Figure 4. Relative abundance of free amino acids in TMV infected Plants. A) Changes in the relative abundance of free amino acids between TMV-inoculated and control plants detected by GC-MS experiments (same data as Figure 2). Pearson correlation between CP and free amino acids levels was calculated and the correlation coefficients R are showed on a blue-red false color scale. Asterisks indicate statistically significant correlations (P,0.01). Amino acid composition of CP is listed as a percentage of abundance (aa %) and CP accumulation during virus infection is shown as line graphic. B) Relative mRNA accumulation of genes involved in amino acids synthesis detected by qPCR: TD1-chPrec = chloroplast threonine deaminase 1 precursor, AAAtransf3 = Branched-Chain Amino Acid Aminotransferase 3, AtKARI = similar to A. thaliana ketol-acid reductoisomerase, Asn2 = As- paragine Synthetase 2, GHmetT = Glycine Hydroxymethyltransferase. doi:10.1371/journal.pone.0028466.g004 reduction of free amino acids is a consequence of the massive CP production. In order to investigate whether the decrease of these amino acids correlated with the expression of their biosynthetic pathway genes, the steady-state mRNA levels of a chloroplast threonine deaminase 1 precursor (TD1-chPrec), branched-chain amino- transferase (AAtransf 3), a ketol-acid reductoisomerase (KARI), asparagine synthase 2 (ASN2) and glycine hydroxymethyltransfer- Metabolic and miR Profiling of TMV Infected Plants ase (GhMetT), were measured by RT-qPCR at 22 dpi (Figure 4B). Those genes were selected based on Arabidopsis amino acids pathways followed by a search for orthologous genes in tobacco data bases to obtain the tobacco sequences. None of the genes analysed displayed statistically significant differences in their mRNA accumulation patterns when comparing TMV-infected versus mock-inoculated leaves. These results suggest that the observed reduction in free amino acids accumulation may be mostly a consequence of an increased use of these pools of amino acids during the massive CP production (Figure 4 A) rather than changes in the rates of their biosynthesis. Biphasic alteration of miRNAs levels during TMV infection To investigate the level of miRNA changes during TMV infection in the exact same samples for the metabolic and ELISA studies (Figure 1A), the accumulation of several miRNAs was measured by using a microArray containing all the known probes for detection of plant miRNAs present in the miRbase release 9 [45]. Small RNAs were extracted from the samples taken at five, 15 and 22 dpi from the TMV- and mock-inoculated plants (Figure 1A). The studies revealed a temporal miRNAs level alteration after the infection unexpectedly showing two clear distinct stages. At five dpi when no virus was detected (Figure 1B and Supplemental Figure S3), several of the analyzed miRNAs were down-regulated compared with mock-inoculated plants (Figure 5 and Supplemental Figure S4) while most of the miRNAs were up regulated at 15 and 22 dpi, as expected, when viral accumulation was high on the sampled leaf. In particular, miR415, mir156/7, mir390, miR398, miR168, miR167, miR171, miR397, miR535, miR165/6 and miR160 form a cluster (named cluster A) that was down- and up-regulated at the early (5 dpi) and later (15 and 22 dpi) stages of infection respectively, when compared with mock-inoculated plants. Within this group miR165/6 and miR160 even thought behave as the other at 5 and 15 dpi show smaller up regulation at 22 dpi. The miRNAs miR172 and miR164, showed a similar tendency considering 5 and 22 dpi. It is noticeably group A mentioned miRNAs are described as responsive to different biotic and abiotic stresses [46,47,48]. Also, these groups show a biphasic alteration similar to that observed for the leaf metabolome after the infection. There are another set of nine miRNAs out of twenty two that also showed changes but without showing this biphasic trend. Finally it worth to mention that miR403 and in stronger manner miR408 were the two unique miRNAs down regulated at late stage (22 dpi) probably indicating a different alteration mechanism. miRNAs and miRNA-targets accumulation during two different tobamovirus infections at early time points In order to provide a validation of the microArray data we measured a set of miRNAs and also some of their mRNA target levels on an independent experiment by means of RT-qPCR. To pursue this analysis a new infection assay was performed on tobacco plants using two viruses, TMV and Sunn-hemp mosaic virus (ShMV), these two viruses differ markedly on the symptoms they produce on tobacco; severe and mild respectively (see Supple- mental Figure S1 for a disease severity comparison). The inoculation and leaf sampling followed same scheme as the previous assay (Figure 1A) but the level of inoculums was reduced (dilution 1/10). The aim of use reduced inoculums was to produce a longer early phase by means of slowing down the initial step of the infection, in agreement with this idea the sampling time was performed at 6 dpi and 11 dpi. To show this effect (extended early phase) the level of TMV-CP of samples at 6 and 11 dpi was analyzed and compared to 22 dpi by means of RT-qPCR assay. PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28466