20.500.12556/RUL-162143 Določanje molekularnega mehanizma bioinsekticidnega delovanja novih glivnih lektinov na invazivne škodljivce Determination of the molecular mode of action of novel fungal lectins as bioinsecticides on invasive pests Svetu naraščajočega prebivalstva sledi potreba po intenzivnem kmetijstvu. Intenzivno kmetijstvo omogoča razvoj visoko specializiranih žuželčjih škodljivcev, kot so na primer koruzni hrošč na koruzi, koloradski hrošč na krompirju in plodova vinska mušica na sadju. Zaradi hitrega pridobivanja odpornosti pri žuželkah se povečuje potreba po insekticidih z novim mehanizmom delovanja. S širokimi presejalnimi testi so bili odkriti trije entomotoksični glivni lektini, ki so specifični za posamezne žuželke; Marasmius oreades aglutinin (MOA) proti koloradskem hrošču (Leptinotarsa decemlineata Say) ter Sclerotinia sclerotiorum aglutinin (SSA) in Agrocybe aegerita galektin (AAG) proti plodovi vinski mušici (Drosophila suzukii). Koruzni hrošč je kljub visoki evolucijski podobnosti s koloradskim hroščem odporen na delovanje lektina MOA. Glavno vprašanje po odkritju toksičnega učinka lektinov je bilo njihovo molekularno delovanje. V sklopu magistrskega dela smo s pomočjo afinitetne kromatografije in masne spektrometrije določili potencialne molekularne tarče lektinov MOA, SSA in AAG v tarčnih organizmih in odkrili potencialen mehanizem odpornosti v koruznem hrošču. Uporaba detekcije z biotiniliranim lektinom po prenosu western se je izkazala za neprimerno pri analizi glikozilacijskega profila tarč lektina MOA. Testi in vivo na tarčnih organizmih z lektini v kombinaciji z enostavnimi inhibitornimi sladkorji in mutanti lektinov so potrdili pomen posameznih regij lektinov za toksičnost. Pokazali smo, da se MOA veže na aminopeptidazo v koloradskem hrošču, medtem ko v koruznem hrošču stik s tarčo verjetno preprečijo mucini. Za tarčo SSA smo potrdili, da gre za glikoprotein, medtem ko vezava glikanov verjetno ni pomembna za toksično delovanje AAG. S pridobitvijo teh rezultatov smo predstavili nov model toksičnosti prehranskih proteinov v koloradskem hrošču in prvič poročamo o zaščitni vlogi mucinov pred entomotoksičnimi proteini pri žuželkah. Poznavanje molekularnih mehanizmov delovanja je pomembno pri razvoju novih bioinsekticidov, saj bo olajšalo nadaljnji razvoj in bolje opredelilo končno uporabo v pridelovalne namene. In a world with a growing population, there is a need for intensive agriculture. Intensive agriculture enables the development of highly specialised insect pests, such as the western corn rootworn on maize, the Colorado potato beetle on potatoes and the spotted wing drosophila on fruit. The rapid acquisition of resistance in insects increases the need for insecticides with new mechanisms of action. Three entomotoxic fungal lectins have been identified through extensive screening tests: Marasmius oreades agglutinin (MOA) against the Colorado potato beetle (Leptinotarsa decemlineata Say) and Sclerotinia sclerotiorum agglutinin (SSA) and Agrocybe aegerita galectin (AAG) against the spotted wing drosophila (Drosophila suzukii). Despite its close evolutionary similarity to the Colorado potato beetle, the western corn rootworm is resistant to the effects of MOA. The most important question after the discovery of the toxic effect of lectins was their molecular mehanism. We used affinity chromatography and mass spectrometry to identify potential molecular targets of the lectins MOA, SSA and AAG in the target organisms and to determine a possible mechanism of resistance in the western corn rootworm. The use of biotinylated lectins to analyse the glycosylation profile of MOA lectin targets proved to be unsuitable. In vivo tests on target organisms with lectins in combination with simple inhibitory sugars and lectin mutants confirmed the importance of specific regions of lectins for toxicity. We were able to show that MOA binds to aminopeptidase in the Colorado potato beetle, whereas in western corn rootworm contact with the target is probably prevented by mucins. For the SSA target we could confirm that it is a glycoprotein, whereas glycan binding is probably not relevant for the toxic effect of AAG. With these results, we present a new model for the toxicity of dietary proteins in the Colorado potato beetle and report for the first time a protective role of mucins against entomotoxic proteins in insects. Knowledge of the molecular mechanisms of action is important for the development of new bioinsecticides as it will facilitate further development and better define their end use in crop production. lektin žuželke bioinsekticid molekularni mehanizem delovanja lectin insect bioinsecticide molecular mechanism of action true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2024-09-19 08:16:13 2024-09-19 08:16:21 2025-08-29 11:05:14 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2025-08-28 208169475 208169475 42534.pdf 42534.pdf 1 E5253D403CEF068EAFD5F9A00D17B3EE cfea73ff963ecc826bea8b3e92a241ed3a3e3489b91205abf4c7e87698259d70 950eca10-764e-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=191305 Biotehniška fakulteta 0 0 0