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Detekce a identifikace patogenu konopí setého (Cannabis sativa)
Jarošová, Natálie
The aim of this work was the detection and identification of Hop Latent Viroid in plants of Cannabis sativa. This Viroid, which may not have any symptoms on plants, as the name suggests. One of the few detection options is based on the principle of the polymerase chain reaction - PCR. Using the chloroform-phenol reaction, it was possible to isolate RNA of the Viroid, which was used as a template for cDNA and subsequently multiplied by the PCR method. After successful PCR, the samples were applied to an agarose gel and evaluated by comparison with a size marker. For subsequent testing of the pathogen detection and identification protocol, positive samples from Genetia BioSciences were added, and for testing real samples from the genotype collection from the Department of Plant Biology, plants with the presence and absence of Hop Latent Viroid were identified.
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Function analysis of overexpression of gene for a Myb transcription factor from tomato, potential target of viroid infection
ONDRUŠ, Jaroslav
The main aim of this thesis was to find out whether gene Trifoliate, encoding a transcription factor from Myb family could be target of viroid PSTVd and is involved in its unknown interactions with host plant molecules, responsible for PSTVd pathogenesis. For this purpose, transformation of wild type plants Arabidopsis thaliana, Nicotiana benthamiana, Nicotiana tabacum, and mutant lof-1 Arabidopsis thaliana with Trifoliate was made, changed anatomy and morphogenesis of transformants has been observed. Due to our research and already known facts we suggest Trifoliate has a great importance in regulation of specific organs development. Manipulation with expression of this gene in some cases leaded to triggering similar sympthoms as PSTVd causes.
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Expression analysis of selected regulation factors in hop with relation to symptoms of viroid pathogenesis
FÜSSY, Zoltán
The aim of this work was to determine whether there are besides morphogenetic and metabolomic changes in viroid-infected plants also some alterations in the expression of transcription factors (TFs) known from our previous work to be involved in the secondary metabolites production, namely HlMyb1, HlMyb3, HlbHLH, HlbZIPA and HlbZIP2 TFs. Infectious vectors were prepared containing dimers of two closely related viroids- hop stunt viroid (HSVd) and cucumber pale fruit viroid (CPFVd). To achieve infection of hop (Humulus lupulus L.), biolistic inoculation of young shoots was performed. Hop cv. Admiral was chosen as a model for our experiments, because of its high flavonoid content in leaves. Infection of hop with both of these viroid species was proven by means of Northern hybridization and dot-blot techniques. Plants infected with HSVd showed serious symptoms such as stunted growth, epinasty and rugosity of leaves. Interestingly, decoloration of the petioles of the plants infected with HSVd was observed, maybe as a result of lower anthocyanins production. These symptoms were similar but milder in CPFVd-infected hops. On plants bearing symptoms, HPLC analyses were performed and compared to controls to detect changes in the levels of flavonol glycosides, phenolic acids, bitter acids and xanthohumol. In HSVd-infected hop leaves and petioles, significant decrease in the contents of flavonol glycosides and phenolic acids was observed. On the other hand, an increase in the amount of xanthohumol and bitter acids was detected in HSVd-infected tissues compared to healthy controls. Unlike to HSVd infection, the decrease of all analyzed secondary metabolites was observed in CPFVd-infected material. This difference suggests an alternative response of metabolome pathways to CPFVd-caused pathogenesis in comparison to HSVd. Semiquantitative RT PCR was performed to assay levels of TFs in healthy and infected hop tissues. Quantitative RealTime analyses of putative hop transcription factors HlbZIPA and HlbZIP2 were carried out using RNA isolated from HSVd-infected petioles. Increased mRNA levels of bZIP TFs were detected in infected material, suggesting an involvement of these factors in the response of the host plants to HSVd infection. Using thermodynamic methods of TGGE and heteroduplex analysis, several sequence variants of HlbZIP2 were idetified. According to aminoacid sequence alignment, this putative factor belongs to a group of bZIP proteins known for ABA/stress signalling in A. thaliana.
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