underlying mechanisms of these effects are unclear. In plants, several groups have demonstrated that viral suppressors of post- transcriptional gene silencing (PTGS) interfere with miRNA- mediated regulation of host genes [28,33,34,35,36]. In the case of tobamoviruses, the replicase protein interferes with PTGS [37,38,39]. Work from our laboratory indicated that co-expression of the TMV movement and coat proteins in transgenic plants does not suppress PTGS, yet interferes with miRNAs accumulation [22]. Along the same line, it has been shown that Turnip mosaic virus (TuMV) induces bra-miR1885 accumulation resulting in cleavage of a TIR-NBS-LRR disease resistance target gene [40]. Here, we used a systemic approach by employing, both liquid chromatography (LC) and gas chromatography (GC), coupled to mass spectrometry (MS) to determine the relative levels of a large set of metabolites across the viral infection process, in parallel with an miRNA profiling using a micro-array based procedure. Identification of metabolites and miRNAs differing in abundance between control and infected samples may provide information concerning the pathways involved in signaling and defense which orchestrate and execute the response in plant–pathogen interac- tions. A clear biphasic response in both, metabolic and miRNA profiles was observed in accordance with the various stages of infection. Data obtained allowed us to search for correlation networks between both molecule types in response to the infection. These data thus suggest that there is an early systemic signaling stage independent of the virus’s presence and a late second phase that correlates with virus accumulation. Results Experimental design To establish differences in metabolites and miRNAs accumu- lation occurring during plant-virus compatible infections, the pathosystem selected in the present study consisted of N. tabacum plants (Xhanti, nn) grown under greenhouse controlled conditions and infected with Tobacco mosaic virus (TMV). The fifth leaf from five-week-old plants were either TMV- or mock-inoculated. The third leaves above from the inoculated one (leaf number eight) were sampled at one, five, eight, 15 and 22 days post-inoculation (dpi) (10 plant/treatment and time point) (Figure 1A). Viral accumulation was followed by an ELISA assay detecting the TMV coat protein (CP) in the sampled leaves (Figure 1B). Non- detectable virus was found at either one or five dpi. At eight dpi the percentage of plants with virus accumulation in the sampled leaf reached 60%; however CP accumulation levels were very low (4% of the maximum level reached at 22 dpi) at this stage. The CP levels increased rapidly at 15 dpi (32% of the maximum level reached at 22 dpi), reaching the maximum at 22 dpi (Figure 1B). Symptoms were first noticed at eight dpi in only 20% of the plants with very mild mosaic in leaves. At 22 dpi all the inoculated plants showed characteristically severe TMV symptoms (Supplemental Figure S1). Viral infections produce a systemic biphasic alteration on leaf metabolism GC- and LC-MS analyses were both performed with the aim of detecting a relatively comprehensive overview of metabolic alterations following viral infection. The relative levels of 64 primary (GC-MS) and 34 secondary (LC-MS) metabolites were analyzed in split samples obtained from the same TMV- and mock-inoculated plants across the 22 day period (Figures 1, 2 and 3). Figure 2A (see Supplemental Figure S2A for detailed data) shows primary metabolite changes detected in systemic leaves from TMV- and mock-inoculated plants. The TMV panel (left Metabolic and miR Profiling of TMV Infected Plants Figure 1. Experimental Design. A) Schematic representation of the experimental design. More than 100 five-week-old tobacco plants were inoculated with TMV or with mock solution in the fifth leaf. Third upper leaves (leaves number 8) were sampled at 1, 5, 8, 15 and 22 days post inoculation (dpi). Each sample was ground and the powder divided in 4 parts for virus detection by ELISA, miRNA detection by microarray and for primary and secondary metabolites detection by GC-MS and LC-MS respectively. B) TMV infection progression along the experiment in virus-inoculated plants. Bar graph: percentage of TMV-inoculated plants systemically accumulating virus as detected by ELISA in the sampled leaf along the infection. Squares line: Relative systemic accumulation of viral CP quantified by ELISA assays for samples at each time point. Diamonds line: percentage of TMV-inoculated plants showing charac- teristic symptoms under naked eye inspection. n = 10 plants per each time point. doi:10.1371/journal.pone.0028466.g001 side) of Figure 2A shows the logarithmic ratio between the levels of each metabolite in TMV- versus mock-inoculated plants at each time point whereas the mock-inoculated panel (right side) shows the ratio between data of each time point of mock- inoculated plants versus the first dpi of mock-inoculated. These two relative values indicated the metabolic alteration produced by the virus infection at each time point (TMV panel) and the metabolic changes produced as a consequence of the develop- ment along the time of the experiment (mock-inoculated panel). The virus-mediated metabolic alterations showed two temporal phases distinct in both the trend and number of altered metabolites (Figure 2AB, TMV panel). An early first phase (one dpi) was mainly characterized by a rapid and significant increment in the level of a number of metabolites whilst the second phase, at later stages of infection (specifically at 15 and 22 dpi), revealed both increases and decreases in metabolite PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28466