Activation of plant toxins by herbivorous insects

有毒的次生代谢物是保护植物免受食草动物侵害的重要物质。许多毒素被储存为前体，即所谓的原毒素，并在组织破坏时被分隔的酶激活。奇怪的是，原毒素并不总是被植物来源的酶激活：在许多情况下，它们似乎被食草动物的消化酶分解。到目前为止，这种现象的遗传基础及其对生态的影响还没有被很好地理解。在以前的工作中，我研究了蒲公英(Taraxacum office inale agg.)及其主要天敌--金龟子幼虫。这些实验确定蒲公英的主要抗性因子是蒲公英的主要抗性因子蒲公英酸β-D-葡萄糖酯(TA-G)，并表明该化合物在自然界中受到甜菜夜蛾的正向选择。我最近未发表的工作表明，TA-G在甜菜夜蛾的肠道中被昆虫衍生的β-葡萄糖苷酶迅速脱糖。通过转录组筛选，我鉴定了19种表达模式与TA-G脱糖基化相匹配的黑曲霉肠道葡萄糖苷酶。虽然初步证据表明TA-G是一种原毒素，它的水解会导致更有毒的苷元的形成，但TA-G脱糖对昆虫行为和行为的影响尚不清楚。基于最新技术和我们的初步结果，我提出了一系列实验来回答关于食草动物消化酶在植物原毒素激活中的作用的三个主要悬而未决的问题：1)在甜菜夜蛾的肠道中，哪些酶能使TA-G脱糖？2)消化的β-葡萄糖苷酶对食草动物的表现和行为有什么影响？3)TA-G激活的B-葡萄糖苷酶是否具有任何主要的消化功能？为了回答这些问题，我们将在昆虫表达系统(High Five细胞)中表达所有19种黑曲霉葡萄糖苷酶，并对它们进行TA-G脱糖活性筛选。在第二步中，将使用一种有效的环境RNA干扰方法来沉默甜瓜中活跃的TA-G？葡萄糖苷酶，并将评估TA-G？葡萄糖苷酶沉默对野生型和TA-G缺陷蒲公英植株上食草动物行为和表现的影响。最后，我们将在体外研究已鉴定的TA-Gβ-葡萄糖苷酶的替代底物，并使用基因沉默平台结合人工饲料试验来确定消化性β-葡萄糖苷酶对草食动物的潜在益处。我们的项目将是第一批研究草食动物消化酶激活原毒素对草食动物行为的影响，并阐明生产相关酶的成本和收益的项目之一。因此，该项目将阐明植物-食草动物相互作用的一个重要但未被充分研究的方面。

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 My recent unpublished work shows that TA-G is rapidly deglycosylated in the gut of M. melolontha by insect-derived ß-glucosidases.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.