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Figure S1. WW or WOS-induced transcriptabundance of NaJIH1 in treated leaves and roots of WTN.  attenuata plants. Five fully elongated leavesof WT N.  attenuata plants were elicited bywounding (WW) or wounding plus M.  sexta OSapplication (WOS) and pooled before RNA extraction and cDNAsynthesis. Mean ± SE transcript abundances ofNaJIH1 (technical replicates n = 3)were determined by qPCR for treated leaves (a) and roots (b1) or bymicroarrays for roots (b2; biological replicates= 3).

Figure S2. Expression of JA biosynthetic genesin WW or WOS-induced leaves of WT N.  attenuataplants as determined by cDNA microarrays. After WW or WOStreatments of fully elongated leaves of WTN.  attenuata plants (= 3), RNAextraction and cDNA synthesis were performed with pooled samplesand the relative transcript accumulation(mean ± SE) of JA biosynthetic genes weredetermined by microarray in treated and systemic leaves.

Figure S3. Nucleotide and amino acid sequencesof the Nicotiana attenuata JIH1. (a)Sequence-verified, full-length coding sequence of the NaJIH1gene (stop codon is highlighted in green bold). The predictedsignal peptide sequence that was removed from invitro–expressed protein is indicated in red bold;277 bp-long underlined sequence was introduced as an invertedrepeat into an RNAi silencing construct. (b) Amino acid sequence ofthe full-length NaJIH1 protein; consensus tetrapeptide sequence(HDEL) known to function as an ER localization and retention signalis located at the C-terminus of the NaJIH1 the sequence(underlined). Predicted signal peptide at the N-terminus ofthe protein is highlighted in red bold.

Figure S4. Phylogeny of JIH1 and ILR-likehydrolases. (a) IAR3 sequences from several plant species wereBLAST-retrieved from NCBI using the tBLASTX program. After aligningthe sequences using the CLUSTAL W package, Maximum Likelihood treeswere re-constructed and tested by bootstraping in MEGA5. (b) NaJIH1was compared with other members of theA.  thaliana ILR1-like IAA amidohydrolases (sequence retrieval, alignment and phylogeny reconstruction was performed as described in a).

Figure S5. Enzymatic activity of JIH1 againstother substrates. The activity of JIH1 was tested against (a)equimolar mixture of JA-Ile and IAA-Ala (b) JA-Glu, JA-Met andJA-Val using Mn2+ as a co-factor. In controls, nosignificant amounts of products were detected while JA and IAA werereleased from the respective conjugates in the presence ofrecombinant NaJIH1-GST protein. Quantification of thereleased JA/IAA was performed using known amounts ofD2-JA/13C6-IAA standards. (c)Relative transcript abundance of the putativeN.  attenuata homologue of theA.  thaliana CYP94B3 gene, and threonine deaminase(TD), jasmonic acid resistant 4 (JAR4) and JAR6 fromN.  attenuata in WOS-induced leaves of WT and irJIH1-silenced plants. Statistical differences are described with different letters (<SMALLCAPS>ANOVA</SMALLCAPS>; P < 0.05).

Figure S6. WOS-induced in vivoaccumulation of other JA-amino acid conjugates in WT and irJIH1plants. Fully elongated leaves of WT and irJIH1 plants(n = 3) were WOS-induced and the levels of JA-Val(a), JA-Glu (b) and JA-Met (c) were measured by LC-MS3.irJIH1 plants accumulated significantly higher amounts of JA-Valthan did WT plants (a), while the levels of other conjugates (b andc) were not significantly different(<SMALLCAPS>ANOVA</SMALLCAPS>,P < 0.05). The relative amounts of other JAconjugates were normalized using the13C6-labeled JA-Ile internal standard.

Figure S7. Accumulation of jasmonates in EV andirJIH1 plants after multiple elicitations. EV and irJIH1 plantswere grown in the field (Santa Clara, Utah, USA) and fullyelongated leaves were elicited either once by WW (1X WW) or threetimes by WOS (3X OS) every h. Samples (n = 7) werecollected and jasmonates were analyzed by LC-MS3.Following multiple elicitation (3X WOS), irJIH1 plants showedsignificantly higher accumulations of JA-Ile (b) and OH-JA-Ile (c)than did WT plants (<SMALLCAPS>ANOVA</SMALLCAPS>;P < 0.05).

Figure S8. Accumulation of herbivory-induceddefense secondary metabolites in field-grown EV and irJIH1 plants.Field-grown (UT, USA) EV and irJIH1 (n = 6) plantswere treated by WOS. Samples were collected after 24 h toanalyze the accumulation of defense secondary metabolites. irJIH1plants accumulated significantly higher levels of(<SMALLCAPS>ANOVA</SMALLCAPS>,P < 0.01) HGL-DTGs (b) and TPI (c) than did EVplants. When individual HGL-DTGs were analyzed by LC-MS3(d), irJIH1 plants were found to have more mono-malonylated (V andVI) and dimalonylated (VII, IX and X) HGL-DTGs than did EV plants(<SMALLCAPS>ANOVA</SMALLCAPS>,P < 0.01). Significant differences areindicated by different letters.

Figure S9. Accumulation of other jasmonatesafter exogenous IAA application. WT and irJIH1 plants(n = 5) were wounded by a pattern wheel andtreated with OS that was supplemented with varying concentrationsof IAA. After 1 h, samples were collected and the effect ofexogenous IAA application on the endogenous accumulation of JA-Ile(in Figure 3) and other jasmonates in WOS-induced leaves wasexamined: (a) OH-JA-Ile, (b) COOH-JA-Ile, (c) JA and (d)12-OH-JA.

Figure S10. Volatile organic compounds notdifferentially regulated in irJIH1 plants. WT and irJIH1 plants(n = 12) were induced by WOS treatments and thevolatile organic compounds emitted in the first 3 h, and inthe following 24 h after elicitation were analyzed by GC-MS.Volatile compounds listed in this figure were not significantlydifferent in irJIH1 plants compared to WT plants(<SMALLCAPS>ANOVA</SMALLCAPS>; P < 0.05);volatiles that were released at significantly different amounts areshown in Figure 6.

Table S1. List of primers.

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