Knockout mutations of Nicotiana benthamiana defenses reveal the relative importance of acylsugars, nicotine, and a serine protease inhibitor in a natural setting

Plants produce an immense diversity of defensive specialized metabolites. However, despite extensive functional characterization, the relative importance of different defensive compounds is rarely examined in natural settings. Here, we compare the efficacy of three Nicotiana benthamiana defensive compounds, nicotine, acylsugars, and a serine protease inhibitor, by growing plants with combinations of knockout mutations in a natural setting, quantifying invertebrate interactions, and comparing relative plant performance. Among the three tested compounds, acylsugars had the greatest defensive capacity, affecting aphids, leafhoppers, spiders, and flies. Nicotine mutants displayed increased leafhopper feeding and aphid colonization. Plants lacking both nicotine and acylsugars were more susceptible to flea beetles and thrips. By contrast, knockout of the serine protease inhibitor did not affect insect herbivory in the field. Complementary experiments under controlled laboratory conditions with caterpillars grasshoppers, and aphids confirmed results obtained in a natural setting. We conclude that the three metabolite groups collectively provide broad-spectrum protection to N. benthamiana . However, there is a gradient in their effects on the interacting invertebrates present in the field. Furthermore, we demonstrate that, even if individual metabolites do not have a measurable defensive benefit on their own, they can have an additive effect when combined with other defensive compounds. ### Competing Interest Statement The authors have declared no competing interest.