Common hop (Humulus lupulus L.) is a dioecious perennial dicotyledonous plant that belongs to the Cannabaceae family. Its primary pest is the phytopathogenic fungus Verticillium nonalfalfae. We distinguish mild and lethal forms of verticillium wilt, which can destroy from 10 up to 50% of the entire crop every year. To colonize the plant host and supply the fungus with nutrients, the pathogen secretes effectors that modulate the host's cellular physiology (transcription, signalling, epigenetic regulation, protein synthesis and secretion), or the immune responses (synthesis of secondary metabolites, signalling). Unravelling the structure, translocation mechanism and biological function of fungal effectors is key to understanding the mechanisms of fungal pathogenicity and devising strategies for permanent crop protection in Slovenia, and around the world. The goal of the master's thesis was to obtain purified, fluorescently labelled VnaSSP4.2 and Vna7.443 proteins and to use them in biological and biochemical assays to unveil their functions. Using In-Fusion cloning, we successfully prepared the constructs for Vna7.443::DsRed2, DsRed2::Vna7.443 and the reporter DsRed2, expressed them in E. coli, and purified and stabilized the proteins. Likewise, we expressed eGFP::VnaSSP4.2 and purified it. The recombinant fluorescent protein eGFP::VnaSSP4.2, injected into the leaves of the model plant N. benthamiana, causes chlorosis and localizes in plasma membrane within specific membrane microdomains. The recombinant fluorescent protein DsRed2::Vna7.443 with a free C-terminal end may interact with the genomic DNA of the host plant H. lupulus L. The Vna7.443 protein is structurally similar to proteins containing MORN repeats. The potential binding of Vna7.443 to DNA in vivo, after the formation of the pore induced by VnaSSP4.2, was not performed, although we partially confirmed the hypothesis that such a mechanism could exist.
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