A single Argonaute gene is required for induction of RNA silencing antiviral defense and promotes viral RNA recombination

Abstract

Dicer gene dcl2, required for the RNA silencing antiviral defense response in the chestnut blight fungus Cryphonectria parasitica, is inducible upon mycovirus infection and promotes viral RNA recombination. We now report that the antiviral defense response requires only one of the four C. parasitica Argonaute-like protein genes, agl2. The agl2 gene is required for the virus-induced increase in dcl2 transcript accumulation. Agl2 and dcl2 transcripts accumulated to much higher levels in response to hairpin RNA production or infection by a mutant CHV1-EP713 hypovirus lacking the suppressor of RNA silencing p29 than to wild-type CHV1-EP713. Similar results were obtained for an agl2-promoter/EGFP-reporter construct, indicating that p29-mediated repression of agl2 transcript accumulation is promoter-dependent. Significantly, the agl2 deletion mutant exhibited stable maintenance of non-viral sequences in recombinant hypovirus RNA virus vectors and the absence of hypovirus-defective interfering (DI) RNA production. These results establish a key role for an Argonaute gene in the induction of an RNA silencing antiviral defense response and the promotion of viral RNA recombination. They also provide evidence for a mechanism by which a virus-encoded RNA silencing suppressor represses the transcriptional induction of an RNA silencing component.Cryphonectria parasitica ͉ mycovirus ͉ defective interfering RNA ͉ silencing suppressor ͉ RNA virus vector A n RNA-based antiviral defense response, related to RNA interference (1), serves as a key component of the innate immunity repertoire in plants, invertebrates, and fungi (2, 3). Common elements of this response across Kingdoms include the action of conserved ribonucleases: members of the Dicer-like and Argonaute-like protein families (4). Dicer nucleases recognize viral double-stranded (ds) and structured RNAs and use the associated RNase III-type activity to process these RNAs into small RNAs of 21-24 nts in length, termed virus-derived small (vs) RNAs. The vsRNAs are incorporated into an effector complex with the aid of an Argonaute family protein. One strand of the vsRNA is removed and the remaining guide strand then targets the effector complex to the cognate viral RNA, which is cleaved, or sliced, by the Argonaute-associated RNase H-like activity.Four Dicer proteins drive the RNA silencing pathways in the model plant, Arabidopsis thaliana (5). All four Dicers have been implicated in antiviral RNA silencing (3, 6-8). Studies with Drosophila melanogaster (9, 10) and with mosquitoes (11) have identified a role for Dicer-2, the Dicer involved in siRNA production, in insect antiviral defense. The contribution to antiviral defense of the second Dicer, Dicer-1, remains unclear due to its essential role in microRNA processing and development (12).Antiviral RNA silencing has been demonstrated in the filamentous Ascomycete fungi Cryphonectria parasitica, the chestnut blight fungus (13), and Aspergillus nidulans (14). Only one of the two C. parasitica Dicer genes, dcl2, was shown to be...

A Novel Victorivirus from a Phytopathogenic Fungus, Rosellinia necatrix, Is Infectious as Particles and Targeted by RNA Silencing

“…Nuss and coworkers demonstrated that the RNA silencing components DCL-2 and AGL-2 are required for the generation of CHV1 DI-RNA species (27,28), and our previous study showed that the existing DI-RNA in EP155 rarely moved to the ⌬dcl-2 recipient side along with helper CHV1 in the lateral transmission assay (17). In the present study, we confirmed this with a virus mutant, CHV1-⌬p69, during RnVV1 coinfection assay in which DI-RNAs appeared only in the EP155 host backgrounds (see the asterisked DI-RNAs in Fig.…”

“…It is known that CHV1 interferes with the host RNA silencing associated with the suppression of dcl-2 and agl-2 expression, possibly through the action of p29 of ORF A (26,27). We compared the levels of dcl-2 expression among EP155 colonies infected with different virus strains.…”

“…Like many other filamentous ascomycetous fungi (25), C. parasitica carries two Dicer-like genes (dcl-1 and dcl-2), four Argonaute-like genes (agl-1 to agl-4), and four RdRp genes (rdr-1 to rdr-4) as possible key RNA silencing components. Among these, dcl-2 and agl-2 are required for antiviral defense (26,27). The RNA silencing pathway is able to suppress replication of hypoviruses, mycoreoviruses (26,27), and also a heterologous partitivi-rus (family Partitiviridae) (17).…”

“…The RNA silencing pathway is able to suppress replication of hypoviruses, mycoreoviruses (26,27), and also a heterologous partitivi-rus (family Partitiviridae) (17). Furthermore, such RNA silencing is required for the generation and maintenance of defective interfering (DI)-RNAs of the prototype hypovirus, Cryphonectria hypovirus 1/EP713 (CHV1-EP713) (27,28).…”

The 21-Nucleotide, but Not 22-Nucleotide, Viral Secondary Small Interfering RNAs Direct Potent Antiviral Defense by Two Cooperative Argonautes inArabidopsis thaliana

“…The function of ARGONAUTE (AGO) proteins is clearly essential since genetic inactivation of a single AGO without altering dicing enhances virus susceptibility in fungal, insect, nematode, and plant hosts (Li et al, 2002;Morel et al, 2002;Lu et al, 2005Lu et al, , 2009Schott et al, 2005;Wilkins et al, 2005;van Rij et al, 2006;Zambon et al, 2006;Qu et al, 2008;Sun et al, 2009). In Arabidopsis, hypomorphic ago1 and null ago7 mutants accumulate higher levels of viral RNA, and AGO1, AGO2, and AGO5 bind to viral siRNAs in the infected cells, suggesting an antiviral role for these AGOs (Morel et al, 2002;Zhang et al, 2006;Qu et al, 2008;Takeda et al, 2008;Azevedo et al, 2010).…”