Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. III. Fatty acid-amino acid conjugates in herbivore oral secretions are necessary and sufficient for herbivore-specific plant responses

Abstract

Feeding by the tobacco specialist Manduca sexta (Lepidoptera, Sphingidae) and application of larval oral secretions and regurgitant (R) to mechanical wounds are known to elicit: (a) a systemic release of mono- and sesquiterpenes, (b) a jasmonate burst, and (c) R-specific changes in transcript accumulation of putatively growth- and defense-related mRNAs in Nicotiana attenuata Torr. ex Wats. We identified several fatty acid-amino acid conjugates (FACs) in the R of M. sexta and the closely related species Manduca quinquemaculata which, when synthesized and applied to mechanical wounds at concentrations comparable with those found in R, elicited all three R-specific responses. Ion-exchange treatment of R, which removed all detectable FACs and free fatty acids (FAs), also removed all detectable activity. The biological activity of ion-exchanged R could be completely restored by the addition of synthetic FACs at R-equivalent concentrations, whereas the addition of FAs did not restore the biological activity of R. We conclude that the biological activity of R is not related to the supply of FAs to the octadecanoid cascade for endogenous jasmonate biosynthesis, but that FACs elicit the herbivore-specific responses by another mechanism and that the insect-produced modification of plant-derived FAs is necessary for the plant's recognition of this specialized herbivore.

Figure 1

Scheme of experimental setup of the ion-exchange approach and structures of identified FACs (left) and HPLC-MS-Base peak profiles of 10-μL injections of test solutions (right): A, oral secretions and regurgitant (R) from M. sexta larvae. HPLC gradient (C18): CH₃CN/H₂O; 0.5% (v/v) HAc; 0.7 mL/min: 0% (v/v) CH₃CN, 20 to 25 min 100% (v/v) CH₃CN. Separation of the FACs in M. sexta (B) and M. quinquemaculata (C) R: HPLC gradient (C18): CH₃CN/H₂O; 0.5% (v/v) HAc; 0.7 mL/min: 40% (v/v) CH₃CN, 7 min 68% (v/v) CH₃CN, 18 min 80% (v/v) CH₃CN, 28 min 100% (v/v) CH₃CN. 1, N-linolenoyl-l-Gln; 2, unidentified; 3, N-linolenoyl-l-Glu; 4, N-linoleoyl-l-Gln; 5, N-linoleoyl-l-Glu; 6, N-palmitoyl-l-Gln; 7, N-oleoyl-l-Gln; and 8, N-palmitoyl-l-Glu. Base peak profiles of ion-exchanged M. sexta R (D) and mixture of synthetic FACs at concentrations found in M. sexta R (E) analyzed with the HPLC gradient as in B and C.

Figure 2

Mean (±se) WP cis-α-bergamotene trapped per hour, per liter air sampled from individual (eight per treatment) N. attenuata (A) plants and mean (±se) JA concentrations (n.d., not determined) of node two leaves of four replicate plants per treatment 35 min (time of maximum JA induction; B) after the node two leaf was wounded and treated with 20 μL of the following test solutions: water (W), oral secretions and regurgitant from M. sexta larvae (M.s.) or M. quinquemaculata (M.q.), exR, Triton X-100 in water (T), FAC in concentrations found in R and FA in concentrations found in R in the triton solution or in exR. Control plants (C) remained undamaged. Stars represent significantly (P < 0.05) increased emissions as compared with wounded plants treated with water (W) as determined by Fisher's protected least significant difference from ANOVAs.

Figure 3

Northern analysis of transcript accumulation in response to different test solutions. The node two leaf of five replicate rosette-stage plants was continuously wounded and supplied with water (W), M. sexta larval oral secretions and regurgitant (R), exR, Triton X-100 in water (T), FACs or FAs in concentrations found in R, dissolved in either T or exR for 80 min, creating one row of puncture wounds every 20 min, and harvesting 20 min after the final treatment. Untreated node two leaves were harvested as controls (C). Hybridization with an 18S rRNA probe indicates equal loading.