Intragenic rearrangements of a mycoreovirus induced by the multifunctional protein p29 encoded by the prototypic hypovirus CHV1-EP713

A recombinant strain of Sclerotinia sclerotiorum hypovirus 2 (SsHV2) was identified from a North American Sclerotinia sclerotiorum isolate (328) from lettuce (Lactuca sativa L.) by high-throughput sequencing of total RNA. The 5=-and 3=-terminal regions of the genome were determined by rapid amplification of cDNA ends. The assembled nucleotide sequence was up to 92% identical to two recently reported SsHV2 strains but contained a deletion near its 5= terminus of more than 1.2 kb relative to the other SsHV2 strains and an insertion of 524 nucleotides (nt) that was distantly related to Valsa ceratosperma hypovirus 1. This suggests that the new isolate is a heterologous recombinant of SsHV2 with a yet-uncharacterized hypovirus. We named the new strain Sclerotinia sclerotiorum hypovirus 2 Lactuca (SsHV2L) and deposited the sequence in GenBank with accession number KF898354. Sclerotinia sclerotiorum isolate 328 was coinfected with a strain of Sclerotinia sclerotiorum endornavirus 1 and was debilitated compared to cultures of the same isolate that had been cured of virus infection by cycloheximide treatment and hyphal tipping. To determine whether SsHV2L alone could induce hypovirulence in S. sclerotiorum, a full-length cDNA of the 14,538-nt viral genome was cloned. Transcripts corresponding to the viral RNA were synthesized in vitro and transfected into a virus-free isolate of S. sclerotiorum, DK3. Isolate DK3 transfected with SsHV2L was hypovirulent on soybean and lettuce and exhibited delayed maturation of sclerotia relative to virus-free DK3, completing Koch's postulates for the association of hypovirulence with SsHV2L. IMPORTANCEA cosmopolitan fungus, Sclerotinia sclerotiorum infects more than 400 plant species and causes a plant disease known as white mold that produces significant yield losses in major crops annually. Mycoviruses have been used successfully to reduce losses caused by fungal plant pathogens, but definitive relationships between hypovirus infections and hypovirulence in S. sclerotiorum were lacking. By establishing a cause-and-effect relationship between Sclerotinia sclerotiorum hypovirus Lactuca (SsHV2L) infection and the reduction in host virulence, we showed direct evidence that hypoviruses have the potential to reduce the severity of white mold disease. In addition to intraspecific recombination, this study showed that recent interspecific recombination is an important factor shaping viral genomes. The construction of an infectious clone of SsHV2L allows future exploration of the interactions between SsHV2L and S. sclerotiorum, a widespread fungal pathogen of plants. Sclerotinia sclerotiorum (Lib.) de Bary is a cosmopolitan fungal plant pathogen that causes necrotic diseases (e.g., Sclerotinia stem rot) in more than 400 plant species, which result in yield losses in major crops each year (1-3). However, the diseases caused by S. sclerotiorum have not been adequately controlled by conventional technologies thus far (4, 5). A number of mycoviruses have been molecularly characterized and ...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Transfection of Sclerotinia sclerotiorum with In Vitro Transcripts of a Naturally Occurring Interspecific Recombinant of Sclerotinia sclerotiorum Hypovirus 2 Significantly Reduces Virulence of the Fungus

Marzano

Hobbs

Nelson

et al. 2015

“…Whether these hypoviral DIRNAs affect viral replication and the fungal host phenotype is unknown. DI segments S4ss and S10ss (rearranged dsRNA segments) of a mycoreovirus, Mycoreovirus 1 (MyRV1), have been shown to contribute to altered symptom induction (12,13,43). DI segments were also identified in Saccharomyces cerevisiae infected with dsRNA viruses (totiviruses, family Totiviridae) that were derived from a satellite RNA (called M dsRNA) (44,45).…”

Section: Discussionmentioning

confidence: 99%

“…It has been reported that some members additionally harbor satellitelike dsRNA segments either carrying or lacking a potential open reading frame (ORF) (11). However, unlike other dsRNA viruses, such as mycoreovirus (genus Mycoreovirus), for which DI-RNAs have been reported (12,13), partitiviruses have never been reported to carry DI-RNA species accompanied by a helper virus. Because of the unavailability of reverse genetics for, and the artificial introduction of, most partitiviruses, no clear picture of interactions among subviral RNAs, their helper viruses, and hosts has emerged.…”

mentioning

confidence: 99%

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

Self Cite

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.

“…3C and D). (51), and H. victoriae A-9 (genome of a victorivirus and a chrysovirus) (62) were analyzed comparatively by 1.0% agarose gel electrophoresis (78). The mobilities of dsRNAs are specified to the left of the panel, respectively.…”

Section: W1029 Is Infected With a Novel Partitivirus And A Novel Victmentioning

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

Self Cite

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.