20.500.12556/RUL-184556 Interakcije med potivirusnim proteinom HCPro in vezavnim proteinom iz rastlin Interactions between the potyviral protein HCPro and a binding plant protein Potivirusi predstavljajo obsežen rod rastlinskih RNA virusov, ki z okužbami širokega spektra gostiteljev povzročajo veliko gospodarsko škodo v kmetijstvu po celem svetu. Zaradi majhnega genoma kodirajo nabor večopravilnih proteinov, med katerimi ima osrednjo vlogo protein HCPro (angl. helper component-protease). Prek kompleksnih interakcij z virusnimi in gostiteljskimi proteini HCPro omogoča namnoževanje in razširjanje virusa ter hkrati zavira rastlinski obrambni sistem. Iz literature so že znane nekatere interakcije HCPro iz različnih potivirusov z rastlinskimi molekulami, ki so bile identificirane z uporabo raznolikih metodoloških pristopov. Namen magistrskega dela je bil v nadzorovanih pogojih in vitro kvalitativno in kvantitativno analizirati molekulske interakcije proteina HCPro virusa krompirja A (PVA) s petimi proteini navadnega tobaka (Nicotiana tabacum), in sicer z eIF(iso)4E, CF1β, MinD, WD40 in HIP2. Vse te proteine smo rekombinantno izražali v bakterijskem sistemu Escherichia coli, pri čemer smo zaradi neustrezne ekspresije oziroma topnosti MinD, WD40 in HIP2 nadaljnje eksperimentalno delo omejili na eIF(iso)4E in CF1β. Za slednja smo z uvedbo dodatne polihistidinske oznake uspešno optimizirali protokol izolacije in čiščenja. Njuno čistost smo preverili s poliakrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecil sulfata, strukturno ustreznost in stabilnost pa z nativno poliakrilamidno gelsko elektroforezo, dinamičnim sipanjem svetlobe, cirkularnim dikroizmom in diferenčno dinamično fluorimetrijo. Z metodo pull-down smo potrdili neposredno interakcijo obeh proteinov s HCPro, z metodo termoforeze na mikroskali pa določili afiniteto vezave. Vrednosti disociacijskih konstant v nizkem mikromolarnem območju izkazujejo specifičnost in prehodno naravo teh interakcij. Ugotovitve predstavljajo pomemben temelj za nadaljnje strukturne in funkcijske raziskave interakcij med virusnimi in rastlinskimi (gostiteljskimi) molekulami. Potyviruses constitute a large genus of plant RNA viruses that cause significant economic losses worldwide in agriculture by infecting a wide range of hosts. Due to their small genomes, they encode a set of multifunctional proteins, among which the HCPro protein plays a central role. Through complex interactions with viral and host proteins, HCPro facilitates viral replication and spread while simultaneously suppressing the plant's defense system. Interactions between HCPro from various potyviruses and selected plant molecules, identified through diverse methodological approaches, have already been documented in the literature. The aim of this master's thesis was to qualitatively and quantitatively analyse the interactions of the potato virus A (PVA) HCPro protein with five tobacco (Nicotiana tabacum) proteins, eIF(iso)4E, CF1β, MinD, WD40 and HIP2, under controlled in vitro conditions. Recombinant proteins were expressed in the bacterial system Escherichia coli. Due to inadequate expression or solubility of MinD, WD40 and HIP2, further experimental work was limited to the proteins eIF(iso)4E and CF1β. By introducing an additional polyhistidine tag, we successfully optimized the isolation and purification protocols for both proteins. Their purity was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while structural integrity and stability were assessed using native polyacrylamide gel electrophoresis, dynamic light scattering, circular dichroism and differential scanning fluorimetry. Using the pull-down assay, we confirmed the direct interaction of both proteins with HCPro, and their binding affinity was determined via microscale thermophoresis. Dissociation constant values in the low micromolar range indicate the specificity and transient nature of these interactions. These findings provide an important foundation for further structural and functional research into the interactions between viral and plant (host) molecules. potivirusi protein HCPro proteinske interakcije tobačni proteini pull-down termoforeza na mikroskali potyviruses HCPro protein-protein interactions tobacco proteins pull-down assay microscale thermophoresis true false false [K. Leben] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-07-10 07:15:20 2026-07-10 07:15:33 2026-07-11 04:13:44 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 578.864:543.645.6(043.2) 281156 284366851 58042.pdf 58042.pdf 1 48A5B42733C87933011E6109ED5D42C3 7356f283e5aa3950d8fc788c470f3863916c4face4c88eb264ad03c75be52184 62852149-7c1e-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=237447 Biotehniška fakulteta 0 0 0