Browsing by Subject "RNS-Interferenz"

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Etablierung eines Wirts-induzierten RNAi-Systems für die Kontrolle des Asiatischen Sojabohnenrostes Phakopsora pachyrhizi
(2015) Müller, Manuel; Vögele, Ralf
Phakopsora pachyrhizi, the causal agent of Asian Soybean Rust is a devastating plant pathogen that can cause significant yield losses in soybean production. So far, Phakopsora pachyrhizi is controlled by the use of fungicides and cultivation practices. A future perspective for the control of obligate biotrophic pathogens such as Phakopsora pachyrhizi, is Host-induced Gene Silencing (HIGS), which utilizes the naturally occuring phenomenon of RNA-interference (RNAi). The basic principle of HIGS is the induction of RNAi targeted against RNA of the fungal pathogen by means of transgenic expression of double stranded RNA (dsRNA) in the host plant. HIGS can be performed by either generating stable transgenic plants or using transient expression systems mainly based on recombinant viral vector systems. Recently, the basic principle of HIGS has been demonstrated in a variety of obligate biotrophic fungal pathogens including the powdery mildew fungus Blumeria graminis or the cereal rusts Puccinia striiformis f. sp. tritici and Puccinia triticina. Furthermore, work on different Fusarium spp. clearly indicates that the use of HIGS can be transferred to pertotrophic pathogens. Althought there is remarkable progress in utilizing HIGS in cereal rusts, to date, no such system has been reported for legume rusts. Thus, the work presented was focused on the development and testing of a HIGS system for the Asian Soybean Rust Phakopsora pachyrhizi. An initial set of ten target genes, presumably essential for signaling, nutrient uptake and host-pathogen interaction, was selected from a database reflecting the haustorial transcriptome of Phakopsora pachyrhizi. Expression of dsRNA complementary to the selected target genes was done using a viral vector system based on the Bean Pod Mottle Virus (BPMV). As an alternative method the use of agroinfiltration for the expression of hairpin RNA (hpRNA) was examined. By using the viral vector system silencing effects were observed for the three target genes Pp_contig01251, Pp_contig05320, and Pp_contig3015. Furthermore, the silencing of Pp_contig05320 resulted in inhibited growth of Phakopsora pachyrhizi as indicated by a reduced number of uredia. The use of agroinfiltration for the expression of hpRNA was not successful. Infiltration of soybean using a syringe resulted in deformation and necrosis of the infiltrated leaf areas. Although the expression of hpRNA could not be realized, the transient transformation of Glycine max via the use of agroinfiltration was demonstrated using a marker gene construct. Concerning the analysis of silencing effects via the use of RT-qPCR, the expression stability of 15 genes from Phakopsora pachyrhizi and 10 genes from Glycine max was analyzed to identify stably expressed reference genes. These studies resulted in the identification of several reference genes, suitable for the normalization of expression data collected under different experimental conditions. The results from this work provide a foundation for further examinations and experiments. Open questions especially concern the factors delimiting a gene as a suitable target gene for HIGS and the molecular mechanism behind the uptake and the translocation of silencing signals in Phakopsora pachyrhizi. Answering these questions will promote the establishment of HIGS as a promising perspective for modern plant protection.
Variabilität und Induzierbarkeit von Cytochrom P450 Monooxygenasen in humanen Leberproben und Hepatozyten : Untersuchungen mittels LC-MS/MS Cocktail-Assay und RNA-Interferenz
(2010) Feidt, Diana M.; Graeve, Lutz
The variability of the expression and function of the P450 enzymes (CYPs) is a cause for having different intensities and lengths of effects as well as side effects when patients are given the same dosage of medication. Up to this day, one cannot allover explain this inter individual variability of expression and activity of the enzymes. In general, there are several factors that may affect the variability, including biological factors (such us age, gender, hormonal status), environmental factors (such as nutrition, smoking, medication) as well as genetic factors like polymorphisms In order to solve this problem and to analyze relatively easy and fast activity differences, we developed an LC-MS/MS based P450 activity cocktail assay to quantify and detect simultaneously the seven most important CYPs as judged by their roles in the metabolism of clinically used drugs. The assay was established for use in in human hepatocytes as well as in recombinant and microsomal enzymes. We used the newly developed model-substrate cocktail assay to analyze the time-dependent induction of seven drug metabolizing cytochrome P450 activities as response to treatment of primary human hepatocytes with different statins. The strongest induction was observed for amodiaquine N-desalkylation of CYP2C8, which was induced up to 20-fold by atorvastatin and approximately 10-fold by simvastatin and lovastatin. Enzymes CYP3A4, CYP2B6 and 2C9 showed lower, but also significant induction after treatment with atorvastatin and simvastatin (4-11-fold). lovastatin and rosuvastatin demonstrated minor effects. Quantitative RT-PCR confirmed corresponding changes on the mRNA level with even more dramatic induction up to almost 100-fold. These data suggest a broader inducing effect of statins on cytochrome P450 expression and activity than previously known, thus further emphasizing their drug-drug interaction potential, especially for CYP2C8. Based on correlation analysis with P450 enzyme activity to their specific protein amount in human liver samples (liverbank IKP) were different functional results observed. Enzymes like CYP3A4 with atorvastatin hydroxylation or CYP1A2 with formation of acetaminophen showed very good correlations. Others like i.e. CYP2C9 with specific substrate diclofenac correlated much lower. Besides biological and environmental factors could these differences based on variability of the enzymes NADPH:P450 oxidoreductase (POR), cytochrome b5 or the two progesterone receptor-membrane components PGRMC1 and PGRMC2. After all, they would take active part in the metabolism of the xenobiotica as possible electron donors of the CYP enzymes. In order to analyze what kind of influence these proteins have on CYP enzyme activity, we developed a lentiviral based RNA-interference (RNAi) method in human hepatocytes and determined P450 activity with cocktail-assay after knocking down these genes. For the P450 reductase, we achieved a successful gene silencing of about 85% on mRNA level. The expression of cytochrome b5 was reduced by 51%, PGRMC1 about 30%. So far, it has not been possible to prove a significant knock down of PGRMC2. After silencing of reductase, an average light decline of about 10-30% of P450 enzyme activities was observed after 4 days. After a period of 7 days, a rest activity of CYP3A4 of only about 5% was detected. For both other potential electron donators cytochrome b5 and PGRMC1 was a reduced activity of 85% and 75% determined. These first results indicate a clear interaction of the enzymes POR, cytochrome b5 and PGRMC1 with the drug metabolizing enzymes.