The mediator (MED) represents a large, conserved, multi-subunit protein complex that regulates gene expression through interactions with RNA polymerase II and enhancer-bound transcription factors. Expanding research accomplishments suggest the predominant role of plant MED subunits in the regulation of various physiological and developmental processes, including the biotic stress response against bacterial and fungal pathogens. However, the involvement of MED subunits in virus/viroid pathogenesis remains elusive. In this study, we investigated for the first time the gene expression modulation of selected MED subunits in response to five viroid species (Apple fruit crinkle viroid (AFCVd), Citrus bark cracking viroid (CBCVd), Hop latent viroid (HLVd), Hop stunt viroid (HSVd), and Potato spindle tuber viroid (PSTVd)) in two model plant species (Nicotiana tabacum and N. benthamiana) and a commercially important hop (Humulus lupulus) cultivar. Our results showed a differential expression pattern of MED subunits in response to a viroid infection. The individual plant MED subunits displayed a differential and tailored expression pattern in response to different viroid species, suggesting that the MED expression is viroid- and plant species-dependent. The explicit evidence obtained from our results warrants further investigation into the association of the MED subunit with symptom development. Together, we provide a comprehensive portrait of MED subunit expression in response to viroid infection and a plausible involvement of MED subunits in fine-tuning transcriptional reprogramming in response to viroid infection, suggesting them as a potential candidate for rewiring the defense response network in plants against pathogens.

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.

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.