Metabolic and miR Profiling of TMV Infected Plants Figure 8. Non-parametric Spearman’s correlation analysis. Metabolites-metabolite (AB) or miRNA-metabolites level (CD) correlation was calculated between inoculated plants (AC) or mock-inoculated plants (BD). Significant correlation (p = 0.001, for AB and p = 0.01 for CD; R.0.5) between two metabolites was drawn with color lines. Positive correlations were drawn with red lines, negative with blue lines. Correlations present in both TMV inoculated (AC) and mock-inoculated plants (BD) were drawn in black, regardless the sense of the correlation. doi:10.1371/journal.pone.0028466.g008 targets, as previously reported [22,51,62,63], as well as viral proteins accumulation, suggesting that viral proteins inhibit miRNA-activity, for example, by sequestering sRNAs through the PTGS suppression activity of the replicase [37,38,39]. The early stage may include a transcriptional component, as implied by the coordinated reduction of the pre-miR166 and mature miR166 levels (Figure 7) also previously demonstrated by our group for the miR164a precursor on Arabidopsis thaliana [21]. The correlation analyses revealed a dense correlation network (metabolites and miRNAs) (Figure 8AC) in infected plants compared to the mock-inoculated ones (Figure 8BD). This information may indicate that the virus infection invokes the operation of, or increased flux through, a set of metabolic pathways that are highly interconnected. Consequently, it can be postulated that the infection modifies metabolite levels by means of key players which coordinate, or modulate, the levels of these components, for example miRNAs. Moreover, a few correlation modules were constant irrespective of the degree of infection. For example a secondary metabolism module, represented by nicotianoside and chlorogenic acid derivatives and flavonoids (Quercetin, Rutin and Kaempferol), was maintained across the experimental conditions described here (Figure 2A and 8AB), suggesting that the levels of these metabolites are either unaffected by viral infection or are modified in concert. However, comparison of the relative levels of the metabolites in question reveals that the first hypothesis is in fact correct. This is in close agreement with a previous proposal that these defense compounds are part of the autonomous defense mechanism [43]. Correlation analysis among miRNAs and metabolites revealed similar results. In particular, two correlation groups were observed in infected and mock-inoculated plants (Figure 8 CD). One constituted only by miRNAs and the second one involving both miRNA and metabolites. Interestingly, miR168, which regulates Ago1 (involved in the miRNA function process) [64,65], in the mock-inoculated samples displayed several connections with the rest of the miRNA core. However, in the infected plant, miR168 only correlated with miRs 156/7 and 390, most probably as a consequence of the viral infection altering specific miRNA levels/ activity. MiRNAs 397, 398 and 408 respond to nutrient deprivation, most particularly under copper and phosphate limitation [66]. miR408 (that together with miR403 are the only two down-accumulated miRNAs at late stage) can be considered a hub of our network analysis and, in infected leaves, it is linked to the TCA cycle intermediates citrate, isocitrate, glutarate and pyruvate. This latter association may imply a connection to the cellular respiration process through one of the miR408 targets (a plantacyanin-like protein (AT2G02850) that belongs to the phytocyanin family of secreted blue copper proteins) and/or reflex the copper homeostasis alteration on the miR398 regulating their target gene the COX5b protein, which is part of the respiratory chain complex III [47,67]. Further experiments are required to establish this connection. The basal defenses and the systemic signaling of viral associated molecular patterns (VAMPs) triggering innate immunity may be playing a role in the initiation of this early phase of miRNA alteration. In turn, this early phase may be orchestrating the antiviral defense in agreement to the suggestion of Ruiz-Ferrer and Voinnet [27]. Moreover, there are previous studies showing links between miRNA and hormones crostalk [12,21,56,68] therefore allowing us to propose that changes in the mentioned metabolites (JA and SA precursors) that are infection-specific hubs on the response network (Figure 8 and 2A) may directly or indirectly trigger the alteration in systemic miRNA accumulation observed in the absence of virus (Figure 5, 5 dpi). In agreement with the proposal that the early phase should be mostly transcriptional, previous work of our and other laboratories detected elements responsive to SA and/or JA in several promoters of the miRNAs that compose the cluster altered at the beginning of the infection stage [21,46]. It is important to mention that the miRNAs that form cluster A are part of a miRNAs group described as stress regulated miRNAs, responsive to several different kind of stresses [46,47,48,69,70]. The fact that ShMV (mild virus) produces the early miRNA alteration at a delayed stage in some of the five tested miRNAs in comparison to TMV (severe virus) (Figure 6) and the fact that ShMV produces light symptoms may indicate that this early alteration is important to either the severity itself or to the timing required for symptom development. In conclusion we have showed a global and more importantly bi-phased series of changes in metabolites and several miRNAs produced following TMV infection, in systemic leaves. Several of the metabolites increased at this first stage were related to hormone production and some of them could be proposed to propagate the phloem signal within the systemic leaf cells. In the case of miRNA the early phase, without virus accumulation, may respond to a systemic signal that probably acts transcriptionally on miRNA genes to produce the mature miRNAs alteration. However, further experiments are required in order to uncover which metabolites/miRNAs play causal biological roles during the virus infection. As a consequence, further knowledge of the role of miRNA in regulating metabolic pathways and/or vice versa will likely be a great aid in understanding the role of miRNAs during host-pathogen interactions. Materials and Methods Plants growing conditions, viral infections and quantification All N. tabacum plants (Xhanti nn) were grown in a greenhouse with temperatures ranging from 20 to 26uC. A single expanded leaf of each plant (leaf 4 to 6 stage) was dusted with carborundum, 20 ml of semi-purified TMV virus in 20 mM NaHPO4 (pH 7) was added, and the surface of the leaf was gently abraded. Two level of inoculums were used, the experiment mentioned in figure 1, 2, 3, 4, 5 and 8 used more than 200 Local Lesion (LL) inoculums, the experiment of figure 6 and 7 used approximately 20 LL inoculums for both viruses TMV and ShMV. The third leaf above the inoculated one was collected (leaf number eight) (Figure 1). For quantification of viral proteins, total proteins were extracted and quantified using Quick Start Bradford Protein Assay (Bio-Rad). For viral detection ELISA experiments were performed as described previously [71]. Metabolic analysis Metabolite extractions were done as previously described [72]. For GC-MS, derivatization and GC-time of flight-MS (GC-TOF- MS) analyses were carried out as described previously [73]. The PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e28466