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

Abstract

Japan bA novel victorivirus, termed Rosellinia necatrix victorivirus 1 (RnVV1), was isolated from a plant pathogenic ascomycete, white root rot fungus Rosellinia necatrix, coinfected with a partitivirus. The virus was molecularly and biologically characterized using the natural and experimental hosts (chestnut blight fungus, Cryphonectria parasitica). RnVV1 was shown to have typical molecular victorivirus attributes, including a monopartite double-stranded RNA genome with two open reading frames (ORFs) encoding capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), a UAAUG pentamer presumed to facilitate the coupled termination/reinitiation for translation of the two ORFs, a spherical particle structure ϳ40 nm in diameter, and moderate levels of CP and RdRp sequence identity (34 to 58%) to those of members of the genus Victorivirus within the family Totiviridae. A reproducible transfection system with purified RnVV1 virions was developed for the two distinct fungal hosts. Transfection assay with purified RnVV1 virions combined with virus elimination by hyphal tipping showed that the effects of RnVV1 on the phenotype of the natural host were negligible. Interestingly, comparison of the RNA silencing-competent (standard strain EP155) and -defective (⌬dcl-2) strains of C. parasitica infected with RnVV1 showed that RNA silencing acted against the virus to repress its replication, which was restored by coinfection with hypovirus or transgenic expression of an RNA silencing suppressor, hypovirus p29. Phenotypic changes were observed in the ⌬dcl-2 strain but not in EP155. This is the first reported study on the host range expansion of a Totiviridae member that is targeted by RNA silencing.

Differential Inductions of RNA Silencing among Encapsidated Double-Stranded RNA Mycoviruses in the White Root Rot Fungus Rosellinia necatrix

“…7. approach. For example, several mycoviruses, including a hypovirus (CHV1), a mycoreovirus (MyRV1), a partitivirus (RnPV2), and a victorivirus (RnVV1), more effectively replicated in an RNA silencing-defective, dicer-like protein 2 knockout mutant strain of C. parasitica (⌬dcl-2) than in the wild-type strain of the fungus (11,33,34). Notably, both dcl-2 and agl-2 are upregulated by infection with CHV1 and MyRV1 and also by constitutive expression of dsRNA in C. parasitica (31,32).…”

“…6Q). It has been predicted that sense-strandspecific inverted repeat and panhandle structures exist between the 5= and 3= termini of genome segments of RnQV1, RnVV1, and RnMyRV3 (29,34,40,61); therefore, these secondary structures might be diced. In contrast, we found that vsRNA peaks were densely distributed at the 5= region of both RnPV1-S1 and RnPV1-S2 (Fig.…”

“…Rosellinia necatrix W97 strains infected with Rosellinia necatrix partitivirus 1 (RnPV1)-W8 (43), Rosellinia necatrix mycoreovirus 3 (RnMyRV3)-W370 (44), Rosellinia necatrix megabirnavirus 1 (RnMBV1)-W779 (28), Rosellinia necatrix quadrivirus 1 (RnQV1)-W1075 (29), Rosellinia necatrix victorivirus (RnVV1)-W1029 (34), and the transgenic W97 strain expressing dsRNA of the green fluorescent protein (irGFP) (45) were used in this study. These strains were cultured on potato dextrose agar (PDA; Difco, Becton Dickinson, Sparks, MD, USA) plates overlaid with sterilized cellophane (6 cm 2 ) for 3 and 6 days at 25°C in the dark.…”

“…However, generally, RNA silencing against encapsidated dsRNA mycoviruses is not well understood. In C. parasitica, some encapsidated mycoviruses, including a mycoreovirus (MyRV1), a partitivirus (Rosellinia necatrix partitivirus 2; RnPV2), and a victorivirus (Rosellinia necatrix victorivirus 1; RnVV1), more effectively replicated in an RNA silencing-defective, dicer-like protein 2 knockout mutant strain of C. parasitica (⌬dcl-2) than in the wild-type strain (11,33,34). This suggests that these encapsidated dsRNA mycoviruses are also sensitive to RNA silencing in C. parasitica.…”

“…It may be infected with several dsRNA mycoviruses (37,38), including partitiviruses (RnPV1 and RnPV2), a mycoreovirus (Rosellinia necatrix mycoreovirus 3; RnMyRV3), a megabirnavirus (Rosellinia necatrix megabirnavirus 1; RnMBV1), a quadrivirus (Rosellinia necatrix quadrivirus 1; RnQV1), a victorivirus (RnVV1), and a novel ssRNA virus (Rosellinia necatrix fusarivirus 1; RnFV1) (28,29,33,34,(39)(40)(41)(42). Among them, RnPV1, RnMyRV3, RnMBV1, RnQV1, and RnVV1 were successfully introduced into the same R. necatrix strain W97, in which RnMyRV3 and RnMBV1 affect mycelial growth and virulence on apples but RnPV1, RnQV1, and RnVV1 were asymptomatic (28,29,34,43,44). Moreover, we showed that the RNA silencing mechanism is present in R. necatrix and is suppressed by RnMyRV3 but not by RnPV1, RnQV1, RnMBV1 (45), or RnVV1 (our unpublished data).…”