Browsing by Subject "RNS"

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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.
Verbreitung, Diversität und Übertragung des Mykovirus PhV und seine Auswirkung auf Plasmopara halstedii, den Falschen Mehltauerreger der Sonnenblume
(2015) Grasse, Wolfgang; Spring, Otmar
The Plasmopara halstedii Virus (PhV) is a ss(+)RNA virus with two segments. It occurs only in its host Plasmopara halstedii, the downy mildew pathogen of the sunflower. The two RNA strands encode for an RNA depending RNA Polymerase (RdRp) and a coat protein (CP), respectively. So far the phylogenetic analysis has shown similarities of the RdRp with the family of Nodaviruses while the CP seems to belong to the family of Tombusviruses. Phylogentic comparison based on both sequences now suggest a new clade, containing PhV and the Sclerophthora macrospora Virus A, which is basal to both mentioned virus families. Studies about diversity and the occurrence of PhV have shown that the virus existed in samples from 17 countries from five continents which were collected over the past 40 years. Its presence in more than 90% of these samples was documented. No correlation was found between the geographic origin and age of the samples, and presence or absence of PhV sequences. The calculated genetic diversity among all samples was surprisingly low. For 22 fully sequenced samples from 13 countries, only 18 SNP positions were reported. Genetic distances were extremely low with means of 0.001 for the RdRp and 0.002 for the CP. Investigations of the influence of PhV on the aggressiveness and pathogenicity of P. halstedii have shown a hypovirulent effect of the virus. In this study, isogenic strains of the Oomycete were infected with PhV and used for a series of bioassays on sunflowers. The production of sporangia was lowered by ca. 30% in case of virus presence and the latent period, i.e. the day of the first observed sporulation, was delayed by one day. The potential for systemic infections of the sunflower was also lowered by one third when PhV was present. Experiments to generate PhV by means of active cDNA clones in P. halstedii were performed with two different vectors and three transformation methods. It was shown that elctroporation techniques were useful to transport plasmids into the zoospores of P. halstedii and that the T7 promotor was able to start the transcription. The following generation of sporangia, however, lacked these sequences. This indicates that there was a transient transformation which produced PhV RNA sequences, but these sequences were unable to rebuild the virus itself.