Supporting Figures

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Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10
Supporting Figure 11

Fig. 6. FAC elicitors do not trigger volatile emission in cowpea. Mean (n = 6; +SEM) DMNT collected from intact 7-day-old soil grown seedlings with two leaves is shown. Both leaves were scratched and treated with either 5 ml of 50 mM phosphate buffer (pH 8.0) containing either H₂O (Buffer) or 1 nmol of N-linoleoyl-L-glutamine (Gln-18:2), N-linolenoyl-L-glutamic acid (Glu-18:3), N-linolenoyl-L-glutamine acid (Gln-18:3), volicitin [N-(17-hydroxylinolenoyl)-L-glutamine], or 2.5 ml of cowpea-derived S. frugiperda OS. Treatments were performed twice the previous day and once at 8 a.m. the day of the volatile collection. Different letters (a and b) represent significant differences. (ANOVA, P < 0.0001; Tukey test correction for multiple comparisons, P < 0.05.)

Fig. 7. S. frugiperda OS and inceptin elicit the same cowpea leaf volatiles. Shown is the mean (n = 6, +SEM) leaf emission of DMNT (A) and minor components (E)-b-ocimene (lane 2), methyl salicylate (lane 3), indole (lane 4), (E)-b-farnesene (lane 5), (E,E)-a-farnesene (lane 6), and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (lane 7) 5 h after treatments including undamaged controls (Control) and razor-damaged leaves plus H₂O (Damage + H₂O), 1 ml of cowpea-derived S. frugiperda OS (Damage + OS), or 450 fmol of cowpea inceptin (Damage + Inceptin) (B). Within individual volatiles, different letters (a-d) represent significant differences. (All ANOVA P values were <0.0001. Tukey test corrections for multiple comparisons: P < 0.0001.)

Fig. 8. Alignment of ATPC and source of inceptin. Predicted chloroplastic (c) and mitochondrial (m) ATPC amino acid sequences from DNA sequences from Z. mays, Glycine max, Pisum sativum, Arabidopsis thaliana, Lycopersicon esculentum, Nicotiana tabacum, Solanum tuberosum, Medicago truncatula, Spinacia oleracea, and Triticum aestivum were identified from the GenBank (GB) or TIGR databases by BLAST analysis. V. unguiculata, P. vulgaris var. black bean, Arachis hypogaea, Cicer arietinum, Lens culinaris, and P. lunatus were sequenced from leaf cDNA as described in Materials and Methods.

Fig. 9. Escherichia coli-expressed cATPC-GST fusion protein is not biologically active. Shown is average (n = 3, +SEM) E production in cowpea leaves damaged with the addition of H₂O alone (D), 12 pmol of cATPC-GST fusion protein (cA), or 12 pmol of cATPC-GST fusion protein plus 4 pmol cowpea inceptin (cA + I). Different letters (a and b) represent significant differences. (ANOVA, P < 0.0004; Tukey test correction for multiple comparisons, P < 0.05.)

Fig. 10. S. frugiperda-induced cowpea leaf phytohormone levels depend on previous larval diet and presence of inceptin precursors. Shown are the average (n = 6, +SEM) JA (A and C) and SA (B and D) levels in cowpea 4 h after leaves were untreated (Con) or were attacked by sixth-instar larvae that had previously fed upon cowpea shoots (S), roots (R), or artificial diet containing either 9 mg of the E. coli-expressed proteins GST (G) and cATPC-GST (cA). In the artificial diet study (C and D), OS from larvae fed GST and cA contained undetectable levels and 270 fmol ml^-1 inceptin, respectively. Different letters (a and b) represent significant differences. (All ANOVA P values were <0.008. Tukey test corrections for multiple comparisons: P < 0.05.)

Fig. 11. Synthetic inceptin [⁺ICDVNGVCIDA^-] induces E and volatiles in bean leaves (P. vulgaris). (A) Mean (n = 8, +SEM) leaf E production after treatments consisting of undamaged controls (white bar), and damaged leaves with the addition of 5 ml of H₂O (gray bar), or inceptin (black bar, 1 pmol). (B) Major volatiles induced include (Z)-3-hexenyl acetate (lane 1) and DMNT (lane 2). (C) Minor volatiles induced include (Z)-3-hexen-1-ol (lane 3), (E)-b-ocimene (lane 4), methyl salicylate (lane 5), (E,E)-a-farnesene (lane 6), and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (lane 7). Different letters (a-c) represent significant differences. (All ANOVA P values were <0.0002. Tukey test corrections for multiple comparisons: P < 0.05.)