During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin-binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases and to interfere with chitin-triggered host immunity.

Viroids are small, naked, infectious single-stranded RNA molecules that exploit host factors to replicate. Some viroids have been linked to severe diseases in agricultural crops, including the recent outbreak of Cocadviroid rimocitri , previously known as Citrus bark cracking viroid (CBCVd), in hop plants ( Humulus lupulus ). Numerous studies have demonstrated the involvement of viroid-derived RNA in viroid pathogenesis through interactions with RNAi host factors, leading to alterations in gene expression, metabolism, and phenotype. Recent research efforts have also focused on elucidating viroid-induced changes in DNA methylation patterns via the RNA-directed DNA methylation pathway. In this study, we conducted an epigenome analysis of CBCVd-infected hop plants to provide novel evidence supporting the putative role of DNA methylation in CBCVd viroid pathogenesis. Our findings revealed that several genes involved in pathogen interaction pathways, such as MAPK signaling and LRR, exhibit hypomethylation, suggesting that their increased transcription enhances the host’s ability to counteract the pathogen. Intriguingly, genes associated with RNA transcription and encoding key proteins, such as POL II, POL IV, and POL V, display hypermethylation, highlighting the significance of DNA methylation as a defense mechanism. IMPORTANCE: Viroids are emerging as a substantial threat to various crops; however, our understanding of the molecular mechanisms governing their pathogenesis and the host’s defense remains incomplete. This knowledge gap leaves crop disease management reliant on unsustainable strategies. Our research seeks to address this issue by examining the complex world of infected hop plants. Specifically, we are investigating the DNA methylation processes, providing insights into the less-explored aspects of the host’s response to viroid interaction. Our aim was to unravel the complexities of how viroids influence the molecular landscape within plants and the corresponding host defenses. By understanding these interactions, we hope to provide insights that lead to more sustainable ways to protect crops and keep agriculture resilient against viroid-related threats.

This study evaluates the susceptibility of various hop varieties to three viroids: Hop Latent Viroid (HLVd), Hop Stunt Viroid (HSVd), and Citrus Bark Cracking Viroid (CBCVd), all of which pose significant threats to Slovenian hop production. The experiment was conducted under in vitro conditions, involving the cultivation of 13 different hop varieties in tissue cultures, alongside the in vitro synthesis of viroid transcripts and subsequent inoculation of the plants. The plants were then monitored for growth, development, and their responses to viroid infection. The findings reveal the complex interactions between hops and viroids, with successful inoculation observed in all varieties. However, infection rates varied, with HSVd showing the highest rates of infection, followed by CBCVd and HLVd. This research enhances the understanding of viroid dynamics in hop plants, which is crucial for developing effective disease management strategies. Despite the insights gained, no resistance or tolerance to the viroids was identified in any of the tested hop varieties.

This study evaluates the susceptibility of various hop varieties to three viroids: Hop Latent Viroid (HLVd), Hop Stunt Viroid (HSVd), and Citrus Bark Cracking Viroid (CBCVd), all of which pose significant threats to Slovenian hop production. The experiment was conducted under in vitro conditions, involving the cultivation of 13 different hop varieties in tissue cultures, alongside the in vitro synthesis of viroid transcripts and subsequent inoculation of the plants. The plants were then monitored for growth, development, and their responses to viroid infection. The findings reveal the complex interactions between hops and viroids, with successful inoculation observed in all varieties. However, infection rates varied, with HSVd showing the highest rates of infection, followed by CBCVd and HLVd. This research enhances the understanding of viroid dynamics in hop plants, which is crucial for developing effective disease management strategies. Despite the insights gained, no resistance or tolerance to the viroids was identified in any of the tested hop varieties.