Antiviral Immunity Directed by Small RNAs

Plants and invertebrates can protect themselves from viral infection through RNA silencing. This antiviral immunity involves production of virus-derived small interfering RNAs (viRNAs) and results in specific silencing of viruses by viRNA-guided effector complexes. The proteins required for viRNA production as well as several key downstream components of the antiviral immunity pathway have been identified in plants, flies, and worms. Meanwhile, viral mechanisms to suppress this small RNA-directed immunity by viruses are being elucidated, thereby illuminating an ongoing molecular arms race that likely impacts the evolution of both viral and host genomes

Biochemical and genetic functional dissection of the P38 viral suppressor of RNA silencing

Phytoviruses encode viral suppressors of RNA silencing (VSRs) to counteract the plant antiviral silencing response, which relies on virus-derived small interfering (si)RNAs processed by Dicer RNaseIII enzymes and subsequently loaded into ARGONAUTE (AGO) effector proteins. Here, a tobacco cell-free system was engineered to recapitulate the key steps of antiviral RNA silencing and, in particular, the most upstream double-stranded (ds)RNA processing reaction, not kinetically investigated thus far in the context of plant VSR studies. Comparative biochemical analyses of distinct VSRs in the reconstituted assay showed that in all cases tested, VSR interactions with siRNA duplexes inhibited the loading, but not the activity, of antiviral AGO1 and AGO2. Turnip crinkle virus P38 displayed the additional and unique property to bind both synthetic and RNA-dependent-RNA-polymerase-generated long dsRNAs, and inhibited the processing into siRNAs. Single amino acid substitutions in P38 could dissociate dsRNA-processing from AGO-loading inhibition in vitro and in vivo, illustrating dual-inhibitory strategies discriminatively deployed within a single viral protein, which, we further show, are bona fide suppressor functions that evolved independently of the conserved coat protein function of P38.ISSN:1355-8382ISSN:1469-900

Molecular analysis of post-transcriptional gene silencing: mechanisms and roles

This work is an investigation of post-transcriptional gene silencing (PTGS) in plants, a process that mediates sequence-specific degradation of RNA. Initially discovered in transgenic plants, PTGS has been long regarded as a curiosity, or even as an artefact of transgenesis. It is shown here that virus-induced gene silencing, in which recombinant viruses carrying element of the host genome trigger PTGS of the corresponding plant gene (Chapter one), is a manifestation of a defence system. This defence is remarkable in its ability to adapt to potentially any virus because its specificity is not genetically programmed by the host but, instead, is dictated by the genome sequence of the viral intruder itself. It is demonstrated in chapters 4 and 5 that PTGS of a transgene can spread in plants from one part to another, indicating the existence of a systemic, sequences-specific silencing signal that is likely to have a nucleic acid component. From the demonstration that replication of potato-virus X also triggers production of a silencing signal in non-transgenic plants (Chapter 8), it is proposed that this long-distance signalling process represents the systemic arm of the host PTGS defence response. Collectively, these findings define the existence of a previously uncharacterised antiviral mechanism in higher plants, which may also operate in animals. This defence holds key features of an elaborate immune system, as it is adaptive, mobile and specific. It is also shown, here, that plant viruses have elaborated counter-defensive measures to overcome the host PTGS response, by producing suppressor proteins that target various steps of the silencing mechanism (Chapters 6, 7). One of these factors, the PYX-encoded p25 protein, had been previously characterised as a facilitator of viral cell-to-cell movement. The finding that p25 specifically inhibits the signalling step of PTGS (Chapter 8) provides a new ground for the investigation of virus movement in plants. In chapter 9, the role of PTGS in plants and its suppression by viruses is discussed in the broader context of plant development and biotechnological applications

Grafting the Way to the Systemic Silencing Signal in Plants

Grafting is a powerful but complex means to study the spread of RNA silencin

A mobile signal transported over a long distance induces systemic transcriptional gene silencing in a grafted partner

Transcriptional gene silencing (TGS) can be induced by promoter-targeted small interfering RNA (siRNA). Long-distance transmission of TGS by viral infection in plants has been reported. However, systemic TGS has not been observed in the case of using an inverted repeat transgene as the silencing trigger. Here it is reported that a mobile signal, presumably the siRNA, produced from a hairpin structure transgene controlled by a companion cell-specific promoter can also induce transmissible TGS in both a modified agroinfiltration and a grafting system. Although the transmissible TGS occurred only in cells located in the vicinity of a leaf vein in the scion, very strong silencing was observed in the root system, especially the lateral roots, including the root apical meristem. The transmissible TGS was maintained through tissue culture and subsequently inherited by the progeny. The results suggest the potential application of mobile promoter-targeting siRNA in horticulture for improvement of plant cultivars by grafting

Functional Analysis of Gene-Silencing Suppressors from Tomato Yellow Leaf Curl Disease Viruses

Tomato yellow leaf curl disease (TYLCD) is caused by a complex of phylogenetically related Begomovirus spp. that produce similar symptoms when they infect tomato plants but have different host ranges. In this work, we have evaluated the gene-silencing-suppression activity of C2, C4, and V2 viral proteins isolated from the four main TYLCDcausing strains in Spain in Nicotiana benthamiana. We observed varying degrees of local silencing suppression for each viral protein tested, with V2 proteins from all four viruses exhibiting the strongest suppression activity. None of the suppressors were able to avoid the spread of the systemic silencing, although most produced a delay. In order to test the silencing-suppression activity of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) proteins in a shared (tomato) and nonshared (bean) host, we established novel patch assays. Using these tools, we found that viral proteins from TYLCV were able to suppress silencing in both hosts, whereas TYLCSV proteins were only effective in tomato. This is the first time that viral suppressors from a complex of diseasecausing geminiviruses have been subject to a comprehensive analysis using two economically important crop hosts, as well as the established N. benthamiana plant model

Asymmetric expression of Argonautes in reproductive tissues

The Arabidopsis genome encodes ten Argonautes proteins showing distinct expression pattern as well as intracellular localisation during sexual reproduction