Analysis of viroid replication and genome DNA methylation of infected hop (Humulus lupulus L.)

Hmelj je pomembna kmetijska poljščina, ki se jo v glavnem uporablja za varjenje piva. Hmelj je dovzeten za okužbo z različnimi patogeni, tako tudi z viroidi. Viroidi so majhne, krožne, enoverižne molekule RNA, ki lahko okužijo različne rastlinske vrste, na katerih se razvijejo bolezenska znamenja, kar se lahko prevesi v hudo gospodarsko škodo. Hmeljarji uporabljajo različne preventivne ukrepe za omejevanje vpliva viroidov na gojenje hmelja. Mnogo raziskovalcev se ukvarja s študijem patologije patogeneze viroidov, vseeno pa molekularni mehanizmi razvoja bolezenskih znamenj ostajajo nepojasnjeni. Zaradi tega smo v pričujočem doktorskem delu proučevali podvojevanje viroidov na eni in spremembe v metilaciji DNA okuženega hmelja na drugi strani. Rezultati, pridobljeni s tehnologijo Nanopore, kažejo na njeno ustreznost za analizo intermediatov podvojevanja, saj omogoča neposredno določanje nukleotidnega zaporedja RNA. Pokazali smo tudi, da viroidi lahko vplivajo na metilacijo DNA hmelja. Rezultati proučevanja metilacije DNA nam v prvi vrsti zagotavljajo nove namige za prihodnje raziskave v povezavi z rastlinsko imunostjo in viroidno patogenezo. Prav tako smo med drugim pokazali, da je diferenčna metilacija DNA povezana z rastlinskim hipersenzitivnostnim odzivom, obrambnim in hormonskim odzivom ter z ribosomskimi procesi. Viroid CBCVd zelo verjetno vpliva na od RNA-odvisno metilacijo DNA, kar je skladno z rezultati nekaterih dosedanjih študij viroidov in metilacije DNA.Hop plants serve as a vital agricultural crop primarily used in beer production. However, its susceptibility to various pathogens, including viroids, poses significant challenges. Viroids are small, circular, single-stranded RNAs capable of infecting diverse plant species, which develop disease symptoms that can translate into substantial economic losses. To mitigate the detrimental effects of viroids on hop cultivation, growers employ a range of preventive measures. While considerable research efforts have focused on elucidating the pathology of viroids, the underlying molecular mechanisms remain largely elusive. In this PhD thesis, we aimed to investigate two key aspects: viroid replication and alterations in DNA methylation patterns in infected hop plants. Notably, we employed the Nanopore sequencing technology, which enabled direct RNA sequencing, making it a suitable tool for analyzing viroid replication intermediates. In addition, we observed that viroids can affect DNA methylation in hop plants. Our investigation of DNA methylation patterns yielded significant outcomes, offering promising directions for future research on plant immunity, and viroid pathogenesis. Our research has unveiled differential DNA methylation patterns associated with viroid presence, implicating their role in plant hypersensitive responses, defensive mechanisms, hormonal signaling, and ribosomal processes. Specifically, CBCVd displayed a strong likelihood of impacting RNA-dependent DNA methylation pathway, corroborating findings from previous studies on viroids and methylation