Figure 3. Relative metabolite content of TMV infected plants detected by LC-MS. Heat map representing changes in relative metabolite contents of TMV-inoculated and control plants detected by LC-MS experiments. On the TMV panel the logarithmic ratio between the levels of each metabolite in TMV-inoculated versus the mock- inoculated plants for each time point are shown (similar to Figure 2). On the mock-inoculated panel, the ratios were calculated between data of each time point of mock-inoculated samples versus the first dpi of mock-inoculated (similar to Figure 2). Asterisks indicate statistically significant differences (P,0.01) by Student’s t test. doi:10.1371/journal.pone.0028466.g003 In the first stage there were three groups of metabolites that are of high interest from the host-pathogen interaction perspective: ascorbate pathway-related metabolites, fatty acids and phenolic compounds (Figure 2A). For the ascorbate acid pathway dehydroascorbate, gulonate and 2-oxo-gulonate were altered (for details see Supplemental Figure S2A). Several of the detected altered fatty acids (palmitate, myristate, pelargonate and stearate) are precursors of the JA biosynthetic pathway and all of them co- ordinately increased at this first phase. Similarly, regarding phenolic compounds, one of the salicylate precursors, benzoate, also showed significantly increased levels both at very early (one dpi) and later (15 and 22 dpi) stages. In addition, c-amino butyric acid (GABA) is another stress-related molecule which was Metabolic and miR Profiling of TMV Infected Plants statistically increased during this early phase. Moreover, a group of sugars (ribose, rhamnose, and sorbose), and several organic acids associated with the tricarboxylic acid cycle (TCA) pathway (malate, isocitrate and pyruvate), displayed a rapid increase in their contents. At this early time point secondary metabolites were not significantly altered (Figure 3 TMV, left panel). Despite the clear metabolic alterations observed in systemic leaves after 24 hours of infection, four days later only a few metabolites displayed significant alterations in their contents (five dpi). Higher numbers of statistically significant changes in metabolite levels were, however, observed at eight dpi and especially at 15 and 22 dpi (Figure 2B). This second phase of metabolic changes strongly correlated with the level of virus accumulation (Figures 1B and 2B). The most altered metabolite groups in this phase were amino acids, since more than half of them showed significant changes at 15 and 22 dpi. However, there were also changes in fatty acids levels which may be related to the disruption or production of endomembrane components which is known to occur during virus replication, movement and spread [41,42]. In contrast to the above described metabolic changes detected in systemic tissues following TMV infection, the heat maps shown in Figures 2A and 3, mock-inoculated panels (right side) dissected metabolic changes during leaf development along the experiment. Figures 2A and 3, mock-inoculated panels displayed metabolite levels as logarithmic ratios between one dpi and each of the subsequent time points sampled. The TCA-related metabolites (citrate, fumarate, isocitrate, 2-oxoglutarate and succinate (see Supplemental Figure S2B) as well as several amino acids such as proline, alanine, phenylalanine and valine were strongly reduced in older leaves. Moreover, massive inductions in secondary metabolites accumulation such as, chlorogenate and flavonoid-related metabolites and nicotianoside related com- pounds were registered upon aging of the leaves (Figure 3 and Supplemental Figure S2B). It is known that as tobacco leaves become older they increase resistance to viral infection in comparison to younger leaves. Chlorogenate and flavonoid compounds have previously been reported to confer pathogen resistance [43,44], hence it is possible that the elevated natural resistance on development is due to the increased accumulation of those metabolites. However, neither flavonoids nor chlorogenate compounds were induced by the infection (Figure 3) suggesting that these metabolites do not belong to inducible defence pathways in accordance with results reported by Maher et al [43], similar behaviour were shown by the large number of nicotianosides detected. Virus accumulation correlates with amino acid depletion in leaves As mentioned above several amino acids showed a marked reduction in their contents particularly at later stages of infection (15 and 22 dpi) (Figure 2A, TMV panel). Moreover, free amino acids residues present on the TMV CP decreased significantly in the infected plants at later stages (Figure 4A). More precisely, negative correlations between free amino acid contents in the TMV-inoculated plants and the accumulation of virus CP were found on most of the amino acids that compose CP (Figure 4A, see R panel). Furthermore, when a second correlation was calculated between the mentioned correlation coefficients (amount of CP and amino acid level) and the percentages of each amino acid on the CP composition (Figure 4A) an R = 20.82 with a p-value of 0.002 was obtained, indicating that the patterns of amino acid changes also strongly correlated with the frequency of these amino acids in the TMV CP composition. These results suggested that the PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e28466