Metabolic and miR Profiling of TMV Infected Plants Figure 7. Pre-miRNA166 is reduced after virus infection. A) Predicted fold-back secondary structures of tobacco and tomato miRNA166a precursors as determined by the RNAfold program. Mature miRNA sequences are highlighted in green. Clustal alignment between Pre-miRNA166a sequences from tobacco and tomato. (BC) Pre-miRNA166a qPCR quantification in plants infected with TMV or ShMV, (B) 6 dpi and (C) 11 dpi. ** means p-value,0,05 and *** means p-value,0,01. doi:10.1371/journal.pone.0028466.g007 propagators of the systemic signal that may orchestrate the plant defence after pathogen attack. Within the early stage group, there are at least four sets of molecules related with signalling that were altered by the infection. The hormone precursors benzoic acid and fatty acids (SA and JA respectively) were induced at 1 dpi, and it has been well demonstrated hormones changes after virus infection triggering stress responses [8,11,12,56,57]. GABA, a signalling molecule in mammals, was strongly up regulated at this phase and it has been postulated to play a similar role in plants [58]. However, it is important to note that in plants it also plays a highly important role in energy metabolism [59]. The fourth set of compounds were those of the ascorbate biosynthesis pathway implying an altered redox state, a change that has also previously been indicated to play an important role in the pathogen response [60,61]. In agreement, altered levels of ascorbate derivatives (transient increase) and fatty acids were previously shown following TMV infection on tobacco plants on inoculated and systemic leaves by using an NMR based approach [52]. Following the initial phase (one dpi), the subsequent time point (five dpi) displayed the smallest number of statistical changes and eight, 15 and 22 dpi time points displayed increasing number of metabolic alterations in correlation with the level of infection. This result suggests that changes in late stages of infection depend upon the accumulation of virus and most likely are the consequence of the elevated level of resources required for the production of viral proteins [3,42]. In full agreement, the observed negative correlation between CP levels and the levels of free amino acids plus the correlation between this results and the frequency of each amino acid in the CP composition (Figure 4A) strongly supports the hypothesis that virus accumulation modulates the metabolome to obtain resources. Our observations of the miRNAs behavior (Figures 5 and 6) lead us to propose that two possibly different mechanisms could be acting at the early and late stage of infection to alter miRNAs accumulation, representing five dpi and 15–22 dpi respectively on the microarray data. In agreement to this hypothesis Hu et al [51] proposed recently the existence of at least more than one mechanism to explain the sRNAs alteration mediated by virus. The early stage (no virus presence) shows a cluster of miRNAs with down-accumulation and a concomitant increment in the mRNA level of one target gene tested, and the second, later stage of infection, includes higher levels of both miRNAs and miRNA- PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28466