Using different bioinformatics tools to analyze data from previous studies of Verticillium nonalfalfae interaction with hop, we predicted that the V. nonalfalfae secretome is rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling. It includes 263 candidate secreted effector proteins (CSEPs), among which several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants (Avr-Pita1 and MgSM1), while the majority of CSEPs were non-annotated. 44 top priority candidates were examined by spatiotemporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed. Several putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease.
An insoluble complex carbohydrate, chitin makes up fungal walls and plays a significant role in the interaction between fungal pathogens and their plant hosts. Plant cells harbor immune receptors that perceive chitin and work to stop fungal infection. However, fungal plant pathogens then release chitin-binding proteins that perturb the chitin-triggered immunity. Article studies one of these chitin-binding proteins from a soilborne fungus (Verticillium nonalfalfae) that causes vascular wilt in plants. This fungus binds a particular protein (VnaChtBP) to chitin in order to abolish the host plant's chitin-triggered burst of reactive oxygen species and shield the fungus from being digested by the plant chitinases. Using 3D homology modelling, molecular docking, CD measurements, and a Y2H assay we determined, for the first time, the probable molecular mechanism of chitin-binding to carbohydrate-binding module family 18 (CBM18)-containing fungal effectors. In addition, our research highlights that, apart from the well-studied Avr4 (CBM14) and LysM (CBM50) fungal effectors, which can interfere with plant chitin perception and activation of immune responses, other structurally unrelated fungal effectors with CBM18 domains have evolved with similar function, suggesting a convergent evolution.
Molecular mechanisms of verticillium wilt resistance in hop are complex, consisting of preformed and induced defense responses, including the synthesis of various phenolic compounds. Article studies the total polyphenolic content at two phenological stages in roots and stems of 14 hop varieties differing in VW resistance, examines changes in the total polyphenols of VW resistant variety Wye Target (WT) and susceptible Celeia (CE) on infection with V. nonalfalfae, and assesses the antifungal activity of six commercial phenolic compounds as well as total polyphenolic extracts from roots and stems of VW resistant WT and susceptible CE on the growth of two different V. nonalfalfae hop pathotypes. Generally, total polyphenols were higher in roots than stems and increased with maturation of the hop. Before flowering, the majority of VW resistant varieties had a significantly higher content of total polyphenols in stems than susceptible varieties. At the symptomatic stage of VW disease, total polyphenols decreased in VW resistant WT and susceptible CE plants in both roots and stems. The antifungal activity of total polyphenolic extracts against V. nonalfalfae was higher in hop extracts from stems than those from roots. Among the tested phenolic compounds, only p-coumaric acid and tyrosol markedly restricted fungal growth. Although the correlation between VW resistance and total polyphenols content is not straightforward, higher levels of total polyphenols in the stems of the majority of VW resistant hop varieties at early phenological stages probably contribute to fast and efficient activation of signaling pathways, leading to successful defense against V. nonalfalfae infection.
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 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. RNAi is a major contributor to the virulence of fungal pathogens as a result of so-called trans-kingdom RNA silencing. The article reports 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 characterized. Using phylogenetic analysis, we 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. With expression analysis we demonstrate the role of RNAi in the pathogenicity of the fungi, since all the RNAi genes were highly upregulated in the aggressive isolate T2 and differentially expressed in the V. nonalfalfae-susceptible Celeia and V. nonalfalfae-resistant Wye Target hop cultivars.
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. In this study, we investigated the differential accumulation of phenolics in the susceptible “Celeia” and resistant “Wye Target” hop cultivars during the infection with soilborne vascular pathogen Verticillium nonalfalfae. 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 post inoculation (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.