Small RNA modification associated with suppression of RNA silencing by tobamovirus replicase protein

Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plantdefense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production ofvirus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is aTMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotianabenthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production.On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggestingthat the viral silencing suppressor cannot revert silencing that is already established. Although previousevidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa smallreplicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connectthe silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirusinfection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). Wedemonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of smallRNAs and that this interference and the formation of virus-induced disease symptoms are linked to thesilencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interfereswith the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA.