Metabolic and miRNA Profiling of TMV Infected Plants Reveals Biphasic Temporal Changes Ariel A. Bazzini1¤, Carlos A. Manacorda1, Takayuki Tohge3, Gabriela Conti1, Maria C. Rodriguez1,2, Adriano Nunes-Nesi3, Sof´ıa Villanueva1, Alisdair R. Fernie3, Fernando Carrari1,2, Sebastian Asurmendi1,2* 1 Instituto de Biotecnolog´ıa, CICVyA-INTA, Hurlingham, Buenos Aires, Argentina, 2 Consejo Nacional de Investigaciones Cient´ıficas y Te´cnicas (CONICET), Buenos Aires, Argentina, 3 Max Planck Institute for Molecular Plant Physiology, Wissenschaftspark Golm, Potsdam-Golm, Germany Abstract Plant viral infections induce changes including gene expression and metabolic components. Identification of metabolites and microRNAs (miRNAs) differing in abundance along infection may provide a broad view of the pathways involved in signaling and defense that orchestrate and execute the response in plant-pathogen interactions. We used a systemic approach by applying both liquid and gas chromatography coupled to mass spectrometry to determine the relative level of metabolites across the viral infection, together with a miRs profiling using a micro-array based procedure. Systemic changes in metabolites were characterized by a biphasic response after infection. The first phase, detected at one dpi, evidenced the action of a systemic signal since no virus was detected systemically. Several of the metabolites increased at this stage were hormone-related. miRs profiling after infection also revealed a biphasic alteration, showing miRs alteration at 5 dpi where no virus was detected systemically and a late phase correlating with virus accumulation. Correlation analyses revealed a massive increase in the density of correlation networks after infection indicating a complex reprogramming of the regulatory pathways, either in response to the plant defense mechanism or to the virus infection itself. Our data propose the involvement of a systemic signaling on early miRs alteration. Citation: Bazzini AA, Manacorda CA, Tohge T, Conti G, Rodriguez MC, et al. (2011) Metabolic and miRNA Profiling of TMV Infected Plants Reveals Biphasic Temporal Changes. PLoS ONE 6(12): e28466. doi:10.1371/journal.pone.0028466 Editor: Miguel A. Blazquez, Instituto de Biolog´ıa Molecular y Celular de Plantas, Spain Received June 27, 2011; Accepted November 8, 2011; Published December 12, 2011 Copyright: 2011 Bazzini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from Instituto Nacional de Tecnolog´ıa Agropecuaria (INTA) PE243532 (Argentina), Agencia Nacional de Promocio´ n Cient´ıfica y Tecnolo´ gica (ANPCyT) (Argentina) Proyecto de Investigacio´ n Cient´ıfica y Tecnolo´ gica (PICT) 2005 32598, and Max Planck Society (Germany). A.B. is INTA research assistant and held a fellowship from European Molecular Biology Organization (EMBO) and Consejo Nacional de Investigaciones Cient´ıficas y Te´cnicas (CONICET). C.A.M. holds a fellowship from INTA and G.C. holds an ANPCyT fellowship. T.T. is a recipient of a fellowship from the Alexander von Humdoldt foundation. M.C.R. hold a CONICET Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sasurmendi@cnia.inta.gov.ar € Current address: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, United States of America Introduction Plants defend themselves against pathogens using a number of strategies and over the years much effort has been made to understand plant-pathogen interactions. The induction of genes and metabolites acting as defense or counter-defense compounds is a common feature that either enhances virus disease by suppressing host defense mechanisms or attenuate the infection orchestrating defense mechanisms [1,2]. Furthermore, the emerg- ing virus-host interaction picture suggests a highly complex network of plant responses and viral counter-responses which greatly impact in the plant physiology [3]. However, despite our broad knowledge concerning the infection effect at the whole plant level, documentation of the metabolic changes associated to this response remains scarce [4]. Systemic acquired resistance (SAR) and induced systemic resistance are enhanced states of broad-spectrum disease resistance in response to signaling and amplification processes triggered by pathogen infection [5,6,7,8]. During these processes salicylic acid (SA), as methyl salicylate, and jasmonates (JA) have been proposed to play crucial roles during long-distance signaling [8,9,10,11,12]. It is well established that several other players are required to induce SAR such as hormones, lipids or proteins. However, how the systemic response is orchestrated still remains poorly characterized [13]. Plants produce an exceptionally large quantity of metabolites, which display a very broad chemical diversity [14,15]. Thus, comparing metabolic profiles of infected plants versus their corresponding controls conceivably represents a powerful tool by which to unravel the biochemical pathways involved in multi- factorial disorders. microRNAs (miRNAs) are small, endogenous RNAs that regulate gene expression in plants and animals by promoting cleavage or inhibiting translation of mRNAs coded by specific target genes [16,17,18,19,20]. miRNAs are involved in regulation of plant development, signal transduction, expression of transcrip- tion factors, protein degradation and interestingly are part of the response to biotic and abiotic stresses. We and others have demonstrated that after virus infection, miRs levels are altered and their alteration correlates with symptoms suggesting an important role of miRNAs in the manifestation of pathogen symptoms [21,22,23,24]. It was also proposed that miRNAs function is exploited by the pathogens to regulate host gene expression for their own benefit [25,26,27,28,29,30,31,32]. However, the PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28466