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Vpliv z redoks stanjem posredovane signalizacije na redoks stanje v kloroplastih in nastanek stromulov pri preobčutljivostnem odgovoru krompirja na okužbo z virusom
ID Golob, Nuša (Author), ID Lukan, Tjaša (Mentor) More about this mentor... This link opens in a new window

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Abstract
Krompirjev virus Y (PVY) na okuženih listih rastlin krompirja kultivarja Rywal povzroči nastanek nekrotičnih območij, lezij, ki nastanejo kot posledica sprožitve s preobčutljivostnim odgovorom pogojene odpornosti. V magistrski nalogi smo po okužbi rastlin krompirja, ki izražajo na redoks stanje občutljiv zeleni fluorescenčni protein, s PVY spremljali nastanek stromulov ter spremembe kloroplastnega redoks stanja z uporabo konfokalnega mikroskopa in skripte za določanje redoks stanja. Primerjali smo rezultate pri genetsko spremenjenih rastlinah krompirja z znižano akumulacijo apoplastnih ROS (apoROS) ter kloroplastnih ROS (chlROS) in spremenjeno akumulacijo salicilne kisline (SA) v različnih kombinacijah. Ugotovili smo, da imajo omenjeni dejavniki časovno-prostorsko pogojen vpliv na kloroplastno redoks stanje ter nastanek stromulov, pri čemer se najbolj oksidirani kloroplasti z največ stromuli nahajajo bližje leziji. Pokazali smo, da na nastanek stromulov bistveno vplivata SA in chlROS, saj višja akumulacija SA nastanek inducira, nižja akumulacija SA in chlROS pa ga zavre. Da bi ocenili uporabnost in stabilnost na novo razvitega virusnega klona PVY-mTurquoise2, smo z njim okužili rastline tobaka (Nicotina clevelandii) ter zgoraj opisane genetsko spremenjene rastline krompirja. Potrdili smo, da je dinamika širjenja virusnega klona PVY-mTurquoise2 primerljiva PVY-GFP, ter da je stabilnost PVY-mTurquoise2 v rastlinah tobaka dolgoročna, saj virus ostaja stabilen po večih pasažah. Potrdili smo, da lahko v okuženih rastlinah krompirja s konfokalnim mikroskopom določimo s PVY-mTurquoise2 okužene celice ter na njihovem območju raziskujemo nastanek stromulov ter kloroplastno redoks stanje. V nalogi smo prikazali tudi učinkovit in optimiziran način spremljanja virusnih klonov PVY-GFP in PVY-mTurquoise2 z uporabo luminometra.

Language:Slovenian
Keywords:krompirjev virus Y, preobčutljivostni odgovor, redoks stanje, stromuli, virusni klon
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Publisher:[N. Golob]
Year:2024
PID:20.500.12556/RUL-153519 This link opens in a new window
UDC:578.8:663.491(043.2)
COBISS.SI-ID:180871427 This link opens in a new window
Publication date in RUL:12.01.2024
Views:487
Downloads:133
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Secondary language

Language:English
Title:Impact of redox-related signaling on chloroplast redox state and stromules formation in potato hypersensitive response-conferred resistance to virus
Abstract:
Potato virus Y (PVY) induces the formation of lesions on infected leaves of potato plants cv. Rywal as a part of defense mechanism, called hypersensitive response conferred resistance. In this master thesis, we followed stromule formation and changes in chloroplastic redox state in pt-roGFP expressing potato plants after infection with PVY using confocal microscope and image analysis script for detection of redox state. We compared the response of genetically modified potato plants with reduced accumulation of apoplastic ROS (apoROS), chloroplastic ROS (chlROS) as well as varying levels of salicylic acid (SA) in different combinations. Carrying a detailed spatiotemporal analysis, we revealed that that the most oxidized chloroplasts accompanied by higher stromule count were predominantly located in the proximity of lesions. Furthermore, we confirmed the pivotal role of SA and chlROS in regulation of stromule formation, as increased SA levels induce it, while reduced SA levels and chlROS accumulation inhibit it. In order to asses the applicability and stability of the newly developed PVY-mTurquoise2 viral clone, we infected tobacco plants (Nicotina clevelandii) as well as genetically modified potato plants, described above. We confirmed that the dynamics of PVY-mTurquoise2 spread closely resembles the one of PVY-GFP and that PVY-mTurquoise remains stable in tobacco plants after several passages. Moreover, we demonstrated the precise identification of PVY-mTurquoise2 infected cells in inoculated potato plants using a confocal microscope, which allowed us to investigate stromule formation and chloroplastic redox state in immediate vicinity of those cells. Additionally, we presented an effective and optimized method for tracking PVY-GFP and PVY-mTurquoise2 viral clones using a luminometer.

Keywords:potato virus Y, hypersensitive response, stromules, redox state, viral clone

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