Extending the Fungal Host Range of a Partitivirus and a Mycoreovirus from <i>Rosellinia necatrix</i> by Inoculation of Protoplasts with Virus Particles

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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.

Section: Discussionsupporting

confidence: 84%

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

Chiba

Lin

Kondō

et al. 2013

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“…In the present study, the protocol has been applied to RnVV1, the first victorivirus whose host range has been expanded experimentally. As in the case for the partitiviruses (RnPV1 and RnPV2) from R. necatrix (17,65), C. parasitica, belonging to an order Diaporthales different from the Xylariales accommodating R. necatrix, was shown by transfection assay to support the replication of RnVV1. We are now attempting to determine whether RnVV1 can infect organisms differing at taxon levels higher than class.…”

Section: Discussionmentioning

confidence: 73%

“…However, experiments using infectious cDNA clones or synthetic transcripts have shown that the prototype hypovirus replicates in fungal species of the same family (63) or a different one within the same order as that of its natural host, the chestnut blight fungus C. parasitica (64). The virion transfection protocol has also provided chance to expand the host range of mycoviruses, such as RnPV1 and MyRV3 (65). In the present study, the protocol has been applied to RnVV1, the first victorivirus whose host range has been expanded experimentally.…”

Section: Discussionmentioning

confidence: 99%

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

Chiba

Lin

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.

“…Recently, phylogenetic analysis of the coat protein sequences of some partitiviruses revealed that incidences of horizontal gene transfer had occurred between viruses and eukaryotic organisms (18,19). A system in which partitivirus particles (i.e., Rosellinia necatrix partitivirus 1-W8 [RnPV1-W8]) can be transfected into fungal protoplasts has been successfully established and was utilized to confirm that RnPV1-W8 has a broad host range in Sordariomycetes (23,24). Particle structures determined by cryo-electron microscopy (cryo-EM) and three-dimensional (3D) image reconstruction, as well as one determined by X-ray crystallography, have shown three different partitivirus capsids to have a so-called "Tϭ2" organization comprising 60 capsid protein (CP) dimers arranged on a Tϭ1 icosahedral lattice (25)(26)(27).…”

mentioning

confidence: 99%

A Novel Partitivirus That Confers Hypovirulence on Plant Pathogenic Fungi

Xiao

Chéng

Tang

et al. 2014

131

103

Members of the family Partitiviridae have bisegmented double-stranded RNA (dsRNA) genomes and are not generally known to cause obvious symptoms in their natural hosts. An unusual partitivirus, Sclerotinia sclerotiorum partitivirus 1 (SsPV1/WF-1), conferred hypovirulence on its natural plant-pathogenic fungal host, Sclerotinia sclerotiorum strain WF-1. Cellular organelles, including mitochondria, were severely damaged. Hypovirulence and associated traits of strain WF-1 and SsPV1/WF-1 were readily cotransmitted horizontally via hyphal contact to different vegetative compatibility groups of S. sclerotiorum and interspecifically to Sclerotinia nivalis and Sclerotinia minor. S. sclerotiorum strain 1980 transfected with purified SsPV1/WF-1 virions also exhibited hypovirulence and associated traits similar to those of strain WF-1. Moreover, introduction of purified SsPV1/WF-1 virions into strain KY-1 of Botrytis cinerea also resulted in reductions in virulence and mycelial growth and, unexpectedly, enhanced conidial production. However, virus infection suppressed hyphal growth of most germinating conidia of B. cinerea and was eventually lethal to infected hyphae, since very few new colonies could develop following germ tube formation. Taken together, our results support the conclusion that SsPV1/WF-1 causes hypovirulence in Sclerotinia spp. and B. cinerea. Cryo-EM (cryo-electron microscopy) reconstruction of the SsPV1 particle shows that it has a distinct structure with similarity to the closely related partitiviruses Fusarium poae virus 1 and Penicillium stoloniferum virus F. These findings provide new insights into partitivirus biological activities and clues about molecular interactions between partitiviruses and their hosts. IMPORTANCEMembers of the Partitiviridae are believed to occur commonly in their phytopathogenic fungal and plant hosts. However, most partitiviruses examined so far appear to be associated with latent infections. Here we report a partitivirus, SsPV1/WF-1, that was isolated from a hypovirulent strain of Sclerotinia sclerotiorum and describe its biological and molecular features. We have demonstrated that SsPV1 confers hypovirulence. Furthermore, SsPV1 can infect and cause hypovirulence in Botrytis cinerea. Our study also suggests that SsPV1 has a vigorous ability to proliferate and spread via hyphal contact. SsPV1 can overcome vegetative incompatibility barriers and can be transmitted horizontally among different vegetative compatibility groups of S. sclerotiorum, even interspecifically. Cryo-EM reconstruction of SsPV1 shows that it has a distinct structure with similarity to closely related partitiviruses. Our studies exploit a novel system, SsPV1 and its hosts, which can provide the means to explore the mechanisms by which partitiviruses interact with their hosts.