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Priprava flourescenčno označenih efektorskih proteinov VnaSSP4.2 in Vna7.443 z namenom razumevanja njihove funkcije
ID Bole, Martin (Avtor), ID Berne, Sabina (Mentor) Več o mentorju... Povezava se odpre v novem oknu, ID Volk, Helena (Komentor)

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Izvleček
Navadni hmelj (Humulus lupulus L.) je dvodomna trajna dvokaličnica, ki pripada družini konopljevk. Njegov primarni škodljivec je fitopatogena gliva Verticillium nonalfalfae. Ločujemo blago in letalno obliko verticilijske uvelosti, ki vsako leto lahko uniči od 10 do 50 % vsega pridelka. Patogen z namenom kolonizacije rastlinskega gostitelja in preskrbe glive s hranili izloča efektorje, s katerimi vpliva na celično fiziologijo gostitelja (transkripcija, signalizacija, epigenetska regulacija, sinteza in izločanje proteinov), ali modulira njegov imunski odgovor (sinteza sekundarnih metabolitov, signalizacija). Poznavanje strukture, mehanizma translokacije in biološke funkcije glivnih efektorjev je ključno za razumevanje mehanizmov patogenosti glive in oblikovanje strategij za trajno zaščito pridelka v Sloveniji in po svetu. Cilj magistrske naloge je bil pridobiti očiščena, fluorescenčno označena proteina VnaSSP4.2 in Vna7.443 ter ju uporabiti v bioloških in biokemijskih testih z namenom razumevanja njune funkcije. S kloniranjem In-Fusion smo uspešno pripravili konstrukte Vna7.443::DsRed2, DsRed2::Vna7.443 ter reporter DsRed2, jih izrazili v E. coli ter proteine očistili in stabilizirali. Prav tako smo v E. coli izrazili konstrukt eGFP::VnaSSP4.2 in rekombinantni protein očistili. Rekombinantni fluorescenčni protein eGFP::VnaSSP4.2 v listih modelne rastline N. benthamiana povzroči kloroze in se v plazmalemi specifično lokalizira v membranskih mikrodomenah. Rekombinantni fluorescenčni protein DsRed2::Vna7.443 s prostim C- terminalnim koncem morda interagira z genomsko DNA gostiteljske rastline H. lupulus L. Protein Vna7.443 je strukturno podoben proteinom, ki vsebujejo MORN ponovitve. Potencialne vezave Vna7.443 na DNA in vivo, po nastanku por, ki jih oblikuje VnaSSP4.2, nismo preverili, smo pa delno potrdili hipotezo, da bi tak mehanizem lahko obstajal.

Jezik:Slovenski jezik
Ključne besede:hmelj, fitopatogena gliva, Verticillium nonalfalfae, efektor, heterologna ekspresija, Vna7.443, VnaSSP4.2, MORN 1, tandemske ponovitve
Vrsta gradiva:Magistrsko delo/naloga
Tipologija:2.09 - Magistrsko delo
Organizacija:BF - Biotehniška fakulteta
Leto izida:2022
PID:20.500.12556/RUL-139106 Povezava se odpre v novem oknu
COBISS.SI-ID:119806723 Povezava se odpre v novem oknu
Datum objave v RUL:31.08.2022
Število ogledov:355
Število prenosov:42
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Sekundarni jezik

Jezik:Angleški jezik
Naslov:Preparation of fluorescently tagged effector proteins VnaSSP4.2 and Vna7.443 as tools in confirming their functions
Izvleček:
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.

Ključne besede:hop, phytopathogenic fungus, Verticillium nonalfalfae, effector, heterologous expression, Vna7.443, VnaSSP4.2, MORN 1, tandem repeats

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