Evolution der Wirtspflanzenanpassung in Pieridae. Untersuchungen zur möglichen Rolle cyanidentgiftender Enzyme

in dieser Arbeit erstmalig Rhodanesen aus Insekten identifiziert. Die Lokalisation in Mitochondrien und die hohe Affinität für Cyanid stützt eine Rolle von Rho1 in der Cyanidentgiftung. Rho2 hat vermutlich eine andere Funktion. Zur Etablierung der RNAi in P. rapae wurden transient mit RNAi-Konstrukten transformierte Pflanzen verfüttert. Die Expression des Zielgens PrNSP wurde hierdurch um etwa 50 % vermindert, dies hatte jedoch keinen Einfluss auf das Gewicht und das Überleben der Raupen auf glucosinolathaltigen Pflanzen. Die Anwendung alternativer Ansätze wird notwendig sein, um die PrNSP-Expression vollständig zu unterbinden. The Small Cabbage White, P. rapae (Lepidoptera: Pieridae), is a specialist on glucosinolate-containing plants. It belongs to the subfamily Pierinae which performed a host shift from Fabales to Brassicales about 80 million years ago. The nitrile-specifier protein (NSP), which leads to the formation of nitriles upon glucosinolate breakdown in the larval gut, is regarded as essential for this host shift. Upon metabolism of aromatic nitriles in the larvae, cyanide is formed. As aromatic glucosinolates were widespread in ancient Brassicales, an efficient cyanide detoxification might have been a prerequisite for the host shift. The goal of this work was to identify cyanide detoxification enzymes, namely ß-cyanoalanine-synthases and rhodaneses, from P. rapae and other lepidopteran species to investigate the role of cyanide detoxification for the host shift. Previous work had identified three -cyanoalanine synthases, PrBSAS1-PrBSAS3, from P. rapae. To further elucidate the physiological function of these enzymes, we attempted to establish RNAi as a method in P. rapae. Analysis of lepidopteran species with and without specialization on glucosinolate-containing plants showed that ß-cyanoalanine-synthase-activity is widespread while rhodanese-activity was only found in a few species. Using a PCR strategy with degenerate primers and database information, 14 ß-cyanoalanine-synthases were identified. All investigated species possessed at least one BSAS2-homolog. In addition, Pierid species contained a BSAS3-homolog and species of the Pierinae an extra BSAS1-homolog. Characterization of PrBSAS1-PrBSAS3 after heterologous expression in E. coli showed highest affinity for cyanide for PrBSAS2 and highest efficiency for PrBSAS1. PrBSAS1-PrBSAS3 were expressed mainly in the larval gut with highest expression levels for PrBSAS2. Characterization of the enzymes from other lepidopteran species after expression in E. coli showed that all BSAS2-homologs possessed high affinity to cyanide. As BSAS2-homologs seem to be widespread in Lepidoptera, they could play a role in the basal detoxification of cyanide. The BSAS1-homologs converted cyanide with high efficiency, which could point at a function in the detoxification of high amounts of cyanide. Taken together, these results support a role of cyanide detoxification by ß-cyanoalanine synthases for host plant adaptation in Pieridae. With the mainly gut-expressed rhodaneses Rho1 and Rho2 from P. rapae, the present study identified the first rhodaneses from insects. Mitochondrial localization and high affinity to cyanide are in agreement with a role of Rho1 in cyanide detoxification. Rho2 is likely to possess a different function. To establish RNAi in P. rapae, two methods were tested which were based on feeding of dsRNA using PrNSP as a target gene. The feeding of leaf disks with added dsRNA did not result in a reduction of PrNSP expression. The feeding of larvae with plants transiently transformed to express PrNSP-dsRNA resulted in a 50 %-reduction of PrNSP expression, but did not affect larval performance and survival on glucosinolate-containing plants. Alternative methods need to be tested in order to obtain completely abolished PrNSP expression.