Geminiviruses and viroids: it's all about methylation

Mixed infections are common in nature and can result in synergistic or antagonistic interactions. Studying mixed infections can be of help to untangle infection processes and defense responses. DNA methylation and post-transcriptional gene silencing are critical for the infection of geminiviruses and ssRNA viroids, respectively, and both pathogens can counteract these defense mechanisms to promote their infectivity. During a survey concerning the response of plants to double infection by geminiviruses and viroids, we found that when plants were experimentally co-inoculated with Tomato yellow leaf curl Sardinia virus (TYLCSV) and Potato spindle tuber viroid (PSTVd), TYLCSV infectivity and accumulation were strongly reduced, indicative of an antagonistic action of PSTVd. A similar antagonistic interaction also occurred towards other geminiviruses.Using methylation-sensitive restriction enzymes and bisulfite conversion assays, we discovered that PSTVd promoted a strong hyper methylation of TYLCSV DNA in tomato plants co-infected by both pathogens. Concomitantly, PSTVd alone or in double infection with TYLCSV significantly upregulated the expression of key genes governing DNA methylation in plants, thus supporting a mechanistic link with the antagonism of the viroid on the virus during co-infection. This study opens new perspectives concerning the outcome of multiple biotic stresses in plants and may reveal novel regulatory networks in the interplay between two nuclear-replicating pathogens and their host.