Potato spindle tuber viroid upregulates DNA methylation-related genes and antagonizes the infectivity and the accumulation of a Geminivirus

Tomato is a natural host of Potato spindle tuber viroid (PSTVd) and Tomato yellow leaf curl Sardinia virus (TYLCSV), which are representative members of pospiviroids (infectious non-coding circular RNAs) and geminiviruses (single-stranded DNA viruses), respectively. While molecular events during infection have been explored separately for each one of these two nuclear replicating pathogens, plant responses during mixed infections are unknown. In this context, dissection of DNA methylation pathway is particularly interesting because it is well known that plants may methylate viral DNA to impair geminivirus infection, while whether viroids interfere with host DNA methylation pathways is unknown. Exploiting an experimental system based on PSTVd and TYLCSV co-infecting the same tomato plant, and applying qRT-PCR, methylation-sensitive restriction and bisulfite conversion assays, we found that: i) when plants were co-infected, TYLCSV infectivity and accumulation were strongly impaired, indicating an antagonistic action of PSTVd; ii) PSTVd alone or in double infection with TYLCSV significantly upregulated the expression of key genes governing DNA methylation in plants; iii) PSTVd promoted a strong hypermethylation of TYLCSV DNA in tomato plants co-infected by both pathogens, thus supporting a mechanistic link with the antagonism of the viroid on the virus during co-infection. Besides providing the first solid evidence that a viroid may interfere with host regulatory networks involved in DNA methylation, these data open new perspectives on the possible involvement of viroid-induced epigenetic changes in plant responses against other biotic and abiotic stresses.