Determination of the activity properties of some Nep1-like protein mutants from oomycete Pythium aphanidermatum

Proteini, podobni Nep1 ali NLP (ang. (Nep-1)-like protein) so posebna družina proteinov, ki se pojavlja pri fitopatogenih bakterijah, glivah in oomicetah. Delujejo kot elicitorji rastlinskega imunskega sistema ter imajo citotoksično delovanje na rastlinske celice. Prvi koraki interakcije proteinov NLP s plazemsko membrano so znani, vemo pa tudi, da se NLP vežejo na sfingolipid glikozil inozitol fosforilceramid (GIPC), ki je gradnik rastlinske plazemske membrane. Nedavno je prišlo do razjasnitve mehanizma delovanja proteinov NLP: po vezavi proteina na sfingolipidni receptor pride do prerazporeditve receptorjev in nastanka majhnih por v membrani, kar posledično vodi v smrt celic in nekrozo tkiv. V nalogi smo želeli opredeliti nekatere lastnosti cisteinskih mutant proteina NLPPya iz oomicete Pythium aphanidermatum. Cisteinske mutante so locirane na C-terminalnem koncu proteina, in sicer na α-vijačnici, z namenom vezave fluorescenčne sonde na mutirano tiolno skupino. Fluorescenčna sonda IANBD amid omogoča razumevanje sprememb okolja, ki se zgodijo pri interakciji proteina z modelno membrano, ki vsebuje GIPC. S testom infiltracije mutant v liste tobaka, merjenjem sproščanja ionov iz infiltriranega območja in testom kosedimentacije z multilamelarnimi vezikli z GIPC, smo preverili ohranjenost citotoksične aktivnosti mutant glede na divji tip proteina. Uvedba dotičnih mutacij ne vpliva na zvitje proteina, saj imajo mutante podobno temperaturo tališča kot divji tip. Fluorimetrične meritve kažejo na stik fluorescenčnega označevalca s hidrofobnim območjem, kar pomeni da C-terminalni del proteina interagira z membrano.NLP proteins are a protein family that occurs in phytopathogenic bacteria, fungi and oomycetes. They act as elicitors of the plant immune system and have a cytotoxic effect on plant cells. The first steps of the interaction of NLPs with the plasma membrane are known, they bind to the sphingolipid glycosyl inositol phosphorylceramide, a common plant membrane lipid. Recently, the mechanism of action of NLPs has been elucidated: they act on the redistribution of the sphingolipid receptor, leading to small pores in the membrane which cause leakage and necrosis. In the present thesis we wanted to define some properties of NLPPya cysteine mutants from oomycete Pythium aphanidermatum. Cysteine mutants are located at the C-terminal end on the α-helix, for the purpose of binding the fluorescent probe to the mutated thiol group. The fluorescent probe IANBD amide helps us to understand the environmental changes of the protein when an interaction with a model membrane containing GIPC occur. By testing the mutant infiltration into tobacco leaves, measuring the release of ions from the infiltrated area, and the GIPC multilamellar vesicle cosedimentation test, we examined the conservation of mutant cytotoxicity in comparison with the wild-type. The introduction of these mutations do not affect protein folding, as mutants have a similar melting point as the wild-type. Fluorimetric measurements indicate the incorporation of the fluorescent probe into the hydrophobic range, which means that the C-terminal part interacts with the membran