Study of DNA methylation changes upon PSTVd infection of N. benthamiana. Investigating epigenetic phenomena in PSTVd infected Nicotiana benthamiana plants

Viroids are small circular RNAs infecting a wide range of plants. They do not encode for any protein or peptide therefore they rely in their structure for their biological cycle. Observed phenotypes of viroid infected plants are thought to occur through changes at the transcriptional/translational level of the host. A mechanism involved in such changes is RNA-directed DNA methylation (RdDM). Till today, there are contradictory works about viroids interference of RdDM. In this study we investigated the epigenetic effect of viroid infection in Nicotiana benthamiana plants. Using potato spindle tuber viroid (PSTVd) as the triggering pathogen and via bioinformatic analyses we identified endogenous gene promoters and transposable elements targeted by 24 nt host siRNAs that differentially accumulated in PSTVd-infected and healthy plants. The methylation status of these targets was evaluated following digestion with methylation-sensitive restriction enzymes coupled with PCR amplification, and bisulfite sequencing. In addition, we used Methylation Sensitive Amplification Polymorphism (MSAP) followed by sequencing (MSAP-seq) to study global DNA methylation upon infection. The study indicated that although a small number of targets are differentially methylated upon PSTVd infection, the overall methylation status did not significantly change. These results enhance our understanding on the epigenetic host changes as a result of Pospiviroid infection.