Abstract
The conserved RNA interference mechanism (RNAi) in the fungal kingdom has become a focus of intense scientific investigation. The three catalytic core components, Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RdRP), and their associated small interfering RNA molecules (siRNAs) have been identified and characterised in several fungal species. Recent studies have proposed that RNAi is a major contributor to the virulence of fungal pathogens as a result of so-called trans-kingdom RNA silencing. In the present study, we report on the existence of three core RNAi proteins in the pathogenic plant fungus Verticillium nonalfalfae, which is a soilborne plant pathogen that causes severe wilting disease in hops (Humulus lupulus L.). Two DCL proteins, two AGO proteins, and two RdRP proteins were identified, and their conserved RNAi domains were characterised. Our phylogeny results confirm the existing taxonomic relationships in the Ascomycete fungal phylum and show that the fungi of the Hypocreomycetidae subclass of the Sordariomycetes class have high amino acid sequence similarity. The expression analysis revealed a potential role of RNAi in the pathogenicity of the fungi, since all the RNAi genes were highly upregulated in the highly virulent isolate T2 and were also differentially expressed in the V. nonalfalfae-susceptible Celeia and V. nonalfalfae-resistant Wye Target cultivars.
Highlights
The molecular mechanism of RNA interference (RNAi) was first identified in Caenorhabditis elegans[1] and has been described as a negative regulator of gene expression driven by small non-coding RNA molecules
The identification of the core components of RNA interference mechanism (RNAi) and their associated small interfering RNA molecules (siRNAs) is associated with the advancement and implementation of next-generation sequencing (NGS) technologies[26], which enables the generation of a large number of sequenced reads for use in the construction of detailed genomes and transcriptomes to allow the identification of major proteins and their gene sequences and of shorter and less abundantly represented RNA molecules in samples and organisms[27]
All obtained hits were within the predicted gene models[37], which were subsequently manually curated based on the BLAST results and RNA sequencing (RNA-seq) data[37]
Summary
The molecular mechanism of RNA interference (RNAi) was first identified in Caenorhabditis elegans[1] and has been described as a negative regulator of gene expression driven by small non-coding RNA molecules (siRNAs). In the conserved RNAi pathway, the Dicer ribonuclease III enzymes (DCR and DCL in fungi) cleave double-stranded RNA precursors (dsRNAs) to generate short dsRNAs that are subsequently loaded into the RNA-induced silencing complex (RISC), which contains an Argonaute (AGO) protein as the core catalytic component. The most severe outbreaks in hops are caused by highly virulent V. nonalfalfae pathotypes that produce infections that induce rapid plant dieback and lethal verticillium wilt symptoms. Disease management relies mainly on phytosanitary measures, soil disinfestation and the use of resistant varieties[36] At this point in time, it is of utmost importance to study and dissect the pathogenicity and all the related biological processes at many different levels to better understand the pathogen, the host and their interactions to develop new strategies for the defence against verticillium wilt disease. Expression analysis was performed to examine expression patterns in the highly virulent and less virulent fungal pathotypes and to predict the potential effects of fungal RNAi in vivo during V. nonalfalfae infection in hop host plants
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