Mycoreovirus genome rearrangements associated with RNA silencing deficiency

Abstract

Mycoreovirus 1 (MyRV1) has 11 double-stranded RNA genome segments (S1 to S11) and confers hypovirulence to the chestnut blight fungus, Cryphonectria parasitica. MyRV1 genome rearrangements are frequently generated by a multifunctional protein, p29, encoded by a positive-strand RNA virus, Cryphonectria hypovirus 1. One of its functional roles is RNA silencing suppression. Here, we explored a possible link between MyRV1 genome rearrangements and the host RNA silencing pathway using wild-type (WT) and mutant strains of both MyRV1 and the host fungus. Host strains included deletion mutants of RNA silencing components such as dicer-like (dcl) and argonaute-like (agl) genes, while virus strains included an S4 internal deletion mutant MyRV1/S4ss. Consequently, intragenic rearrangements with nearly complete duplication of the three largest segments, i.e. S1, S2 and S3, were observed even more frequently in the RNA silencing-deficient strains Δdcl2 and Δagl2 infected with MyRV1/S4ss, but not with any other viral/host strain combinations. An interesting difference was noted between genome rearrangement events in the two host strains, i.e. generation of the rearrangement required prolonged culture for Δagl2 in comparison with Δdcl2. These results suggest a role for RNA silencing that suppresses genome rearrangements of a dsRNA virus.

Highly activated RNA silencing via strong induction of dicer by one virus can interfere with the replication of an unrelated virus

“…Various interactions have been reported in the C. parasitica/virus system: one-way synergism between CHV1 [nonsegmented (+) RNA genome] and MyRV1 (11-segmented dsRNA genome) (36), and the generation of MyRV1 genome rearrangements by CHV1 coinfection; transgenic expression of its RSS, p29; or deletion of host dcl2 or agl2 (37,38). This study focused on different virus/ virus interplays involving RnVV1 (undivided dsRNA genome) and showed that (i) MyRV1 and an RSS-lacking CHV1 mutant, CHV1-Δp69, interfere with the replication and lateral transmission of unrelated RnVV1 in WT C. parasitica; (ii) this interference is also mediated by transgene-derived dsRNA; (iii) disruption of dcl2 completely abolished the interference, whereas disruption of agl2 did not always do so; and (iv) the interference coincided with high levels of accumulation of the dcl2 and agl2 transcripts.…”

Novel, diverse RNA viruses from Mediterranean isolates of the phytopathogenic fungus, Rosellinia necatrix: insights into evolutionary biology of fungal viruses

“…Total nucleic acids were extracted from the mycelia of fungal strains cultured in 20 ml PDB and dsRNA fractions were isolated as described by (Eusebio‐Cope and Suzuki, ). All strains were cultured on PDA overlaid with cellophane (PDA‐cellophane) until they covered the plate.…”

SAGA complex mediates the transcriptional up-regulation of antiviral RNA silencing

“…Total RNA and genomic DNA were extracted as described previously (56). Identification of genomic regions flanked a NeoR cassette sequence was performed using fusion primer and nested integrated-PCR method, as described previously (21).…”

“…Total RNA was isolated from two fungal strains, DK80 and Δsgf73, infected with CHV1-Δp69, as described by EusebioCope and Suzuki (56). Small RNA cDNA library construction and sequencing were performed by Macrogen Japan using the Illumina TruSeq Small RNA Library Preparation Kit and the HiSeq 2000 system (50-bp single-end reads).…”