Phenolic compounds are involved in plant responses to various biotic and abiotic stress factors, with many studies suggesting their role in defense mechanisms against fungal pathogens. Soilborne vascular pathogen Verticillium nonalfalfae causes severe wilting and consequent dieback in a wide range of economically important crops, including hops (Humulus lupulus L.). In this study, we investigated the differential accumulation of phenolics in the susceptible “Celeia” and resistant “Wye Target” hop cultivars during the pathogenesis of Verticillium wilt. Quantitative polymerase chain reaction showed that colonization in the roots of both cultivars was intensive, but decreased continuously throughout the experiment in the resistant cultivar, while the relative fungal amount continuously increased in the stems of the susceptible cultivar. In response to colonization in the roots of the resistant cultivar, a significant increase in total flavanols was detected at three days postinoculation (dpi), suggesting a possible role in preventing fungus spread into the stems. The accumulation of phenolic compounds was less pronounced in the stems of the resistant cultivar since, compared to the latter, significant increases in flavonols at 3 and 15 dpi and hydroxycinnamic acids at 6 dpi were observed in the stems of the susceptible cultivar.

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.

Abiotic and biotic stresses can lead to changes in host DNA methylation, which in plants is also mediated by an RNA-directed DNA methylation mechanism. Infections with viroids have been shown to affect DNA methylation dynamics in different plant hosts. The aim of our research was to determine the content of 5-methylcytosine (5-mC) in genomic DNA at the whole genome level of hop plants (Humulus lupulus Var. ‘Celeia’) infected with different viroids and their combinations and to analyse the expression of the selected genes to improve our understanding of DNA methylation dynamics in plant-viroid systems. The adapted HPLC-UV method used proved to be suitable for this purpose, and thus we were able to estimate for the first time that the cytosine methylation level in viroid-free hop plants was 26.7%. Interestingly, the observed 5-mC level was the lowest in hop plants infected simultaneously with CBCVd, HLVd and HSVd (23.7%), whereas the highest level was observed in plants infected with HLVd (31.4%). In addition, we identified three DNA methylases and one DNA demethylase gene in the hop’s draft genome. The RT-qPCR revealed upregulation of all newly identified genes in hop plants infected with all three viroids, while no altered expression was observed in any of the other hop plants tested, except for CBCVd-infected hop plants, in which one DNA methylase was also upregulated.

MicroRNAs are 21- to 24-nucleotide-long, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. They can modulate various biological processes, including plant response and resistance to fungal pathogens. Hops are grown for use in the brewing industry and, recently, also for the pharmaceutical industry. Severe Verticillium wilt caused by the phytopathogenic fungus Verticillium nonalfalfae, is the main factor in yield loss in many crops, including hops (Humulus lupulus L.). In our study, we identified 56 known and 43 novel miRNAs and their expression patterns in the roots of susceptible and resistant hop cultivars after inoculation with V. nonalfalfae. In response to inoculation with V. nonalfalfae, we found five known and two novel miRNAs that are differentially expressed in the susceptible cultivar and six known miRNAs in the resistant cultivar. Differentially expressed miRNAs target 49 transcripts involved in protein localization and pigment synthesis in the susceptible cultivar, whereas they are involved in transcription factor regulation and hormone signalling in the resistant cultivar. The results of our study suggest that the susceptible and resistant hop cultivars respond differently to V. nonalfalfae inoculation at the miRNA level and that miRNAs may contribute to the successful defence of the resistant cultivar.

RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which Verticillium, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with Verticillium nonalfalfae. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species Cannabis sativa, Morus notabilis and Ziziphus jujuba which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation.

The main challenge associated with genotyping based on conventional length polymorphisms is the cross-laboratory standardization of allele sizes. This step requires the inclusion of standards and manual sizing to avoid false results. Capillary electrophoresis (CE) approaches limit the information to the length polymorphism and do not allow the determination of a complete marker sequence. As an alternative, high-throughput sequencing (HTS) offers complete information regarding marker sequences and their flanking regions. In this work, we investigated the suitability of a semi-quantitative sequencing approach for microsatellite genotyping using Illumina paired-end technology. Twelve microsatellite loci that are well established for grapevine CE typing were analysed on 96 grapevine samples from six different countries. We redesigned primers to the length of the amplicon for short sequencing (~100 bp). The primer pair was flanked with a 10 bp overhang for the introduction of barcodes on both sides of the amplicon to enable high multiplexing. The highest data peaks were determined as simple sequence repeat (SSR) alleles and compared with the CE dataset based on 12 reference samples. The comparison showed that HTS SSR genotyping can successfully replace the CE system in further experiments. We believe that, with next-generation sequencing, genotyping can be improved in terms of its speed, accuracy, and price.