Viroids are circular RNAs of minimal complexity compelled to subvert plant‐regulatory networks to accomplish their infectious process. Studies focused on the response to viroid‐infection have mostly addressed specific regulatory levels and considered specifics infection‐times. Thus, much remains to be done to understand the temporal evolution and complex nature of viroid‐host interactions. Here we present an integrative analysis of the temporal evolution of the genome‐wide alterations in cucumber plants infected with hop stunt viroid (HSVd) by integrating differential host transcriptome, sRNAnome and methylome. Our results support that HSVd promotes the redesign of the cucumber regulatory‐pathways predominantly affecting specific regulatory layers at different infection‐phases. The initial response was characterised by a reconfiguration of the host‐transcriptome by differential exon‐usage, followed by a progressive transcriptional downregulation modulated by epigenetic changes. Regarding endogenous small RNAs, the alterations were limited and mainly occurred at the late stage. Significant host‐alterations were predominantly related to the downregulation of transcripts involved in plant‐defence mechanisms, the restriction of pathogen‐movement and the systemic spreading of defence signals. We expect that these data constituting the first comprehensive temporal‐map of the plant‐regulatory alterations associated with HSVd infection could contribute to elucidate the molecular basis of the yet poorly known host‐response to viroid‐induced pathogenesis.

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.