Activation of plant toxins by herbivorous insects

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Toxic secondary metabolites are of central importance to protect plants against herbivores. Many toxins are stored as precursors, so called protoxins, and are activated by compartmentalized enzymes upon tissue disruption. Curiously, protoxins are not always activated by plant-derived enzymes: in many cases, they seem to be cleaved by digestive enzymes from the herbivores. To date, the genetic basis of this phenomenon as well as its ecological implications are not well understood. During my previous work, I studied the interaction between the common dandelion (Taraxacum officinale agg.) and its major natural enemy, the larvae of the cockchafer (Melolontha melolontha). These experiments led to the identification of taraxinic acid β-D-glucopyranosyl ester (TA-G) as a major resistance factor of dandelion and showed that this compound is under positive selection by M. melolontha in nature. My recent unpublished work shows that TA-G is rapidly deglycosylated in the gut of M. melolontha by insect-derived ß-glucosidases. Through a transcriptome screen, I identified 19 M. melolontha gut ß-glucosidases whose expression pattern matches TA-G deglycosylation. Although preliminary evidence suggests that TA-G is a protoxin whose hydrolysis leads to the formation of the more toxic aglycone, the impact of TA-G deglycosylation on insect performance and behaviour remains unclear.Based on the state-of-the-art and our preliminary results, I propose a series of experiments to answer three major open questions regarding the role of herbivore digestive enzymes in plant protoxin activation: 1) Which enzymes deglycosylate TA-G in the gut of M. melolontha? 2) What is the impact of TA-G deglycosylation by digestive β-glucosidases on herbivore performance and behavior? 3) Do the TA-G activating ß-glucosidases have any primary digestive functions? To answer these questions, we will express all 19 M. melolontha ß-glucosidases in an insect expression system (High Five cells) and screen them for TA-G deglycosylation activity. In a second step, active TA-G ß-glucosidases will be silenced in M. melolontha using an efficient environmental RNA interference approach, and the impact of TA-G ß-glucosidase silencing on the behavior and performance of the herbivore on wild type and TA-G deficient dandelion plants will be assessed. Finally, we will investigate alternative substrates of the identified TA-G β-glucosidases in vitro and determine the potential benefits of digestive ß-glucosidases for the herbivore using the gene silencing platform in combination with artificial diet assays. Our project will be among the first to study the impact of protoxin activation by herbivore digestive enzymes on herbivore behavior and to elucidate both costs and benefits of producing the associated enzymes. Thereby, this project will shed light on an important, but understudied aspect of plant-herbivore interactions.