Knockout mutations ofNicotiana benthamianadefenses reveal the relative importance of acylsugars, nicotine, and a serine protease inhibitor in a natural setting

Abstract

Plants produce an immense diversity of defensive specialized metabolites. However, despite extensive functional characterization, such experiments are seldom performed in natural settings, and the balance between different defensive compounds is rarely examined. 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 confirmed results obtained in a natural setting. We conclude that the three metabolite groups, which provide defense against different herbivores and herbivore classes, collectively provide broad-spectrum protection to N. benthamiana. However, there is a gradient in their effectiveness, with acylsugars providing the strongest protection and nicotine providing a secondary line of defense. Furthermore, we demonstrate that, even if individual metabolites do not have a measurable defensive effect on their own, they can have an additive effect when combined with other defensive compounds.

Catechol acetylglucose: A newly identified benzoxazinoid-regulated defensive metabolite in maize

“…When these two compounds are knocked out individually and in combination, different herbivores are affected. However, there remain herbivores that are tolerant to both of these defenses 64 .…”