An amino acid substitution inhibits specialist herbivore production of an antagonist effector and recovers insect-induced plant defenses

Abstract

Plants respond to insect herbivory through the production of biochemicals that function as either direct defenses or indirect defenses via the attraction of natural enemies. While attack by closely related insect pests can result in distinctive levels of induced plant defenses, precise biochemical mechanisms responsible for differing responses remain largely unknown. Cowpea (Vigna unguiculata) responds to Fall armyworm (Spodoptera frugiperda) herbivory through the detection of fragments of chloroplastic ATP synthase γ-subunit proteins, termed inceptin-related peptides, present in larval oral secretions (OS). In contrast to generalists like Fall armyworm, OS of the legume-specializing velvetbean caterpillar (VBC; Anticarsia gemmatalis) do not elicit ethylene production and demonstrate significantly lower induced volatile emission in direct herbivory comparisons. Unlike all other Lepidoptera OS examined, which preferentially contain inceptin (Vu-In; +ICDINGVCVDA-), VBC OS contain predominantly a C-terminal truncated peptide, Vu-In(-A) (+ICDINGVCVD-). Vu-In(-A) is both inactive and functions as a potent naturally occurring antagonist of Vu-In-induced responses. To block antagonist production, amino acid substitutions at the C terminus were screened for differences in VBC gut proteolysis. A valine-substituted peptide (Vu-In(ΔV); +ICDINGVCVDV-) retaining full elicitor activity was found to accumulate in VBC OS. Compared with the native polypeptide, VBC that previously ingested 500 pmol of the valine-modified chloroplastic ATP synthase γ-subunit precursor elicited significantly stronger plant responses in herbivory assays. We demonstrate that a specialist herbivore minimizes the activation of defenses by converting an elicitor into an antagonist effector and identify an amino acid substitution that recovers these induced plant defenses to a level observed with generalist herbivores.

Figure 1.

The legume specialist VBC elicits weaker defense responses than the generalist FAW on cowpea leaves. A and B, Average (n = 4; +se) ET production (A) at 1 h and DMNT tissue pools (B) at 4 h after cowpea leaves were treated as undamaged controls (Con) or damaged and treated with 5 μL of water (Dam), VBC OS, or FAW OS. C, Average (n = 7; +se) cowpea leaf tissue pools of DMNT at 4 h after a single feeding bout by VBC and FAW larvae. D, Average (n = 4; +se) whole-plant cowpea volatile emission of DMNT during continuous herbivore feeding damage by eight early sixth instar VBC and FAW larvae. Different letters above the bars (a–c) represent significant differences (all ANOVA P values were P < 0.007; Tukey’s test corrections for multiple comparisons were at P < 0.05). FW, Fresh weight.

Figure 2.

In contrast with other Lepidopteran pests, VBC OS contain predominantly the inactive inceptin Vu-In^−A. A and B, HPLC-MS selected [M+H]⁺ mass-to-charge ratio ion trace of inceptin-related peptides Vu-In (1,119.5) and Vu-In^–A (1,048.5) in FAW OS (A) and VBC OS (B) following ingestion of the 19-mer-Vu-In peptide precursor (⁺KGEICDINGVCVDAAEDEF⁻). C, Average (n = 4; +se) percentage of active inceptins [(active = Vu-^GE+In + Vu-^E+In + Vu-In)/(total = Vu-^GE+In + Vu-^E+In + Vu-In + Vu-In^−A)] quantified by HPLC-MS in the larval OS of eight different Lepidopteran species following ingestion of 4.5 nmol or less of 19-mer-Vu-In. Different letters above the bars (a–c) represent significant differences (ANOVA P < 0.0001; Tukey’s test correction for multiple comparisons was at P < 0.05).

Figure 3.

Vu-In^−A is a natural antagonist of inceptin elicitation. A, Average (n = 4; ± se) ET production in damaged cowpea leaves treated first with either water (solid line) or Vu-In^−A (dashed line) followed by a subsequent treatment 1 min later with Vu-In. Treatments involving both Vu-In^−A and Vu-In utilized equivalent paired doses separated by time. B, Average (n = 4; ± se) ET production in damaged cowpea leaves treated simultaneously with a fixed amount of Vu-In (1 pmol leaf⁻¹) and increasing levels of Vu-In^−A in the mixture. Vu-In ranged from 100% to 5.8% and included a water control (0%). Different letters above the bars (a–d) represent significant differences (all ANOVA P values were P < 0.0001; Tukey’s test corrections for multiple comparisons were at P < 0.05).

Figure 4.

cATPC amino acid substitutions alter inceptin activity and processing in VBC OS. A, Average (n = 3; +se) ET production in damaged cowpea leaves treated with 5 μL of water containing 10 pmol of C-terminally substituted peptides Vu-In^ΔX (⁺ICDINGVCVDX⁻). Capital letters (D through Y) denote the amino acid analogs of Vu-In. B and C, The 11-mer-Vu-In^ΔX peptides recovered from VBC OS (ng 100 μL⁻¹) after ingestion of active C-terminally substituted 11-mer-Vu-In^ΔX (B) and 19-mer-Vu-In^ΔX (C) analogs (⁺KGEICDINGVCVDXAEDEF⁻). Black and white bars represent C-terminally substituted 11-mer-Vu-In^ΔX analogs and Vu-In^−A, respectively. D, Average (n = 4; +se) ET production in undamaged cowpea leaves (Con) or those damaged and treated with 5 μL of water, Vu-In, 19-mer-Vu-In, or 19-mer-Vu-In^ΔV, all at 10 pmol leaf⁻¹. Asterisks represent peptides with ET production significantly lower than Vu-In (i.e. C terminal in A). Different letters above the bars (a–c) represent significant differences (all ANOVA P values were P < 0.000; Tukey’s test correction for multiple comparisons was at P < 0.05).

Figure 5.

An inceptin Ala-to-Val C-terminal substitution maintains activity and enables the predominant accumulation of active elicitors in VBC OS. A, Dose response of average (n = 4; ± se) ET production in damaged cowpea leaves treated with either Vu-In or Vu-In^ΔV. B, Average (n = 4; +se) percentage of active inceptins recovered from VBC OS after larvae were fed 4.9 nmol of either 19-mer-Vu-In or 19-mer-Vu-In^ΔV. Different letters above the bars (a and b) represent significant differences (Student’s t test, P < 0.001).

Figure 6.

An Ala-to-Val substitution in cATPC polypeptides recovers induced plant defenses during VBC herbivory. A to D, Average (n = 6; +se) concentrations of JA (A), SA (B), CA (C), and DMNT (D) 4 h after cowpea leaves experienced a single feeding bout from VBC larvae that had previously ingested cowpea leaf discs containing water, 0.5 nmol of 19-mer-Vu-In (A) or 0.5 nmol of 19-mer-Vu-In^ΔV (V). E to H, Average (n = 6; +se) concentrations of JA (E), SA (F), CA (G), and DMNT (H) in a repeated experiment using 2.5 nmol of 19-mer-Vu-In (A) or 19-mer-Vu-In^ΔV (V). Within plots, different letters above the bars (a and b) represent significant differences (all ANOVA P values were P < 0.015; Tukey’s test corrections for multiple comparisons were at P < 0.05). FW, Fresh weight.

Figure 7.

Simplified proposed model for generalist activation, specialist suppression, and engineered recovery of inceptin-elicited plant defenses in cowpea. 1A, FAW and VBC larvae consume cowpea leaves and produce the predominant cATPC digestive fragments Vu-In and Vu-In^−A, respectively. 1B, VBC larvae that consume precursor proteins containing an altered cATPC^ΔV motif produce the active modified inceptin Vu-In^ΔV. 2, Plants indirectly perceive larval attack when Vu-In and Vu-In^ΔV peptides recontact the wounded leaf surface and bind a putative receptor. VBC that contain Vu-In^−A at levels of 66% or greater of the total inceptin-related peptides mediate the antagonism of Vu-In activity. As a preliminary hypothesis, Vu-In^−A antagonism potentially occurs at the receptor-ligand level through competition. 3, Dependent upon the proportions of inceptin-related peptides present, multiple signaling pathways can be elicited, including the phytohormones JA, ET, and SA, or antagonized (ET and SA). 4, Insect OS elicitors drive quantitatively different levels of induced biochemical defenses such as DMNT that can provide reliable cues to facilitate the attraction of natural enemies.