Genetic evidence for the involvement of Dicer-like 2 and 4 as well as Argonaute 2 in the Nicotiana benthamiana response against Pelargonium line pattern virus.

In plants, RNA silencing functions as a potent antiviral mechanism. Virus-derived double-stranded RNAs (dsRNAs) trigger this mechanism, being cleaved by Dicer-like (DCL) enzymes into virus small RNAs (vsRNAs). These vsRNAs guide sequence-specific RNA degradation upon their incorporation into an RNA-induced silencing complex (RISC) that contains a slicer of the Argonaute (AGO) family. Host RNA dependent-RNA polymerases, particularly RDR6, strengthen antiviral silencing by generating more dsRNA templates from RISC-cleavage products that, in turn, are converted into secondary vsRNAs by DCLs. Previous work showed that Pelargonium line pattern virus (PLPV) is a very efficient inducer and target of RNA silencing as PLPV-infected Nicotiana benthamiana plants accumulate extraordinarily high amounts of vsRNAs that, strikingly, are independent of RDR6 activity. Several scenarios may explain these observations including a major contribution of dicing versus slicing for defence against PLPV, as the dicing step would not be affected by the RNA silencing suppressor encoded by the virus, a protein that acts via vsRNA sequestration. Taking advantage of the availability of lines of N. benthamiana with DCL or AGO2 functions impaired, here we have tried to get further insights into the components of the silencing machinery that are involved in anti-PLPV-silencing. Results have shown that DCL4 and, to lesser extent, DCL2 contribute to restrict viral infection. Interestingly, AGO2 apparently makes even a higher contribution in the defence against PLPV, extending the number of viruses that are affected by this particular slicer. The data support that both dicing and slicing activities participate in the host race against PLPV.

A Viral Suppressor of RNA Silencing May Be Targeting a Plant Defence Pathway Involving Fibrillarin.

To establish productive infections, viruses must be able both to subdue the host metabolism for their own benefit and to counteract host defences. This frequently results in the establishment of viral-host protein-protein interactions that may have either proviral or antiviral functions. The study of such interactions is essential for understanding the virus-host interplay. Plant viruses with RNA genomes are typically translated, replicated, and encapsidated in the cytoplasm of infected cells. Despite this, a significant array of their encoded proteins has been reported to enter the nucleus, often showing high accumulation at subnuclear structures such as the nucleolus and/or Cajal bodies. However, the biological significance of such a distribution pattern is frequently unknown. Here, we explored whether the nucleolar/Cajal body localization of protein p37 of Pelargonium line pattern virus (PLPV, genus Pelarspovirus, family Tombusviridae), might be related to potential interactions with the nucleolar/Cajal body marker proteins, fibrillarin and coilin. The results revealed that p37, which has a dual role as coat protein and as suppressor of RNA silencing, a major antiviral system in plants, is able to associate with these cellular factors. Analysis of (wildtype and/or mutant) PLPV accumulation in plants with up- or downregulated levels of fibrillarin or coilin have suggested that the former might be involved in an as yet unknown antiviral pathway, which may be targeted by p37. The results suggest that the growing number of functions uncovered for fibrillarin can be wider and may prompt future investigations to unveil the plant antiviral responses in which this key nucleolar component may take part.