Research ArticlePLANT SCIENCES

Convergent evolution of a metabolic switch between aphid and caterpillar resistance in cereals

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Science Advances  05 Dec 2018:
Vol. 4, no. 12, eaat6797
DOI: 10.1126/sciadv.aat6797
  • Fig. 1 Phenotype of ZmBx12-overexpressing wheat lines.

    (A) Relative (rel.) expression of the maize DIMBOA-Glc OMT ZmBx12 in wild-type (WT) and ZmBx12-overexpressing plants (n = 3). (B) Major benzoxazinoids in the leaves of the different lines (n = 5). FW, fresh weight. (C) Major benzoxazinoids in the phloem of the different lines (n = 3 to 4). (D) Representative photograph of WT and transgenic lines. (E) Chitosan-induced callose deposition (n = 9 to 12). (F) Aphid-induced callose deposition (n = 23 to 39). Because of irregular shapes and brightness of callose induction spots following aphid attack, area*rel. brightness were used to assess callose deposition. Different letters indicate significant differences between wheat lines [analysis of variance (ANOVA) followed by Holm-Sidak post hoc tests, P < 0.05]. Photo credit for (D): B. Li and T. Züst, University of Bern.

  • Fig. 2 Callose suppression through DIMBOA-Glc O-methylation.

    (A) Induction of callose in WT and ZmBx12-overexpressing lines after infiltration with DIMBOA, DIMBOA-Glc, or HDMBOA-Glc (n = 4 to 11). (B) Representative photographs of callose induction in WT plants after aniline blue staining. (C) Callose induction in WT plants treated with different benzoxazinoid mixtures (n = 5 to 11). (D) Concentration of DIMBOA-Glc (dark gray bars) and DIMBOA (light gray bars) in the leaves of WT and transgenic plants infused with DIMBOA-Glc or DIMBOA (n = 3). Different letters and asterisks indicate significant differences between treatments within lines (ANOVA followed by Holm-Sidak post hoc tests, P < 0.05). Photo credit for (B): B. Li, University of Bern.

  • Fig. 3 DIMBOA-Glc O-methylation changes aphid and caterpillar resistance in opposite directions.

    (A) Preference of S. littoralis caterpillars for WT or ZmBx12-overexpressing plants (n = 15 to 21). (B) Leaf damage in a choice situation (n = 8). Asterisks indicate significant differences between lines (one-sample t tests on pairwise differences, *P < 0.05, **P < 0.01, ***P < 0.001). (C) Caterpillar growth in a no-choice experiment. (D) S. avenae aphid reproduction on different lines. Different letters indicate differences between plant lines (repeated-measures ANOVA followed by Holm-Sidak post hoc tests, P < 0.05). Significance levels for repeated-measure ANOVA factors are shown. n.s., not significant.

  • Fig. 4 DIMBOA-Glc O-methylation mediates a trade-off between induced caterpillar and aphid resistance.

    (A) Changes in major benzoxazinoids in WT and ZmBx12-overexpressing lines at different time points following 24 hours of S. littoralis attack (n = 5 to 7). Different letters indicate significant differences in concentrations over time (ANOVA followed by Holm-Sidak post hoc tests, P < 0.05). (B) Callose inducibility in control and S. littoralis–attacked wheat plants (n = 12 to 20). Asterisks indicate significant differences between treatments (two-way ANOVA followed by Holm-Sidak post hoc tests, *P < 0.05, **P < 0.01, ***P < 0.001). (C) Aphid performance on S. littoralis–attacked wheat plants relative to nonattacked controls (n = 9 to 11). Results from two repetitions of the experiments are shown side by side. Stars indicate a significant reduction of aphid growth induced by S. littoralis caterpillar feeding relative to S. littoralis–unattacked plants (two-way ANOVA followed by Holm-Sidak post hoc tests.

  • Fig. 5 Identification and characterization of TaBX10 as a functional DIMBOA-Glc OMT.

    (A) Recombinant TaBX10 methylates DIMBOA-Glc and DIM2BOA-Glc. The enzyme was heterologously expressed in E. coli, purified, and incubated with a mixture of DIMBOA-Glc and DIM2BOA-Glc. Enzyme products were analyzed using liquid chromatography–tandem mass spectrometry (LC-MS/MS). CPS, counts per second (electron multiplier). (B) Phylogenetic tree of Poaceae OMT genes similar to Bx7. Maize Bx7,10,11,14 and wheat TaBx10 are shown in red and green, respectively. The tree represents a subclade of a larger Poaceae OMT tree that is given in fig. S13. The tree was inferred by using the maximum likelihood method based on the Tamura 3-parameter model. Bootstrap values are shown next to each node. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Zm, Zea mays; Sb, Sorghum bicolor; Sv, Setaria viridis; Si, S. italica; Pv, Panicum virgatum; Ph, P. hallii; Ot, Oropetium thomaeum; Ta, T. aestivum; Hv, Hordeum vulgare; Bd, Brachypodium distachyon; Bs, B. stacei; Os, Oryza sativa. The PACMAD lineage–specific Bx10 clade is marked in red, and its sister clade is marked in blue.

  • Fig. 6 Independently evolved O-methylation of benzoxazinoids and glucosinolates regulates defense and resistance.

    DIMBOA-Glc is required for callose induction in wheat and maize. Methylation of DIMBOA-Glc to HDMBOA-Glc reduces the DIMBOA-Glc pool and subsequently suppresses callose formation. The responsible OMTs, TaBX10 and ZmBX10–ZmBX12, evolved independently from each other. HDMBOA-Glc repels caterpillars, while DIMBOA-Glc reduces aphid growth, most likely by promoting callose formation. In Arabidopsis, methylation of the glucosinolate 4OH-I3M by IGMT1 and IGMT2 leads to the formation of 4MO-I3M, which is required for callose deposition in this species. 4MO-I3M increases aphid and pathogen resistance, most likely by promoting callose formation. IGMT1 and IGMT2 are only distantly related to cereal benzoxazinoid OMTs.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/12/eaat6797/DC1

    Fig. S1. Phenotyping of ZmBx12-overexpressing plants I.

    Fig. S2. Phenotyping of ZmBx12-overexpressing plants II.

    Fig. S3. HDMBOA-Glc and MBOA levels upon DIMBOA and DIMBOA-Glc infiltration.

    Fig. S4. Specificity of benzoxazinoid- and glucosinolate-induced callose deposition.

    Fig. S5. Impact of DIMBOA-Glc O-methylation on wheat pathogen resistance.

    Fig. S6. Aphids do not induce benzoxazinoids in wheat leaves.

    Fig. S7. Identification of DIMBOA-Glc OMT candidate genes.

    Fig. S8. Phylogenetic tree of maize OMT genes similar to Bx7 and wheat OMT genes that were found to be up-regulated after herbivory in wheat seedlings (RNA sequencing).

    Fig. S9. Sequence comparison of maize BX7 and BX10 with herbivore-induced OMT proteins from wheat.

    Fig. S10. Phylogenetic tree of maize and wheat OMT genes similar to Bx7.

    Fig. S11. Identification of TaBx10 as a functional DIMBOA-Glc OMT.

    Fig. S12. No influence of ZmBx12 overexpression on TaBx10 expression.

    Fig. S13. Phylogenetic tree of Poaceae OMT genes similar to Bx7.

    Fig. S14. Phylogenetic tree of maize, wheat, and Arabidopsis OMT genes similar to Bx7.

    Table S1. Wheat OMT genes up-regulated after herbivory (RNA sequencing).

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Phenotyping of ZmBx12-overexpressing plants I.
    • Fig. S2. Phenotyping of ZmBx12-overexpressing plants II.
    • Fig. S3. HDMBOA-Glc and MBOA levels upon DIMBOA and DIMBOA-Glc infiltration.
    • Fig. S4. Specificity of benzoxazinoid- and glucosinolate-induced callose deposition.
    • Fig. S5. Impact of DIMBOA-Glc O-methylation on wheat pathogen resistance.
    • Fig. S6. Aphids do not induce benzoxazinoids in wheat leaves.
    • Fig. S7. Identification of DIMBOA-Glc OMT candidate genes.
    • Fig. S8. Phylogenetic tree of maize OMT genes similar to Bx7 and wheat OMT genes that were found to be up-regulated after herbivory in wheat seedlings (RNA sequencing).
    • Fig. S9. Sequence comparison of maize BX7 and BX10 with herbivore-induced OMT proteins from wheat.
    • Fig. S10. Phylogenetic tree of maize and wheat OMT genes similar to Bx7.
    • Fig. S11. Identification of TaBx10 as a functional DIMBOA-Glc OMT.
    • Fig. S12. No influence of ZmBx12 overexpression on TaBx10 expression.
    • Fig. S13. Phylogenetic tree of Poaceae OMT genes similar to Bx7.
    • Fig. S14. Phylogenetic tree of maize, wheat, and Arabidopsis OMT genes similar to Bx7.
    • Table S1. Wheat OMT genes up-regulated after herbivory (RNA sequencing).

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