A Host Factor Involved in Hypovirus Symptom Expression in the Chestnut Blight Fungus, Cryphonectria parasitica

Salaipeth

et al. 2009

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260

190

White root rot, caused by the ascomycete Rosellinia necatrix, is a devastating disease worldwide, particularly in fruit trees in Japan. Here we report on the biological and molecular properties of a novel bipartite double-stranded RNA (dsRNA) virus encompassing dsRNA-1 (8,931 bp) and dsRNA-2 (7,180 bp), which was isolated from a field strain of R. necatrix, W779. Besides the strictly conserved 5 (24 nt) and 3 (8 nt) terminal sequences, both segments show high levels of sequence similarity in the long 5 untranslated region of approximately 1.6 kbp. dsRNA-1 and -2 each possess two open reading frames (ORFs) named ORF1 to -4. Although the protein encoded by 3-proximal ORF2 on dsRNA-1 shows sequence identities of 22 to 32% with RNA-dependent RNA polymerases from members of the families Totiviridae and Chrysoviridae, the remaining three virus-encoded proteins lack sequence similarities with any reported mycovirus proteins. Phylogenetic analysis showed that the W779 virus belongs to a separate clade distinct from those of other known mycoviruses. Purified virions ϳ50 nm in diameter consisted of dsRNA-1 and -2 and a single major capsid protein of 135 kDa, which was shown by peptide mass fingerprinting to be encoded by dsRNA-1 ORF1. We developed a transfection protocol using purified virions to show that the virus was responsible for reduction of virulence and mycelial growth in several host strains. These combined results indicate that the W779 virus is a novel bipartite dsRNA virus with potential for biological control (virocontrol), named Rosellinia necatrix megabirnavirus 1 (RnMBV1), that possibly belongs to a new virus family.

Section: Discussionmentioning

confidence: 99%

A Novel Bipartite Double-Stranded RNA Mycovirus from the White Root Rot Fungus Rosellinia necatrix : Molecular and Biological Characterization, Taxonomic Considerations, and Potential for Biological Control

Salaipeth

et al. 2009

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260

190

“…Furthermore, this study represents an example of a heterologous virus being characterized using an experimental host, C. parasitica. Together with its ease of culture, the availability of genetic manipulation techniques such as transfection and multiple transformation, molecular tools and resources, genome information, and knockout mutants makes C. parasitica amenable to and suitable for studies of virus-host (6,26,(61)(62)(63) and virus-virus interactions (12,13,64). Preliminary data have revealed its susceptibility to other heterologous viruses, such as Rosellinia necatrix megabirnavirus 1 (type species of the family Megabirnaviridae) and Rosellinia necatrix victorivirus 1 (a novel victorivirus) (L. Salaipeth, Y.-H. Lin, S. Chiba, S. Kanematsu, and N. Suzuki, unpublished data).…”

Section: Discussionmentioning

confidence: 99%

Effects of Defective Interfering RNA on Symptom Induction by, and Replication of, a Novel Partitivirus from a Phytopathogenic Fungus, Rosellinia necatrix

Kondō

et al. 2013

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A novel mycovirus termed Rosellinia necatrix partitivirus 2 (RnPV2), isolated from a phytopathogenic fungus, Rosellinina necatrix strain W57, was molecularly and biologically characterized in both natural and experimental host fungi. Three doublestranded RNA (dsRNA) segments, dsRNA1, dsRNA2, and defective interfering dsRNA1 (DI-dsRNA1), whose sizes were approximately 2.0, 1.8, and 1.7 kbp, respectively, were detected in W57. While the dsRNA2 sequence, encoding the coat protein, was reported previously, dsRNA1 and DI-dsRNA1 were shown to encode competent and defective (truncated) RNA-dependent RNA polymerase, respectively. Artificial introduction of RnPV2 into an RNA silencing-defective, Dicer-like 2 knockout mutant (⌬dcl-2) of a nonnatural host, Cryphonectria parasitica (chestnut blight fungus), resulted in successful infection by the DI-dsR-NA1-carrying and -free RnPV2. The DI-dsRNA1-free RnPV2 strain was characterized by a higher ratio of accumulation of the intact dsRNA1 to dsRNA2, enhanced replication and severer symptom expression, compared with the DI-carrying strain. These findings confirmed the nature of DI-dsRNA1 as a DI-RNA. Both viral strains replicated to higher levels in a ⌬dcl-2 mutant than in a wild-type C. parasitica fungal strain (EP155) and induced severe symptoms in the ⌬dcl-2 mutant but subtle symptoms in EP155, indicating that the host RNA silencing targets the partitivirus. No obvious phenotypic effects of infection by either virus strain were detected in the natural host fungus. These combined results represent the first example of a partitivirus with DI-RNA that alters viral symptom induction in a host-dependent manner.

“…Protoplasts were prepared from mycelia of C. parasitica virus-free strain EP155 (see below). Transformation of the resulting protoplasts with a vector, pCPXHY2 (39), carrying a hygromycin B phosphotransferase gene (hph) as a selectable marker, was performed as described previously (40). Four transformant lines with each of the viral suppressor of RNA silencing (VSR) genes, i.e., p19 of tomato bushy stunt virus (TBSV; a plant tombusvirus, EP155/p19), codon-optimized TBSV p19 (EP155/O-p19), 2b of cucumber mosaic virus (CMV; a plant cucumovirus) (EP155/2b), or p29 of CHV1 (EP155/ p29) (16), were generated.…”

Section: Methodsmentioning

confidence: 99%

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

Kondō

et al. 2013

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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.