Development and validation of an analysis method for the detection of altered resistance in transgenic plants to herbivore-pathogen-complexes

摘要

Genetically modified plants (GMPs) have to pass several safety evaluations before they can be approved but identification of undesired changes in metabolism is hard to achieve due to its complexity. Such unexpected changes could be reflected by changes in virus resistance and feeding behaviour of herbivores such as aphids. Altered behaviour can be determined by so- called life table statistics where e. g. the lifetime and the reproduction rate are analysed. Such experiments are time consuming and costly. The behaviour alterations should also be detectable by changes in virus uptake und transmission. Thus, quantitative detection methods are needed in order to measure the absolute virus content in plants and in aphids. _Potato leafroll virus_ (PLRV) and _Potato virus Y_ (PVY) are efficiently transmitted by the green peach aphid _Myzus persicae_. PVY is transmitted non-persistently whereas PLRV is transmitted in a persistent and circulative manner. Consequently, PLRV is especially suited for experiments determining changes in the feeding behaviour. In recent years, several quantitative PCR-assays have been developed for the detection of PLRV in plants. Published assay conditions were not useful for the virus detection in aphids, since primers often bound non-specifically. Furthermore, particular extraction methods had to be developed, since reagents used for plant material were not suitable for aphids with their high content of inhibitory substances. However, RNA extraction is very costly and not suitable for high-throughput screening. Therefore, highly specific assays based on immunocapture-RT-qPCR were developed for the quantitative detection of both PLRV and PVY in aphids and plants. The efficiency of the assays is comparable for the detection of viruses in plants as well as in vectors. In the future, these methods will be applied to investigate the influence of different genetic modifications on virus content and feeding behaviour.