quantification number) was obtained from the equation 2DDCT where DDCT represents DCT(control) - DCT(N-isobutyl decana- mide 60 mM). Each CT was previously normalized using the expression levels of ACT2/7 as internal reference. Expression levels were obtained from four independent replicates, every set of oligonucleotides had an efficiency greater than 99%. Standar deviations were less than 0.1 arbitrary units. (TIF) Figure S3 Local induction of defense genes by N- isobutyl decanamide on detached leaves. Leaves from 20 day-old transgenic LOX2:GUS or WT (Col-0) plants grown in soil were detached and incubated 24 h on solvent (control, white squares), or 30 mM N-isobutyl decanamide containing plates (black squares), transferred to decanamide-free plates and then analized. (A) Dose-response assay with leaves from transgenic Arabidopsis line carrying LOX2:GUS were stained for GUS expression 24 h after transference to agar plates. (B) qRT-PCR analysis of the JA- responsive genes OPR3 and VSP2, and the camalexin biosynthetic marker PAD3 using CT value of ACT2/7 as internal expression reference. Relative expression values were normalized with endogenous levels from each transcript in Col-0 control seedlings. Bars represent 6 SE from three independent biological replicates from 30 leaves each one, and from four technical replicates for the assay. (TIF) Figure S4 Effect of N-isobutyl decanamide on Botrytis cinerea mycelial growth. B. cinerea mycelium excised from a solid culture in Petri dishes was transferred to potato dextrose agar dishes supplemented with N-isobutyl decanamide at the concen- trations indicated. Radial growth of the fungus was measured 24, 48 and 72 h after inoculation (A). Data means average radial References 1. Glazebrook J (2001) Genes controlling expression of defense responses in Arabidopsis - 2001 status. Curr Opin Plant Biol 4: 301–308. 2. Wendehenne D, Courtois C, Besson A, Gravot A, Buchwalter A, et al. (2006) NO-based signaling in plants. In: Lamattina L, Polacco JC, eds. Nitric oxide in plant growth, Plant Cell Monogr vol 6. Springer-Verlag, Berlin Heidelberg New York. pp 35–51. 3. Satner A, Estelle M (2009) Recent advances and emerging trends in plant hormone signaling. Nature 459: 1071–1078. 4. Shah J (2005) Lipids, lipases, and lipid-modifying enzymes in plant disease resistance. Annu Rev Phytopathol 43: 229–260. 5. Upchurch RG (2008) Fatty acid unsaturation, mobilization, and regulation in the response of plants to stress. Biotech Lett 30(6): 967–977. 6. Kachroo A, Kachroo P (2009) Fatty acid-derived signals in plant defense. Ann Rev Phytopathol 47: 153–176. 7. Kachroo A, Lapchyk L, Fukushige H, Hildebrand D, Klessig D, et al. (2003) Plastidial fatty acid signaling modulates salicylic acid- and jasmonic acid- mediated defense pathways in the Arabidopsis ssi2 mutant. Plant Cell 15: 2952–2965. 8. Savchenko T, Walley JW, Chehab EW, Xiao Y, Kaspi R, et al. (2010) Arachidonic acid: an evolutionarily conserved signaling molecule modulates plant stress signaling networks. Plant Cell 22(10): 3193–3205. 9. Grechkin A (1998) Recent developments in biochemistry of the plant lipoxigenase pathway. Prog Lipid Research 37: 317–352. 10. Berger S (2002) Jasmonate-related mutants of Arabidopsis as tools for studying stress signaling. Planta 214: 497–504. 11. Devoto A, Ellis C, Magusin A, Chang H-S, Chilcott C, et al. (2005) Expression profiling reveals COI1 to be a key regulator of genes involved in wound- and methyl jasmonate-induced secondary metabolism, defense, and hormone interactions. Plant Mol Biol 58: 497–513. 12. Browse J (2009) Jasmonate passes muster: a receptor and targets for the defense hormone. Ann Rev Plant Biol 60: 183–205. 13. Turner JG, Ellis C, Devoto A (2002) The jasmonate signal pathway. Plant Cell Suppl. pp S153–S164. 14. Yan J, Zhang C, Gu M, Bai Z, Zhang W, et al. (2009) The Arabidopsis CORONATINE INSENSITIVE1 protein is a jasmonate receptor. Plant Cell 21: 2220–2236. 15. Kazan K, Manners JM (2008) Jasmonate signaling: toward an integrated view. Plant Physiol 146: 1459–1468. Alkamide-Regulated Transcriptional Networks growth from three independent samples 6 SD; no statistical differences were found at any concentration tested. Mycelial growth at 72 h after inoculation on solvent-containing media (Control) and 120 mM N-isobutyl decanamide-supplied media (B). Fungicide Techto 60 was employed at 1 mg/ml as fungal growth inhibition control to compare with the highest concentration of N- isobutyl decanamide in divided Petri dishes (C). (TIF) Table S1 Full list of genes differentially expressed by N-isobutyl decanamide in Arabidopsis. (PDF) Table S2 Functional categories over-represented N-isobutyl decanamide responsive genes. (PDF) in Table S3 Sequences of oligonucleotides used as PCR primers for quantitative expression analysis. (PDF) Acknowledgments We thank Drs Carla Schommer, Detlef Weigel and Frederick M. Ausubel for kind donation of transgenic seeds. We also thank Susana M.L. Fuentes- Guerra, Flor Zamudio-Herna´ndez and Mar´ıa de J. Ortega-Estrada for qRT-PCR analysis and microarray scanning. Author Contributions Conceived and designed the experiments: JLB AMB CCV EIL LHE. Performed the experiments: AMB EIL ERC CCV JRG. Analyzed the data: AMB LHE JLB EIL CCV AGG ERC. Contributed reagents/ materials/analysis tools: LHE JLB AGG JMT. Wrote the paper: AMB LHE JLB. 16. Staswick PE, Su W, Howell SH (1992) Methyl jasmonate inhibition of root growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant. Proc Natl Acad Sci USA 89: 6837–6840. 17. Takahashi F, Yoshida R, Ichimura K, Mizoguchi T, Seo S, et al. (2007) The mitogen-activated protein kinase cascade MKK3–MPK6 is an important part of the jasmonate signal transduction pathway in Arabidopsis. Plant Cell 19: 805–818. 18. Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, et al. (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415: 977–983. 19. Ichimura K, Shinozaki K, Tena G, Sheen J, Henry Y, et al. (2002) Mitogen- activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci 7: 301–308. 20. Qiu JL, Fiil BK, Petersen K, Nielsen HB, Botanga CJ, et al. (2008) Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus. EMBO J 27: 2214–2221. 21. Hu X, Li W, Chen Q, Yang Y (2009) Early signal transduction linking the synthesis of jasmonic acid in plant. Plant Sig & Beh 4: 696–697. 22. Ren DT, Liu Y, Yang KY, Han L, Mao G, et al. (2008) A fungal-responsive MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis. Proc Natl Acad Sci USA 105: 5638–5643. 23. Ram´ırez-Cha´vez E, Lo´pez-Bucio J, Herrera-Estrella L, Molina-Torres J (2004) Alkamides isolated from plants promote growth and alter root development in Arabidopsis. Plant Physiol 134: 1058–1068. 24. Blancaflor EB, Hou G, Chapman KD (2003) Elevated levels of N- lauroylethanolamine, an endogenous constituent of desiccated seeds, disrupt normal root development in Arabidopsis thaliana seedlings. Planta 217: 206–217. 25. Ort´ız-Castro R, Mart´ınez-Trujillo M, Lo´ pez-Bucio J (2008) N-acyl-L- homoserine lactones: a class of bacterial quorum-sensing signals alter post- embryonic root development in Arabidopsis thaliana. Plant Cell Environ 31: 1497–1509. 26. Chapman KD, Venables B, Markovic R, Blair RW, Bettinger C (1999) N- Acylethanolamines in seeds. Quantification of molecular species and their degradation upon imbibition. Plant Physiol 120: 1157–1164. 27. Tripathy S, Venables BJ, Chapman KD (1999) N-acylethanolamines in signal transduction of elicitor perception. Attenuation of alkalinisation response and activation of defense gene expression. Plant Physiol 121: 1299–1308. PLoS ONE | www.plosone.org 13 November 2011 | Volume 6 | Issue 11 | e27251