(Andersen et al 2004) and Bestkeeper v1 (Pfaffl et al, 2004) according to the developer’s instructions. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3(7):RESEARCH0034 Andersen CL, Jensen JL, Orntoft TF: Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 2004, 64:5245– 5250. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP: Determination of stable housekeeping genes, differentially regu- lated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 2004, 26:509– 515. (DOC) References 1. Hull R (2002) Matthews’ Plant Virology: Academic Press. 2. Whitham SA, Yang C, Goodin MM (2006) Global impact: elucidating plant responses to viral infection. Mol Plant Microbe Interact 19: 1207–1215. 3. Culver JN, Padmanabhan MS (2007) Virus-induced disease: altering host physiology one interaction at a time. Annu Rev Phytopathol 45: 221–243. 4. Jahangir M, Abdel-Farid IB, Choi YH, Verpoorte R (2008) Metal ion-inducing metabolite accumulation in Brassica rapa. J Plant Physiol 165: 1429–1437. 5. Metraux JP (2002) Recent breakthroughs in the study of salicylic acid biosynthesis. Trends Plant Sci 7: 332–334. 6. Durrant WE, Dong X (2004) Systemic acquired resistance. Annu Rev Phytopathol 42: 185–209. 7. Mishina TE, Zeier J (2006) The Arabidopsis flavin-dependent monooxygenase FMO1 is an essential component of biologically induced systemic acquired resistance. Plant Physiol 141: 1666–1675. 8. Browse J (2009) Jasmonate passes muster: a receptor and targets for the defense hormone. Annu Rev Plant Biol 60: 183–205. 9. Truman W, Bennett MH, Kubigsteltig I, Turnbull C, Grant M (2007) Arabidopsis systemic immunity uses conserved defense signaling pathways and is mediated by jasmonates. Proc Natl Acad Sci U S A 104: 1075–1080. 10. Park SW, Kaimoyo E, Kumar D, Mosher S, Klessig DF (2007) Methyl salicylate is a critical mobile signal for plant systemic acquired resistance. Science 318: 113–116. 11. Vlot AC, Dempsey DA, Klessig DF (2009) Salicylic Acid, a multifaceted hormone to combat disease. Annu Rev Phytopathol 47: 177–206. 12. Bari R, Jones JD (2009) Role of plant hormones in plant defence responses. Plant Mol Biol 69: 473–488. 13. Vlot AC, Klessig DF, Park SW (2008) Systemic acquired resistance: the elusive signal(s). Curr Opin Plant Biol 11: 436–442. 14. Fernie AR (2007) The future of metabolic phytochemistry: larger numbers of metabolites, higher resolution, greater understanding. Phytochemistry 68: 2861–2880. 15. Yonekura-Sakakibara K, Saito K (2009) Functional genomics for plant natural product biosynthesis. Nat Prod Rep 26: 1466–1487. 16. Ambros V (2004) The functions of animal microRNAs. Nature 431: 350–355. 17. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281–297. 18. Blevins T, Rajeswaran R, Shivaprasad PV, Beknazariants D, Si-Ammour A, et al. (2006) Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing. Nucleic Acids Res 34: 6233–6246. 19. Carrington JC, Ambros V (2003) Role of microRNAs in plant and animal development. Science 301: 336–338. 20. Kurihara Y, Watanabe Y (2004) Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc Natl Acad Sci U S A 101: 12753–12758. 21. Bazzini AA, Almasia NI, Manacorda CA, Mongelli VC, Conti G, et al. (2009) Virus infection elevates transcriptional activity of miR164a promoter in plants. BMC Plant Biol 9: 152. 22. Bazzini AA, Hopp HE, Beachy RN, Asurmendi S (2007) Infection and coaccumulation of tobacco mosaic virus proteins alter microRNA levels, correlating with symptom and plant development. Proc Natl Acad Sci U S A 104: 12157–12162. 23. Cillo F, Mascia T, Pasciuto MM, Gallitelli D (2009) Differential effects of mild and severe Cucumber mosaic virus strains in the perturbation of MicroRNA- regulated gene expression in tomato map to the 39 sequence of RNA 2. Mol Plant Microbe Interact 22: 1239–1249. 24. Tagami Y, Inaba N, Kutsuna N, Kurihara Y, Watanabe Y (2007) Specific enrichment of miRNAs in Arabidopsis thaliana infected with Tobacco mosaic virus. DNA Res 14: 227–233. 25. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAS AND THEIR REGULATORY ROLES IN PLANTS. Annu Rev Plant Biol 57: 19–53. Metabolic and miR Profiling of TMV Infected Plants Figure S9 (DOC) Acknowledgments List of used primers. We especially thank Dr. Mariana del Vas for helpful discussion and Laura Ramos for great bench assistance. F.C. and S.A. are members of CONICET and INTA researchers. Author Contributions Conceived and designed the experiments: AAB SA. Performed the experiments: AAB CAM TT GC MCR AN-N SV SA. Analyzed the data: AAB CAM TT ARF FC SA. Wrote the paper: AAB ARF FC SA. 26. Padmanabhan C, Zhang X, Jin H (2009) Host small RNAs are big contributors to plant innate immunity. Curr Opin Plant Biol 12: 465–472. 27. Ruiz-Ferrer V, Voinnet O (2009) Roles of plant small RNAs in biotic stress responses. Annu Rev Plant Biol 60: 485–510. 28. Silhavy D, Burgyan J (2004) Effects and side-effects of viral RNA silencing suppressors on short RNAs. Trends Plant Sci 9: 76–83. 29. Dunoyer P, Voinnet O (2005) The complex interplay between plant viruses and host RNA-silencing pathways. Curr Opin Plant Biol 8: 415–423. 30. Voinnet O (2005) Induction and suppression of RNA silencing: insights from viral infections. Nat Rev Genet 6: 206–220. 31. Gottwein E, Cullen BR (2008) Viral and cellular microRNAs as determinants of viral pathogenesis and immunity. Cell Host Microbe 3: 375–387. 32. Deleris A, Gallego-Bartolome J, Bao J, Kasschau KD, Carrington JC, et al. (2006) Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science 313: 68–71. 33. Chapman EJ, Prokhnevsky AI, Gopinath K, Dolja VV, Carrington JC (2004) Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev 18: 1179–1186. 34. Dunoyer P, Lecellier CH, Parizotto EA, Himber C, Voinnet O (2004) Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing. Plant Cell 16: 1235–1250. 35. Chen J, Li WX, Xie D, Peng JR, Ding SW (2004) Viral virulence protein suppresses RNA silencing-mediated defense but upregulates the role of microrna in host gene expression. Plant Cell 16: 1302–1313. 36. Kasschau KD, Xie Z, Allen E, Llave C, Chapman EJ, et al. (2003) P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA unction. Dev Cell 4: 205–217. 37. Vogler H, Kwon MO, Dang V, Sambade A, Fasler M, et al. (2008) Tobacco mosaic virus movement protein enhances the spread of RNA silencing. PLoS Pathog 4: e1000038. 38. Csorba T, Bovi A, Dalmay T, Burgyan J (2007) The p122 subunit of Tobacco Mosaic Virus replicase is a potent silencing suppressor and compromises both small interfering RNA- and microRNA-mediated pathways. J Virol 81: 11768–11780. 39. Kurihara Y, Inaba N, Kutsuna N, Takeda A, Tagami Y, et al. (2007) Binding of tobamovirus replication protein with small RNA duplexes. J Gen Virol 88: 2347–2352. 40. He XF, Fang YY, Feng L, Guo HS (2008) Characterization of conserved and novel microRNAs and their targets, including a TuMV-induced TIR-NBS-LRR class R gene-derived novel miRNA in Brassica. FEBS Lett 582: 2445–2452. 41. Reichel C, Beachy RN (1998) Tobacco mosaic virus infection induces severe morphological changes of the endoplasmic reticulum. Proc Natl Acad Sci U S A 95: 11169–11174. 42. Asurmendi S, Berg RH, Koo JC, Beachy RN (2004) Coat protein regulates formation of replication complexes during tobacco mosaic virus infection. Proc Natl Acad Sci U S A 101: 1415–1420. 43. Maher EA, Bate NJ, Ni W, Elkind Y, Dixon RA, et al. (1994) Increased disease susceptibility of transgenic tobacco plants with suppressed levels of preformed phenylpropanoid products. Proc Natl Acad Sci U S A 91: 7802–7806. 44. Shadle GL, Wesley SV, Korth KL, Chen F, Lamb C, et al. (2003) Phenylpropanoid compounds and disease resistance in transgenic tobacco with altered expression of L-phenylalanine ammonia-lyase. Phytochemistry 64: 153–161. 45. Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36: D154–158. 46. Liu HH, Tian X, Li YJ, Wu CA, Zheng CC (2008) Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana. Rna 14: 836–843. 47. Trindade I, Capitao C, Dalmay T, Fevereiro MP, Santos DM (2010) miR398 and miR408 are up-regulated in response to water deficit in Medicago truncatula. Planta 231: 705–716. PLoS ONE | www.plosone.org 13 December 2011 | Volume 6 | Issue 12 | e28466