Novel Identification of the Collection of Pathogenic Fungal Species Verticillium with the Development of Species-Specific SSR Markers.

The genus Verticillium is a group of ascomycete fungi that includes several pathogenic plant species. In 2011, a new taxonomic classification, proposed by Inderbitzin and coworkers (2011), re-defined the genus as Verticillium sensu stricto. The objective of our study was the re-classification of the fungal species held in the culture collection in the Slovenian Institute of Hop Research and Brewing in accordance with the newly established taxonomy. With the PCR marker system proposed by Inderbitzin and coworkers in 2011, we re-classified 88 Verticillium isolates out of the 105 samples that are held in the institute's bank, which were obtained from different geographic locations in Europe, North America, and Japan, and from different host plants, including alfalfa, cotton, hop, olive, potato, and tomato. However, the PCR marker for the V. dahliae identification proved to be less specific, and it resulted in the positive amplification of Gibellulopsis nigrescens, V. isaacii, and V. longisporum. To enable the accurate distinction of the fungi, the SSR and LAMP markers were added to the analyses. The 12 newly identified SSR markers, which were used in simplex PCR reactions or in combination, enabled the accurate identification of all included Verticillium isolates and could potentially be used as biomarkers for rapid and easy species identification.

Discovery of microRNA-like Small RNAs in Pathogenic Plant Fungus Verticillium nonalfalfae Using High-Throughput Sequencing and qPCR and RLM-RACE Validation.

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA interference (RNAi) mechanism has become one of the main areas of focus in plant-fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the V. nonalfalfae species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated V. nonalfalfae genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine V. nonalfalfae milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5' RLM-RACE approach.

RNA interference core components identified and characterised in Verticillium nonalfalfae, a vascular wilt pathogenic plant fungi of hops.

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.

Asthma MicroRNA Regulome Development Using Validated miRNA-Target Interaction Visualization.

Asthma is a common multifactorial complex disease caused by an interaction of genetic and environmental factors. There are no robust biomarkers or molecular diagnostics for asthma or its detailed phenotypic stratification in the clinic. Regulatory and epigenomic factors are priority candidates for asthma biomarker discovery and translational research because this common disease emerges in association with host/environment interactions. In this context, epigenomic molecular events such as microRNA (miRNA) silencing affect asthma susceptibility and severity. We report here an analysis of the miRNAs in the literature, their targets associated with asthma, and present the findings organized as an miRNA-target network, an miRNA regulome of asthma. The miRNA-target interactions in asthma were extracted from the PubMed and the Web of Science databases, while the miRNA-target network was visualized with the Cytoscape tool. Genomic locations of miRNA and target genes were displayed using the Ensembl Whole Genome tool. We cataloged miRNAs associated with asthma and their experimentally validated targets, retrieving 48 miRNAs associated with asthma, and 54 experimentally validated miRNA targets. Four central molecules involved in 34.5% of all interactions were identified in the network. The miRNA-target pairs were constructed as an asthma-associated miRNA-target regulatory network. The network revealed subnetworks pointing toward potential asthma biomarker candidates. The asthma miRNA regulome reported here offers a strong foundation for future translational research and systems medicine applications for asthma diagnostic and therapeutic innovation. Developed protocol for constructing miRNA regulome could now be used for biomarker development in multifactorial diseases.