Modification of Small RNAs Associated with Suppression of RNA Silencing by Tobamovirus Replicase Protein

Abstract

Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, 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, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA.RNA silencing is a posttranscriptional, RNA-guided, gene regulatory mechanism that operates through RNA-mediated sequence-specific interactions in the cytoplasm of eukaryotes, including plants (5,47,57).RNA silencing is generally induced by double-stranded RNA (dsRNA), which can originate from various sources, such as transgenes, viral replication intermediates, or experimentally introduced dsRNA sequences. Central to the silencing process are dicers or "dicer-like" enzymes that cleave dsRNA into small double-stranded fragments, called small interfering RNAs (siRNAs). Single-stranded siRNAs are then incorporated into multicomponent RNA-induced silencing complexes (RISC), which contain an "argonaute" (AGO) family protein (in plants usually AGO1) (3) and inactivate homologous RNA through endonucleolytic cleavage. In addition to siRNAs, which are usually derived from foreign elements such as transgenes and viruses, other small RNA (sRNA) species are encoded by specific noncoding RNA genes. Among these, micro-RNAs (miRNAs) have predominant roles during plant development (28) and are processed from miRNA precursors encoded by miRNA genes. Similarly to siRNAs, miRNAs are incorporated into AGO-containing RISC complexes to guide the recognition of target RNAs. In plants, miRNA-RISC complexes usually cause target RNA cleavage, whereas in most mammalian cases miRNA-RISC inhibits translation of target mRNA (37). Plant siRNAs and miRNAs (commonly referred to as sRNAs) ...

La bataille du silence

“…En effet, les vsARN sont, comme leurs homologues endogènes, stabilisés par l'ajout d'un groupement méthyl à leur extrémité 3', catalysé par la protéine HEN1. L'inhibition de l'activité de cette méthyltransférase, comme cela a été démon-tré entre autres pour la protéine p122 des tobamovirus [37], augmentera donc potentiellement la vitesse de dégradation des vsARN. Une autre stratégie pour supprimer le RNA silencing repose sur la séquestration des sARN, empêchant ainsi leur incorporation dans le complexe RISC.…”

Modulation of host plant immunity by Tobamovirus proteins

“…The silencing suppressing activity is located in the viral 126 kDa small replicase subunit by interfering with HEN1-mediated methylation of small RNAs. This interference is closely associated with the formation of disease symptoms (Vogler et al, 2007). It was also reported that the transgenic expression of the 126 kDa replicase subunit in N. tabacum produced enhanced susceptibility to several viruses (Harries et al, 2008).…”

“…These alterations underlie many disease symptoms typically observed during plant viral infections (Siddiqui et al, 2008;Bazzini et al, 2011;Shimura and Pantaleo, 2011). Thus, the production of symptoms due to the effect of VSRs, including TMV replicase, has been associated with side effects over off-target components shared between pathways involved in silencing mediated by miRNA/trans-acting siRNAs (tasiRNAs) and vsiRNAs (Yu et al, 2006;Vogler et al, 2007).…”

Attenuated plant viruses: preventing virus diseases and understanding the molecular mechanism

“…The propagation of both L and L 11 were inhibited in this NtTOM1-overexpressing line, implying that silencing suppression was apparently caused by the 130-kDa protein in the soluble fraction (HagiwaraKomoda et al 2008). The molecular mechanism underlying the silencing suppression by the tobamovirus 130-kDa protein (replicase protein) (including the 122-kDa protein of cr-TMV and the 126-kDa protein of TMV) is postulated to prevent formation of the RNA-induced silencing complex (RISC) after siRNA and miRNA syntheses (Csorba et al 2007;Ding et al 2004;Kubota et al 2003;Vogler et al 2007). The 130-kDa protein was found to bind to double-stranded RNA (dsRNA) but not to single-stranded Only amino acid substitutions are described on the genome RNA.…”