RNA interference (RNAi) is a conserved mechanism for regulating gene expression in animals, plants and fungi. The RNAi mechanism was studied extensively in the model fungus Neurospora crassa, and these studies led to the identification of the core components of the mechanism, the RNA-dependant RNA polymerase (RdRP), Dicer (DCL) and Argonaut (AGO), that mediate silencing events. In filamentous fungi, including Verticillium nonalfalfae (V. nonalfalfae), the causal agent of wilting disease of hops, the RNAi mechanism was identified as one of the pathogenic mechanisms of these fungi. Small and microRNA-like RNAs (milRNAs) were implicated not only in regulating endogenous genes but also as effectors that fungi can export into host plants, where they regulate the expression of host defence mechanisms. Due to the important contribution of RNAi to the pathogenicity of fungi, in this doctoral dissertation, we characterised the RNAi mecahanism of V. nonalfalfae by identifying the core components of small and milRNA synthesis and the components required for gene silencing. We confirmed the existence and expression of two RdRP, two DCL and two AGO proteins. Furthermore, we identified and validated the expression of nine fungal milRNAs and confirmed the activity of the RNAi by experimental analysis of the target cleavage sites of the target gene models identified in silico in the annotated fungal genome. With the studies conducted in the scope of the doctoral dissertation, we reported on the existence of RNAi in V. nonalfalfae for the first time, and the obtained knowledge will help in the development of new strategies for the management of verticillium wilt disease in hops.
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