Reversing the escape from herbivory: Knockout of cardiac glycoside biosynthesis in wormseed wallflower ( Erysimum cheiranthoides L., Brassicaceae).

Like other members of the Brassicaceae, plants in the wallflower genus ( Erysimum ) produce glucosinolates, which are potent defenses against a wide range of herbivores. As a more recently evolved second line of defense, Erysimum produces cardiac glycosides, which are allosteric inhibitors of Na + ,K + -ATPases in animals. Cardiac glycoside biosynthesis has evolved in diverse lineages including foxglove ( Digitalis , Plantaginaceae) and milkweeds (Apocynaceae), but the full biosynthetic pathway has not been described in any species. We identify and generate CRISPR/Cas9 knockouts of two cytochrome P450 monooxygenases involved in cardiac glycoside biosynthesis in wormseed wallflower ( Erysimum cheiranthoides L.): Ec CYP87A126, which cleaves the side chain from sterol precursors to initiate cardiac glycoside biosynthesis, and Ec CYP716A418, which has a role in cardiac glycoside hydroxylation. In the Ec CYP87A126 knockout lines, cardiac glycoside production is eliminated, effectively reversing Erysimum 's escape from herbivory. For the generalist herbivores green peach aphid ( Myzus persicae Suzler) and cabbage looper ( Trichoplusia ni Hübner), cardiac glycosides appear to be largely redundant with glucosinolates, having some effect in choice assays but little to no effect on insect performance. By contrast, the crucifer-feeding specialist cabbage butterfly ( Pieris rapae L.), which will not oviposit or feed on wildtype E. cheiranthoides , is able to complete its life cycle on cardenolide-free E. cheiranthoides mutant lines. Thus, our study demonstrates in vivo that cardiac glycoside production allows Erysimum to escape from a specialist herbivore.