Browsing by Subject "VIGS"

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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.
Successful silencing of the mycotoxin synthesis gene TRI5 in fusarium culmorum and observation of reduced virulence in VIGS and SIGS experiments
(2022) Tretiakova, Polina; Voegele, Ralf Thomas; Soloviev, Alexander; Link, Tobias Immanuel
Crops constantly experience various biotic stresses during their life cycle, and Fusarium spp. remain one of the most serious groups of pathogens affecting plants. The ability to manipulate the expression of certain microorganism genes via RNAi creates the opportunity for new-generation dsRNA-based preparations to control a large number of diseases. In this study, we applied virus-induced gene silencing (VIGS), and spray-induced gene silencing (SIGS) to silence the trichothecene-producing gene TRI5 in F. culmorum as a means to reduce its aggressiveness on spring wheat. Treatment of the fungus with dsTRI5RNA in vitro reduced deoxynivalenol (DON) and 3-acetyldeoxynivalenol (3-A-DON) accumulations by 53–85% and 61–87%, respectively, and reduced TRI5 expression by 84–97%. VIGS decreased the proportion of infected wheat spikelets by 73%, but upregulation was observed for TRI5. SIGS on wheat leaves and ears using certain dsTRI5RNA amounts negatively impacted F. culmorum growth. However, when performing in vivo analyses of TRI5 mRNA levels, the upregulation of the gene was determined in the variants where fungal colonization was restricted, suggesting a compensatory reaction of the pathogen to RNAi.