Browsing by Subject "SiRNA"

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Entwicklung von „screening“-Methoden zur Analyse von PTGS-basierter Resistenz gegen Nepoviren in Pflanzen
(2006) Winterhagen, Patrick; Reustle, Götz
Nepoviruses are the causal agent of the fanleaf disease which leads to severe loss in viticulture (Raski et al., 1983). To induce virus resistance by post transcriptional gene silencing (PTGS) against Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV) and Raspberry ringspot virus (RpRSV), grapevine rootstocks were transformed with inverted repeat constructs or constructs containing sequences of the target virus and the defective interfering (DI) sequence of Tomato bushy stunt virus (Reustle et al., 2005). The induction und efficiency of PTGS by different constructs were investigated on the model plant Nicotiana benthamiana. Transgene-induced PTGS was demonstrated by the detection of small interfering (si)RNA in N. benthamiana. Using Agrobacterium for infiltration of a GFP-sensor construct, consisting of the GFP expression cassette and the sequence of the target virus, the efficiency of established transgene-induced PTGS was investigated. GFP-expressing plants accumulated mRNA of the sensor construct after the first day post infiltration in infiltrated leaves. After the second day the accumulation of siRNA with GFP- und virus-specific sequences was detected. In plants, which did not show any GFP-fluorescence after infiltration, GFP or viral sequence specific siRNA were not detectable. Generally, in virus resistant plants GFP-fluorescence was absent after infiltration. A correlation of virus resistance und accumulation of virus- or transgene-specific siRNA was not found. Several systems to evaluate PTGS and virus resistance in transgenic grapevine were tested. Transgenic grapevine did not accumulate transgene specific siRNA. An elevated resistance of transgenic grapevine was discovered by grafting experiments onto virus infected rootstocks. Whereas virus infected grapevine accumulated virus- and transgene-specific RNA and siRNA, in the non-infected grafts viral RNA was not detectable. Obviously, degradation of viral RNA in resistant grapevine und N. benthamiana was rapid und highly efficient without leading to accumulation of siRNA. However, due to the high inoculum pressure, grafting experiments are difficult to interprete and a possible field resistance against natural infection by the vector nematodes is probably not detectable. For investigation of PTGS in transgenic grapevine in vivo a system for vacuum infiltration to transfer the GFP-sensor construct into leaf tissue was established. For inoculation of grapevine using the natural GFLV vector nematode Xiphinema index an in vitro dual culture was developed. This space saving system allows analysis of resistance of grapevine under controlled conditions within a short time. An incubation time of about only six weeks was sufficient for the inoculation of control plants.