A pathogenic long noncoding RNA redesigns the epigenetic landscape of the infected cells by subverting host Histone Deacetylase 6 activity.

Viroids - ancient plant-pathogenic long noncoding RNAs - have developed a singular evolutionary strategy based on reprogramming specific phases of host-metabolism to ensure that their infection cycle can be completed in infected cells. However, the molecular aspects governing this transregulatory phenomenon remain elusive. Here, we use immunoprecipitation assays and bisulfite sequencing of rDNA to shown that, in infected cucumber and Nicotiana benthamina plants, Hop stunt viroid (HSVd) recruits and functionally subverts Histone Deacetylase 6 (HDA6) to promote host-epigenetic alterations that trigger the transcriptional alterations observed during viroid pathogenesis. This notion is supported by the demonstration that, during infection, the HSVd-HDA6 complex occurs in vivo and that endogenous HDA6 expression is increased in HSVd-infected cells. Moreover, transient overexpression of recombinant HDA6 reverts the hypomethylation status of rDNA observed in HSVd-infected plants and reduces viroid accumulation. We hypothesize that the host-transcriptional alterations induced as a consequence of viroid-mediated HDA6 recruitment favor spurious recognition of HSVd-RNA as an RNA Pol II template, thereby improving viroid replication. Our results constitute the first description of a physical and functional interaction between a pathogenic RNA and a component of the host RNA silencing mechanism, providing novel evidence of the potential of these pathogenic lncRNAs to physically redesign the host-cell environment and reprogram their regulatory mechanisms.

Changes in the DNA methylation pattern of the host male gametophyte of viroid-infected cucumber plants.

Eukaryotic organisms exposed to adverse conditions are required to show a certain degree of transcriptional plasticity in order to cope successfully with stress. Epigenetic regulation of the genome is a key regulatory mechanism allowing dynamic changes of the transcriptional status of the plant in response to stress. The Hop stunt viroid (HSVd) induces the demethylation of ribosomal RNA (rRNA) in cucumber (Cucumis sativus) leaves, leading to increasing transcription rates of rRNA. In addition to the clear alterations observed in vegetative tissues, HSVd infection is also associated with drastic changes in gametophyte development. To examine the basis of viroid-induced alterations in reproductive tissues, we analysed the cellular and molecular consequences of HSVd infection in the male gametophyte of cucumber plants. Our results indicate that in the pollen grain, accumulation of HSVd RNA induces a decondensation of the generative nucleus that correlates with a dynamic demethylation of repetitive regions in the cucumber genome that include rRNA genes and transposable elements (TEs). We therefore propose that HSVd infection impairs the epigenetic control of rRNA genes and TEs in gametic cells of cucumber, a phenomenon thus far unknown to occur in this reproductive tissue as a consequence of pathogen infection.

A pathogenic non-coding RNA induces changes in dynamic DNA methylation of ribosomal RNA genes in host plants.

Viroids are plant-pathogenic non-coding RNAs able to interfere with as yet poorly known host-regulatory pathways and to cause alterations recognized as diseases. The way in which these RNAs coerce the host to express symptoms remains to be totally deciphered. In recent years, diverse studies have proposed a close interplay between viroid-induced pathogenesis and RNA silencing, supporting the belief that viroid-derived small RNAs mediate the post-transcriptional cleavage of endogenous mRNAs by acting as elicitors of symptoms expression. Although the evidence supporting the role of viroid-derived small RNAs in pathogenesis is robust, the possibility that this phenomenon can be a more complex process, also involving viroid-induced alterations in plant gene expression at transcriptional levels, has been considered. Here we show that plants infected with the 'Hop stunt viroid' accumulate high levels of sRNAs derived from ribosomal transcripts. This effect was correlated with an increase in the transcription of ribosomal RNA (rRNA) precursors during infection. We observed that the transcriptional reactivation of rRNA genes correlates with a modification of DNA methylation in their promoter region and revealed that some rRNA genes are demethylated and transcriptionally reactivated during infection. This study reports a previously unknown mechanism associated with viroid (or any other pathogenic RNA) infection in plants providing new insights into aspects of host alterations induced by the viroid infectious cycle.