Institut für Sonderkulturen und Produktionsphysiologie

Permanent URI for this collectionhttps://hohpublica.uni-hohenheim.de/handle/123456789/43

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Development of a strategy to induce RNA-silencing in squash against virus diseases by genetic transformation
(2007) Khidr, Yehia; Reustle, Götz
Viral diseases of Cucurbits are an important limitation in the production of the crop. Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus-2 (WMV-2) are the most important squash (Cucurbita pepo L.) infecting viruses. Mixed infections with these viruses are deleterious for cucurbitaceous plants leading annually to significant yield losses world wide. All varieties of economical importance are susceptible for these viruses and classical breeding did not yield resistance. Therefore, a transgenic approach was chosen to induce resistance against both viruses by post-transcriptional gene silencing (PTGS). Highly conserved regions in the coat protein genes of ZYMV and WMV-2 were chosen to establish an inverted repeat construct. This construct was cloned into binary vector under control of the 35S promoter. Embryogenic callus was induced from different organs of three squash cultivars as target tissues for Agrobacterium transformation. The embryogenic callus was developed within 13-20 weeks incubation on MS medium containing different plant growth regulator combinations of auxin and cytokinin. Induction of embryogenesis in different explants ranged from 5 to 100 % depending on the organ and genotype used. Efficiency of embryo maturation, conversion and germination into entire plants from squash embryogenic callus was found to be callus age depended. Regeneration with young (2 months old) material was efficient, whereas regeneration of material maintained under in vitro conditions for more than 2 years was not possible. Agrobacterium-mediated transformation of squash embryogenic callus was established using transient GUS-gene expression. The highest transformation efficiency was obtained with the supervirulent ATHV strain, bacterial density of 0.85, washing procedure of the embryogenic material prior to Agrobacterium co-culture, application of 1 mM Acetosyringone in induction medium and sub-culturing of embryogenic callus on fresh MS medium 5-9 days prior co-culturing with the Agrobacterium. Selection strategy was optimized using GFP as reporter gene. For the genotype CX3006 300 mg/l Kanamycin showed the highest number of green areas but most efficient selection agent was Paromomycin 150 mg/l. For the genotype Dundoo 200 mg/l Paromomycin was the effective selection agent and showed the highest number of green areas. Selection of transformed calli could be efficient with the used selection agents but regeneration of transgenic plant was not possible because the old material was only one to be used for transformation experiments. It seems that these old materials may have lost their competency when they were maintained for long term in tissue culture. Therefore, the functionality of the inverted repeat construct was evaluated in Nicotiana benthamiana as a model plant. Transgenic lines were analyzed by PCR, Southern blot analysis and segregation analysis of T1 offspring. The transgene-induced PTGS in transgenic lines was confirmed by infiltration of GFP-sensor constructs containing viral derived sequences as silencing target and /or a construct containing the p19 silencing suppressor. In all transgenic lines tested, GFP fluorescence in infiltrated leaves was extinguished three days post-infection with GFP-sensor constructs. In contrast, all transgenic lines showed GFP fluorescence in infiltrated leaves when GFP-sensor constructs were co-infiltrated with a binary vector containing the viral silencing suppressor p19. With this work, tools have been developed to engineer virus resistance in squash. Using the optimized Agrobacterium-mediated transformation procedure together with the efficient RNA-silencing of the inverted inverted repeat construct and freshely induced embryogenic material it is quite possible to establish virus resistance in squash.
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.
Molekulare Analyse des Himbeerringflecken Nepovirus (RpRSV) und Herstellung eines Konstrukts zur Induktion von RpRSV?Resistenz in Reben
(2003) Ebel, Rainer; Reustle, Götz
The Raspberry Ringspot Nepovirus (RpRsV) is one of the viruses responsible for the fanleaf disease of grapevine. Two different serological strains of RpRSV exist: the grapevine strain (RpRSV-g) and the cherry strain (RpRSV-ch), which occurs in Germany and Switzerland. RpRSV has two RNAs (RNA1 and RNA2). Both have a genome-linked protein at the 5' ends and are polyadenylated at the 3' ends. RNA1 and RNA2 have one open reading frame flanked by 5' and 3' non-coding regions. The ORFs encode for one large polyprotein which is proteolytically cleaved in smaller functional proteins. The aims of the work were to produce RpRSV grapevine plants and the closer characterisation of RpRSV. The strategy was to create a gene construct, which should induce a gene silencing against the viruses in the grapevine rootstocks. Nicotiana benthamiana was chosen as a test system for the constructs. Because there was no sequence data available both strains were sequenced. The RpRSV strains were propagated in Chenopodium quinoa. The viral RNAs were purified, cDNA synthesized, cloned and sequenced. The sequences were compared and multiple alignments were performed. A sequence from the RNA2 3' non-coding region of RpRSV-ch was selected for the gene construct. An inverted repeat of this sequence was generateted, separated by a plant intron sequence. This construct under the control of a 35S promotor was cloned in the transformation vectors pPZPnptII (antibiotic resistance) and pPZPbar (herbicide resistance). Agrobacterium mediated transformation of Nicotiana benthamiana has been carried out. The T2 generation of the regenerated transgenic tobacco lines were tested for RpRSV resistance. Furthermore full-length clones of the RNA1 and RNA2 of RpRSV-g were produced. Therefore the full-length ds cDNA of both RNA strains were cloned under the control of a 35S promotor. Infection experiments in Chenopodium quinoa with the full clones have successfully been carried out.

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