Hypovirus Papain-Like Protease p29 Suppresses RNA Silencing in the Natural Fungal Host and in a Heterologous Plant System

Abstract

Virulence-attenuating hypoviruses of the species Cryphonectria hypovirus 1 (CHV1) encode a papain-like protease, p29, that shares similarities with the potyvirus-encoded suppressor of RNA silencing HC-Pro. We now report that hypovirus CHV1-EP713-encoded p29 can suppress RNA silencing in the natural host, the chestnut blight fungus Cryphonectria parasitica. Hairpin RNA-triggered silencing was suppressed in C. parasitica strains expressing p29, and transformation of a transgenic green fluorescent protein (GFP)-silenced strain with p29 resulted in an increased number of transformants with elevated GFP expression levels. The CHV1-EP713 p29 protein was also shown to suppress both virus-induced and agroinfiltration-induced RNA silencing and systemic spread of silencing in GFP-expressing transgenic Nicotiana benthamiana line 16c plants. The demonstration that a mycovirus encodes a suppressor of RNA silencing provides circumstantial evidence that RNA silencing in fungi may serve as an antiviral defense mechanism. The observation that a phylogenetically conserved protein of related plant and fungal viruses functions as a suppressor of RNA silencing in both fungi and plants indicates a level of conservation of the mechanisms underlying RNA silencing in these two groups of organisms.RNA-mediated, sequence-specific silencing of gene expression, termed RNA silencing, has been reported for plants (33,48), fungi (39), and animals (16) and variously referred to as posttranscriptional gene silencing, quelling, and RNA interference, respectively. A common feature of RNA silencing is the processing of structured or double-stranded RNA into small interfering RNAs (siRNAs) of 21 to 25 nucleotides by members of the RNase III family of double-stranded RNA-specific endonucleases (Dicers). These siRNAs are then incorporated into an RNA-induced silencing complex that guides sequencespecific degradation of homologous RNA (reviewed in reference 56).RNA silencing plays a key antiviral defense role in plants (reviewed in reference 52) and has been demonstrated to influence virus replication in animal cells (31). Viruses, in turn, produce proteins capable of suppressing host cell RNA silencing (reviewed in reference 46). RNA silencing is part of a larger set of regulatory pathways involving small RNAs that include microRNA (miRNA)-mediated developmental regulation in plants and animals (reviewed in reference 20). Recent findings (29) indicate that some viral suppressors of RNA silencing also contribute to virus-induced disease symptoms by interfering with miRNA-controlled developmental pathways.Although RNA silencing in fungi is generally portrayed as having originated as an ancient antiviral defense mechanism, there is currently no experimental evidence to support this assumption. Our understanding of RNA silencing in fungi comes primarily from studies with the model filamentous fungus Neurospora crassa (4, 6, 11). However, no experimental virus system is available for this organism. In this regard, the chestnut blight fungus, Cryphonectria ...

Virus World as an Evolutionary Network of Viruses and Capsidless Selfish Elements

“…Moreover, hypoviruses and potyviruses are unique among the members of the picornavirus-like superfamily in possessing a superfamily 2 helicase instead of a superfamily 3 helicase as is typical of this vast virus group (23). Both potyviral and hypoviral proteases are involved in suppression of the host RNA interference defense (55,56), in agreement with the common ancestry of these virus families.…”

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

“…Only one of the two C. parasitica Dicer genes, dcl2, was shown to be required for antiviral RNA silencing (13) and the production of vsRNAs (15), while the second Dicer gene, dcl1, was shown to be dispensable. Expression of the C. parasitica dcl2 gene was also shown to be significantly induced in response to mycovirus infection by a mechanism that was repressed (15) by the hypovirus CHV1-EP713-encoded suppressor of RNA silencing p29 (16). A similar induction and repression of Dicer gene expression has not yet been reported for infected plants or animals.…”

“…Plasmid pGSN2GFP is similar to the hairpin structure producing plasmid pGS-2GFP (described in ref. 16), except that it has a neomycin resistance gene cassette in place of the benomyl resistance cassette (31). Neomycin-resistant transformants were selected on G418-containing PDA plates and single spore isolates were obtained for each transformant and grown on PDA (20 mL/plate) for 5 days.…”

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

“…A one-way synergistic interaction between the prototypes of hypovirus and mycoreovirus (CHV1 and MyRV1) has been noted (Sun et al [16]). The mechanism of this synergism involves elevation of the MyRV1 accumulation level by CHV1, conferred by a multifunctional hypoviral RNA silencing suppressor, p29, which was the first of its kind identified (56). Thus, it was anticipated that the very low level of RnVV1 in EP155 might be similarly elevated by coinfection with CHV1.…”