Summary: Viroids are pathogenic noncoding RNAs that completely rely on their host molecular machinery to accomplish their life cycle. Several interactions between viroids and their host molecular machinery have been identified, including interference with epigenetic mechanisms such as DNA methylation. Despite this, whether viroids influence changes in other epigenetic marks such as histone modifications remained unknown. Epigenetic regulation is particularly important during pathogenesis processes because it might be a key regulator of the dynamism of the defense response. Here we have analyzed the changes taking place in Cucumis sativus (cucumber) facultative and constitutive heterochromatin during hop stunt viroid (HSVd) infection using chromatin immunoprecipitation (ChIP) of the two main heterochromatic marks: H3K9me2 and H3K27me3. We find that HSVd infection is associated with changes in both H3K27me3 and H3K9me2, with a tendency to decrease the levels of repressive epigenetic marks through infection progression. These epigenetic changes are connected to the transcriptional regulation of their expected targets, genes, and transposable elements. Indeed, several genes related to the defense response are targets of both epigenetic marks. Our results highlight another host regulatory mechanism affected by viroid infection, providing further information about the complexity of the multiple layers of interactions between pathogens/viroids and hosts/plants.

Sharka disease, caused by the plum pox virus (PPV), negatively impacts stone fruit production, resulting in economic losses. It has been demonstrated that grafting the almond (Prunus dulcis (Miller) D.A. Webb) variety ‘Garrigues’ into susceptible peach (Prunus persica (L.) Batsch) rootstocks can result in PPV resistance. The molecular circuits related to grafting in Prunus species, however, have not been fully investigated. In this study, susceptible peach rootstocks ‘GF305’ were either heterografted with ‘Garrigues’ almond or homografted with the same cultivar. Peach samples were collected at two stages of scion development, with ungrafted plants utilized as controls. Profiles of transcripts, small RNAs (sRNAs), and DNA methylation were obtained and analyzed on a genome-wide scale. Homografting and heterografting significantly altered the transcriptome and methylome of peach rootstocks, with these modifications being more pronounced during the early stages of scion development. The profiles of sRNAs were significantly more impacted when almonds were used as a scion as opposed to peaches, likely due to the transmission of PPV-unrelated viral sequences. Gene expression differences resulting from DNA methylation alterations are more thoroughly documented at the promoter sequences of genes than within their bodies. This study suggests that the ‘Garrigues’ almond variety triggers a complex defense response in the peach rootstock, potentially involving the interplay of epigenetic modifications and small RNA-mediated priming of antiviral defenses, which ultimately may contribute to PPV resistance.

Constricted by an extreme biological simplicity, viroids are compelled to subvert host regulatory networks in order to accomplish their infectious process. Most of the studies focused on the response to viroid infection have only addressed a specific host regulatory level and considered a unique infection time. Thus, much remains to be done if we want to understand the temporal evolution and complex nature of viroid-host interactions. Here we present an integrative analysis of the timing and intensity of the genome-wide alterations in cucumber plants infected with Hop stunt viroid (HSVd). Differential host transcriptome, sRNAnome and methylome were integrated to determine the temporal response to viroid-infection. Our results support that HSVd promotes a dynamic redesign of the cucumber regulatory pathways predominantly affecting specific regulatory layers at different infection phases. Remarkably, the initial response was characterized by a reconfiguration of the host transcriptome by differential exon usage, followed by a predominant down-regulation of the transcriptional activity possibly modulated by the host epigenetic changes associated to infection and characterized by an increased hypermethylation. The silencing of at least three cucumber transcripts potential targets of vd-sRNAs was also observed. The alteration in host sRNA and miRNA metabolism was marginal. We expect that these data constituting the first comprehensive map of the cucumber-response to HSVd could contribute to elucidate the molecular basis of the host alterations triggered by viroid infection.

Viroids are pathogenic non-coding RNAs that completely rely on their host molecular machinery to accomplish their life cycle. Several interactions between viroids and their host molecular machinery have been identified, including an interference with epigenetic mechanisms such as DNA methylation. Despite this, whether viroids influence changes in other epigenetic marks such as histone modifications remained unknown. Epigenetic regulation is particularly important during pathogenesis processes because it might be a key regulator of the dynamism of the defense response. Here we have analyzed the changes taking place in Cucumis sativus facultative and constitutive heterochromatin during hop stunt viroid (HSVd) infection using chromatin immunoprecipitation (ChIP) of the two main heterochromatic marks: H3K9me2 and H3K27me3. We find that HSVd infection is associated with changes in both H3K27me3 and H3K9me2, with a tendency to decrease the levels of repressive epigenetic marks through infection progression. These epigenetic changes are connected to the transcriptional regulation of their expected targets, genes and transposable elements. Indeed, several genes related to the defense response are targets of both epigenetic marks. Our results highlight another host regulatory mechanism affected by viroid infection, providing further information about the complexity of the multiple layers of interactions between pathogens/viroids and hosts/plants.