Identification of micro RNAs and their targets in defense response of hop (Humulus lupulus L.) after infection with fungus Verticillium nonalfalfae

V okviru raziskav doktorske disertacije smo izvedli umetno inokulacijo odpornega in občutljivega kultivarja hmelja s fitopatogeno glivo Verticillium nonalfalfae ter v različnih časovnih točkah po inokulaciji določili profil fenolnih spojin in glivne kolonizacije. Ugotovili smo, da se v koreninah občutljivega kultivarja pojavlja bimodalen vzorec kolonizacije, prav tako pa se je gliva razširila v stebla, medtem ko se je relativna količina glive v odpornem kultivarju tekom eksperimenta zmanjševala. V koreninah odpornega kultivarja smo tretji dan po inokulaciji zaznali statistično značilno povečanje skupnih flavanolov. V steblih je bila akumulacija fenolov v občutljivem kultivarju v primerjavi z odpornim višja, saj smo zaznali znatno povečanje flavonolov 3. in 15. dan ter hidroksicimetnih kislin 6. dan po inokulaciji. Študija identifikacije, karakterizacije, filogenetske analize in analize izražanja treh genov Argonavt ter dveh genov Dicerju-podobnih proteinov in dveh genov od RNA-odvisne RNA polimeraze je pokazala grozdenje z njihovimi ortologi iz ožje sorodnih vrst Cannabis sativa, Morus notabilis in Ziziphus jujuba. Ugotovili smo, da se tretji dan po inokulaciji znatno zniža izražanje AGO2 pri obeh kultivarjih, kar lahko povzroči zmanjšano post-transkripcijsko utišanje genov, ki ga posredujejo AGO2 vezane male RNA. Prav tako smo opazili zmanjšano izražanje AGO7 pri občutljivem kultivarju prvi dan po inokulaciji in zmanjšano izražanje RDR6 pri odpornem kultivarju tretji dan. Slednje nakazuje na morebitno zavrtje biogeneze trans-delujočih malih RNA. V študiji identifikacije in diferenčnega izražanja miRNA smo odkrili 56 znanih in 43 novih miRNA. Kot odziv na okužbo z V. nonalfalfae smo zaznali spremembe v izražanju petih znanih in dveh novih miRNA v občutljivem kultivarju ter šestih znanih miRNA v odpornem kultivarju. Diferenčno izražene miRNA predvidoma utišajo 49 transkriptov, ki so pri občutljivem kultivarju vključeni v lokalizacijo proteinov in sintezo pigmentov, medtem ko so pri odpornem kultivarju vključeni v regulacijo transkripcijskih faktorjev in hormonsko signalizacijo.In the studies conducted as part of the doctoral dissertation, a resistant and a susceptible hop cultivar were artificially inoculated with the phytopathogenic fungus Verticillium nonalfalfae and the profiles of phenolic compounds and fungal colonization were determined at different sampling times after inoculation. We observed a bimodal pattern of colonization in the roots of the susceptible cultivar and the fungus spread to the stems, while the relative amount of fungus in the resistant cultivar decreased over the course of the experiment. In the roots of the resistant cultivar, a significant increase in total flavanols was observed on the third day after inoculation. In the stems, the accumulation of phenolics was higher in the susceptible cultivar than in the resistant cultivar as a significant increase in flavonols was observed on days 3 and 15 and in hydroxycinnamic acids on day 6 after inoculation. A study on the identification, characterization, phylogenetic, and expression analyses of three Argonaute genes, two Dicer-like protein genes and two RNA-dependent RNA polymerase genes in hops showed that they clustered with their orthologues from Cannabis sativa, Morus notabilis and Ziziphus jujuba. We found that AGO2 was significantly downregulated in both cultivars at day 3 post-inoculation, which may result in reduced post-transcriptional gene silencing mediated by AGO2-bound small RNAs. We also observed decreased expression of AGO7 in the susceptible cultivar on day 1 post-inoculation and decreased expression of RDR6 in the resistant cultivar on day 3, the latter possibly indicating suppression of biogenesis of trans-acting siRNAs. In a study of miRNA in hop, we identified 56 known and 43 novel miRNAs. In response to V. nonalfalfae infection, we detected changes in the expression of five known and two novel miRNAs in the susceptible cultivar and six known miRNAs in the resistant cultivar. The differentially expressed miRNAs silence 49 transcripts involved in protein localization and pigment synthesis in the susceptible variety, whereas they are involved in transcription factor regulation and hormone signaling in the resistant cultivar