Plants and pathogens engage in complex interactions, including cross-kingdom RNA interference, where miRNAs can be transferred between host and pathogen to modulate gene expression. This process has been demonstrated in cotton infected by Verticillium dahliae, where plant-derived miRNAs target fungal virulence genes, reducing disease severity. Whether similar mechanisms occur in other important crops, such as hop (Humulus lupulus), remains unknown. Hop is a key agricultural commodity in brewing, but it is highly susceptible to Verticillium nonalfalfae, the causal agent of Verticillium wilt, which can severely reduce yield. In this Master’s thesis, we aimed to (i) test whether hop-derived miRNAs can be detected in V. nonalfalfae using stem-loop RT-qPCR, and (ii) establish soil-free infection systems for the hop – V. nonalfalfae pathosystem. Of the 19 newly designed stem-loop primer sets for hop’s miRNA identification, we successfully amplified the target hop miRNAs in hop tissue with 17 sets, yielding Ct values between 15.3 and 32.9 and clear, single-peak melt curves consistently observed across replicates. Although we could not confirm the presence of hop miRNAs in V. nonalfalfae, these assays represent a promising toolkit for future studies on hop miRNA responses to biotic or abiotic stress. We also successfully established two infection systems. For in vitro approach, we screened 12 hop genotypes across six media and identified the best media-genotype combinations for future use. In an ex vitro hydroponic infection system, we reached up to 100% of root and shoot development among tested genotypes. 100% of root and stem samples were confirmed infected with V. nonalfalfae at 21 dpi via Vna-ITS-2 PCR, demonstrating the system’s suitability for early-stage infection studies under controlled conditions. Overall, this thesis provides valuable tools and experimental systems to advance our understanding of interactions between hop and the phytopathogenic fungus V. nonalfalfae.
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