Characterization of Hypovirus-Derived Small RNAs Generated in the Chestnut Blight Fungus by an Inducible DCL-2-Dependent Pathway

Abstract

The disruption of one of two dicer genes, dcl-2, of the chestnut blight fungus Cryphonectria parasitica was recently shown to increase susceptibility to mycovirus infection (G. C. Segers, X. Zhang, F. Deng, Q. Sun, and D. L. Nuss, Proc. Natl. Acad. Sci. USA 104:12902-12906, 2007). We now report the accumulation of virusderived small RNAs (vsRNAs) in hypovirus CHV1-EP713-infected wild-type and dicer gene dcl-1 mutant C. parasitica strains but not in hypovirus-infected dcl-2 mutant and dcl-1 dcl-2 double-mutant strains. The CHV1-EP713 vsRNAs were produced from both the positive and negative viral RNA strands at a ratio of 3:2 in a nonrandom distribution along the viral genome. We also show that C. parasitica responds to hypovirus and mycoreovirus infections with a significant increase (12-to 20-fold) in dcl-2 expression while the expression of dcl-1 is increased only modestly (2-fold). The expression of dcl-2 is further increased (ϳ35-fold) following infection with a hypovirus CHV1-EP713 mutant that lacks the p29 suppressor of RNA silencing. The combined results demonstrate the biogenesis of mycovirus-derived small RNAs in a fungal host through the action of a specific dicer gene, dcl-2. They also reveal that dcl-2 expression is significantly induced in response to mycovirus infection by a mechanism that appears to be repressed by the hypovirus-encoded p29 suppressor of RNA silencing.RNA silencing, the RNA-mediated, sequence-specific suppression of gene expression, in animals, plants, and fungi has been described previously (21,45,51,64). The basic elements of the RNA-silencing pathways are conserved across kingdoms and involve the processing of structured or double-stranded RNA (dsRNA) into short duplex RNAs of 21 to 24 nucleotides (nt) by RNase III-like endonucleases termed dicers. With the aid of the Argonaute family of proteins, these short RNA duplexes are then incorporated into RNA-induced silencing complexes (RISC) that guide the sequence-specific cleavage or translational repression of homologous RNA molecules or transcriptional silencing through the modification of homologous DNA (reviewed in references 63 and 68).RNA silencing serves a crucial role as an antiviral defense mechanism in plants (reviewed in references 16, 17, 37, and 65). Multiple mechanisms appear to be available for the generation of virus-specific small RNAs (vsRNAs) that guide the subsequent degradation of cognate viral RNA in plants. vsRNAs are derived primarily from highly structured regions of viral single-stranded RNA in the cases of Cymbidium ringspot virus (43), Turnip crinkle virus (27), Potato virus X (43), and Tobacco mosaic virus (43). In contrast, vsRNAs are derived equally from both strands in the cases of Turnip mosaic virus (27) and Cucumber yellows virus (67), indicating the processing of complementary viral dsRNAs. vsRNAs are also generated from branched stem-loop regions of 35S leader RNA produced by the plant DNA virus Cauliflower mosaic virus (42) and from structured regions of viroid RNA (29). The clarification of th...

Multiple approaches for the detection and characterization of viral and plasmid symbionts from a collection of marine fungi

“…In facts, as already shown by the results obtained with different techniques using exactly the same RNA sample as template, in the isolate MUT4330, the majority of viruses are detectable with the sRNA technique but one, the PaBv1, seems to be able to escape almost completely the quelling machinery since only for this virus, the number of sequenced sRNA was minimal ( Table 2). Accumulation of vsRNA is characteristic of RNA silencing-based antiviral defense, but in case of dicer-like or argonaute mutants the accumulation of this vsRNA is greatly affected (Segers et al, 2007;Zhang et al, 2008). Moreover some fungi have completely lost the genes for the RNA silencing machinery but do not seem to suffer high incidence of virus-induced symptoms suggesting the possibility of additional and yet uncharacterized mechanism used by fungi to mitigate viral replication (Nakayashiki and Nguyen, 2008).…”

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

“…In this antiviral defense, small interfering RNAs (siRNAs) are processed from a viral RNA precursor and used to guide specific silencing of the cognate viral RNAs in an Argonaute-containing effector complex. In fruit flies, nematodes, and fungi, doublestranded RNA (dsRNA) replicative intermediates of viral RNA genomes are recognized and processed into virus-derived siRNAs by a single host Dicer (Schott et al, 2005;Wilkins et al, 2005;Galiana-Arnoux et al, 2006;Wang et al, 2006;Aliyari et al, 2008;Zhang et al, 2008;Lu et al, 2009;Wu et al, 2010). However, production of viral siRNAs in plants involves multiple Dicer-like proteins (DCLs) (Blevins et al, 2006;Bouché et al, 2006;Deleris et al, 2006;Fusaro et al, 2006;Moissiard and Voinnet, 2006;Diaz-Pendon et al, 2007).…”

A host dicer is required for defective viral RNA production and recombinant virus vector RNA instability for a positive sense RNA virus

“…We subsequently showed that dcl-2 functions to process mycovirus RNAs into virus-derived small interfering RNAs (vsRNAs) as part of an inducible RNA silencing antiviral response (15). Cloning and characterization of vsRNAs generated from hypovirus CHV1-EP713 revealed a nonrandom distribution of vsRNAs along the 12.7-kb genome RNA.…”

“…Cloning and characterization of vsRNAs generated from hypovirus CHV1-EP713 revealed a nonrandom distribution of vsRNAs along the 12.7-kb genome RNA. Conspicuous was the very low representation of vsRNA mapping to an internal portion of the 12.7-kb genome RNA that encodes the viral polymerase and helicase domains, from map position 7500 to 11000 (15). It has been postulated that the propensity of hypoviruses to generate internally deleted DI RNAs results in a lower level of substrate for vsRNA biogenesis from this region (15).…”

“…Conspicuous was the very low representation of vsRNA mapping to an internal portion of the 12.7-kb genome RNA that encodes the viral polymerase and helicase domains, from map position 7500 to 11000 (15). It has been postulated that the propensity of hypoviruses to generate internally deleted DI RNAs results in a lower level of substrate for vsRNA biogenesis from this region (15). Here we confirm that the region extending from map position 7348 to 11267 is absent in the DI RNA population.…”