Browsing by Subject "Phakopsora pachyrhizi"

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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.
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    In vivo und molekularbiologische Untersuchungen zur Sensitivität von Phakopsora pachyrhizi gegenüber Demethylierungs-Inhibitoren und Qo-Inhibitoren
    (2013) Schmitz, Helena Katharina; Vögele, Ralf
    Soybeans are one of the most important crops worldwide. Since 1980, soybean production attained increasing distinction in Brazil. Following the leading producer USA, Brazil counted as the second biggest soybean producer in 2010. A number of threats are involed reducing soybean yield, rating the Asian Soybean Rust, Phakopsora pachyhrizi, as one of the worst pathogens since its invasion in 2001. Until this date the American Soybean Rust, Phakopsora meibomiae, was known in Brazil only, which is of minor importance. Not only did P. pachyrhizi reduce soybean yield in Asia, but also in Brazil considerable additional costs were caused by yield reduction and disease management. Control is mainly based on fungicide treatment, demethylation inhibitors (DMI) and quinone outside inhibitors (QoI) being the most important and effective classes used. Both fungicide groups are frequently applied in combinations to ensure prolonged effects. While efficiency of QoIs remained unchanged, protection by DMIs was significantly narrowed. The primary objective of the recent study was to survey the sensitivity of P. pachyrhizi isolates towards fungicides and to analyse the genetical background of a conceivable adaption. Indeed, an adaption towards DMIs could be observed, while efficiency of QoIs was stable. Due to the P. pachyrhizi genetical structure of the cytochrome b (cyt b) gene, which corresponds to the QoI fungicide target protein, resistance towards QoIs based on the most important mechanism known from other pathogens is rather unlikely. The major resistance mechanism of phytopathogenic fungi against QoIs is an alteration of the cyt b-sequence, in particular point mutations F129L, G137R and G143A, whereas G143A results in highest resistance factors. An intron after codon 143 of the cyt b-gene prevents the development of G143A-mutation. In contrast, genetical analyses of the cyp51-gene, which corresponds to the target protein of DMI fungicides, revealed that adaption is based on different resistance mechanisms which have an additive or synergistic impact. In P. pachyrhizi, point mutations within the cyp51-gene and a modified expression of cyp51 were involved. An altered expression of cyp51 was due to a selective expression of a mutated cyp51-allele and due to up-regulation of cyp51. Six point mutations (F120L, Y131H, Y131F, K142R, I145F and I475T) which appeared in defined combinations, except for one mutation which was found as a single character, correspond to a reduced sensitivity. Additionaly, in some of the isolates cyp51 was up-regulated three- to thenfold compared to the reference strain, leading to decreased efficiency of DMIs. Indications were found, assuming that the copy number of the cyp51-gene in P. pachyrhizi is responsible for the observed alterations in the expression. Other resistance mechanisms than described in the recent study (such as expression of efflux transporters) may additionally play a role now or in future. Resistance towards DMIs evolved by P. pachyrhizi was caused by nesting mechanisms which appeared in quite a short time period due to enormous selection pressure by extensive DMI-use in large areas and the clonal, dicaryotic live cycle with short generation times. Thus, applications of fungicides with different modes of actions are recommended for management of P. pachyrhizi to prevent further resistance development and -distribution.