Different Dicer-like protein components required for intracellular and systemic antiviral silencing in <em>Arabidopsis thaliana</em>

Abstract

RNA silencing is a small RNA-mediated mechanism to down-regulate gene expression and is conserved in various eukaryotic organisms.¹ One of the most important roles of RNA silencing is to serve as an innate defense system against viral infection.² A vast number of genetic studies have uncovered various key protein components required for RNA silencing pathways. In antiviral silencing in plants, members of the plant Dicer-like (DCL) protein family play crucial roles along the pathway. First, they act as a primary sensor for recognizing double-stranded (ds) viral RNA and then process it into viral primary small interfering RNAs (siRNAs) that are loaded into an Argonaute (AGO)-containing effector complex to guide the cleavage of homologous viral RNAs. Next, to further intensify the antiviral silencing response, DCL proteins also biosynthesize viral secondary siRNAs from the dsRNA substrates produced by the activity of plant RNA-dependent RNA polymerases (RDRs) that convert viral single-stranded RNA into dsRNA.³

Plants encode more DCL, AGO and RDR proteins compared to other eukaryotes, thus allowing functional specification and a more diverse RNA silencing pathway.⁴ The model plant Arabidopsis thaliana contains 4 DCL genes.⁵ Except for DCL1, which primarily functions in the biosynthesis of micro-RNAs, the other 3 DCLs play direct roles in antiviral defense. While DCL3 particularly contributes to the antiviral response against DNA viruses through the generation of 24-nt viral siRNAs, DCL4 and DCL2 are involved in antiviral responses against RNA viruses. Earlier works showed that the antiviral function of DCL4 and DCL2 appear to be hierarchical or redundant, in which antiviral silencing is abolished only in the absence of both DCL4 and DCL2. Specifically, DCL4, which generates 21-nt viral siRNAs, functions as a primary DCL component, while DCL2, which generates 22-nt viral siRNAs, serves as a backup to functionally compensate for DCL4 when it is absent.^6-9

Depending on the virus-plant combination, viruses commonly spread throughout the entire plant from the primary infected tissue or organ.¹⁰ After virus transmission by the natural vector or mechanical inoculation, the virus initially infects a limited number epidermal or mesophyll cells and then spreads locally (cell-to-cell movement) to neighboring cells symplastically via plasmodesmata, crossing the mesophyll, bundle sheath and parenchyma cells to finally reach phloem cells. However, some viruses can be directly introduced into the phloem by insect vectors. Once the virus enters companion cells and sieve elements, the virus is systemically transported with the flow of photoassimilates to reach distal tissues and organs. The virus is then unloaded from sieve elements and establishes an infection at new sites. Thus, by way of these events, the virus is able to invade throughout the entire plant.^11,12

In a recent study, we performed 2 sets of experiments to demonstrate the differential requirement for DCL protein components in the intracellular and systemic antiviral defense against an RNA virus, potato virus X (PVX) in A. thaliana.¹³ In the first experiment, PVX RNA accumulation was examined in inoculated leaves, upper leaves and roots of wild type and dcl mutant plants following the mechanical inoculation of leaves. In inoculated leaves, inactivation of DCL4 alone was sufficient to enable the maximal levels of PVX RNA accumulation, whereas inactivation of both DCL4 and DCL2 was required for effective viral systemic infection in the upper leaves and roots. In the second experiment, we used transgenic A. thaliana carrying a replication-competent PVX cDNA transgene with GFP insertion (AMP243 line)¹⁴ that, due to the activity of intracellular antiviral silencing, is strongly suppressed in the cell. Introduction of the dcl allele into this transgenic plant by crossing showed that the absence of DCL4 but not DCL2 or DCL3 abolishes the suppression of PVX replication in various aerial organs. This indicates that DCL4 is the only essential DCL component for intracellular antiviral silencing against PVX in the aerial parts of the plant and could explain why the inactivation of DCL4 alone allows high levels of PVX accumulation in inoculated leaves. It was also observed that inactivation of DCL4 has a marginal effect on the suppression of PVX multiplication in the roots of AMP243 plants, suggesting that other DCL proteins (most likely DCL2) functionally compensate for DCL4 activity. Thus, functional redundancies among DCL proteins in antiviral defense are different between shoots and roots. Similar with the results of our studies, Garcia-Ruiz et al.,¹⁵ reported that DCL4 is sufficient for antiviral activity against the suppressor (HC-Pro)-defective turnip mosaic virus in inoculated leaves, whereas both DCL4 and DCL2 are required for the inhibition of viral systemic infection. The observation that DCL2 is dispensable for intracellular (local) antiviral silencing is corroborated by another study showing that DCL2-generated 22-nt viral siRNAs do not effectively mediate the defense against the suppressor (2b)-defective cucumber mosaic virus.¹⁶ Indeed, in line with this observation, we detected high levels of PVX RNA accumulation in inoculated leaves of dcl4 mutant plants, even though abundant 22-nt PVX siRNAs were present (Andika et al., unpublished result). Taken together, we propose a model in which DCL4 and DCL2 exhibit functional specification in the inhibition of intracellular (local) and systemic infection of the virus (Fig. 1).

Figure 1.

A cartoon illustration depicting different DCL protein components involved in intracellular (local) and systemic antiviral silencing against PVX in A. thaliana. DCL4 is sufficient and essential for the inhibition of viral multiplication in inoculated leaves (Inoc.), whereas DCL2 specifically functions in preventing viral systemic infection (Sys.), possibly during viral phloem transport and/or unloading from sieve elements. In roots, DCL4 is the primary DCL protein component involved in intracellular antiviral silencing, but can be functionally compensated for by DCL2 or possibly partially, DCL3.¹³

Given that viral systemic invasion could elicit detrimental effects in the host, plants may have evolved distinct defense mechanisms against viral systemic invasion, as exemplified by the function of some genes to inhibit the systemic movement of tobacco etch virus in A. thaliana.^17-19 Since viral systemic movement is a rather complex and prolonged process, the mechanism by which antiviral silencing blocks viral systemic movement is still unclear and needs to be further studied. The following observations argue against the notion that DCL2 may be specifically active in the phloem to prevent the entry or passage of viruses. First, we observed that PVX accumulated in the petiole of inoculated leaves in dcl4 mutants, suggesting that the virus can enter and accumulate in the vascular tissue even with the presence of DCL2.¹³ Second, PVX moved systemically in the form of virions,^20,21 which would be a recalcitrant target of RNA silencing-mediated degradation in the phloem. Third, a recent finding indicates that DCL2 is poorly expressed in the phloem.²² Thus, the manner by which DCL2 is specifically involved in the inhibition of viral systemic movement needs to be characterized in detail. Moreover, the contribution of other cellular factors to systemic antiviral silencing also warrants further investigation.