV Infected Plants miR156a) for every array was calculated. Afterwards a median value was obtained for every single miRNA type and species- specific probe (e.g., ath|miR156) at 5, 15 and 22 days post- inoculation (dpi) for all arrays and each treatment. Median values were only taken into account in the case of the existence of at least three independent arrays showing mean values above the background for each miRNA and probe. The ‘‘no data’’ annotation indicates lack of sufficient mean values. B) Using the data presented in A a median value was then calculated in order to obtain a single value for each miRNA for all probes spotted in the arrays. MiRNAs belonging to closely related families with similar mature sequences were put together for the calculations. There after a log-2 quotient between infected and mock-inoculated was calculated for each miRNA. (TIF) Figure S5 TMV-CP cuantitative detection by RT-qPCR relative quantification analysis at 6, 11 and 22 dpi TMV and mock infected tobacco plants. Ratios were obtained between mock and TMV infected plants from each day post infection. It is observed that at 11 dpi low but significative amounts of CP is detected and at 22 dpi a severe infection shows high amounts of TMV CP. *** indicates p-value,0.001. (TIF) Figure S6 miRNA Targets genes validation. (A–B) Align- ments of the miRNA-target sites between tobacco genes: SGN- U435399, SGN-U439906, and SGN-U428805 mRNAs and reported Arabidopsis miRNA-target genes. (C) miR156, miR166 and miR171 cleavage sites in target genes SGN-U435399, SGN- U439906, and SGN-U428805 mRNAs, respectively, determined by a modified RNA ligase-mediated RACE. The frequency of RACE clones corresponding to each cleavage site (arrows) is shown with the number of clones matching the target message. Aligned are the reported Arabidopsis target sites. Methods: To map the internal cleavage site in putative targets of tobacco mRNA, RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) was done using the GeneRacer Kit (Invitrogen, Carlsbad, CA). A modified procedure for RLM-RACE was carried out as described previously by Llave et al. (Llave C, Xie Z, Kasschau KD, Carrington JC (2002) Science 297:2053– 20562002). Total RNA was isolated from 4-week old plants and Poly(A)+ mRNA was purified using an Oligotex mRNA Midi Kit (Qiagen, Germany). Two nested PCRs were done to amplify the DNA fragment, which was subsequently cloned into pGEM-T Easy vector (Promega, Madison, WI) for sequencing. (TIF) Figure S7 Experimental conditions used in Quantitative Real Time PCR Experiments based on MIQE require- ments. (TIF) Figure S8 Stability analysis of candidate reference genes in 6 and 11 dpi, TMV, SHMV and mock infected tobacco leaves. The housekeeping genes were ranked according to their expression stability by (A) geNorm, (B) Normfinder and (C) BestKeeper statistical tools. In the three plots, genes were ordered from least (left) to most (right) stable. (C, bottom) Pair-wise correlation analysis between the candidate reference genes and the calculated BestKeeper index were highlighted in grey. (D) Ct values of the three houskeeping genes EF-1a, Ubi-3 and Actin, obtained by RT-qPCR and LinReg data analysis. Ct values resulting from RT-qPCR and LinReg software were used for expression stability analysis using the Microsoft Excel based tools geNorm 3.5 (Vandesompele et al 2002), Normfinder 0.953 PLoS ONE | www.plosone.org 12 December 2011 | Volume 6 | Issue 12 | e28466