Diverse Partitiviruses From the Phytopathogenic Fungus, Rosellinia necatrix.

Partitiviruses (dsRNA viruses, family Partitiviridae) are ubiquitously detected in plants and fungi. Although previous surveys suggested their omnipresence in the white root rot fungus, Rosellinia necatrix, only a few of them have been molecularly and biologically characterized thus far. We report the characterization of a total of 20 partitiviruses from 16 R. necatrix strains belonging to 15 new species, for which "Rosellinia necatrix partitivirus 11-Rosellinia necatrix partitivirus 25" were proposed, and 5 previously reported species. The newly identified partitiviruses have been taxonomically placed in two genera, Alphapartitivirus, and Betapartitivirus. Some partitiviruses were transfected into reference strains of the natural host, R. necatrix, and an experimental host, Cryphonectria parasitica, using purified virions. A comparative analysis of resultant transfectants revealed interesting differences and similarities between the RNA accumulation and symptom induction patterns of R. necatrix and C. parasitica. Other interesting findings include the identification of a probable reassortment event and a quintuple partitivirus infection of a single fungal strain. These combined results provide a foundation for further studies aimed at elucidating mechanisms that underly the differences observed.

ICTV Virus Taxonomy Profile: Megabirnaviridae.

Megabirnaviridae is a family of non-enveloped spherical viruses with dsRNA genomes of two linear segments, each of 7.2-8.9 kbp, comprising 16.1 kbp in total. The genus Megabirnavirus includes the species Rosellinia necatrix megabirnavirus 1, the exemplar isolate of which infects the white root rot fungus (Rosellinia necatrix) to which it confers hypovirulence. Megabirnaviruses are characterized by their bisegmented genome with large 5'-untranslated regions (1.6 kb) upstream of both 5'-proximal coding strand ORFs, and large protrusions on the particle surface. This is a summary of the ICTV Report on the family Megabirnaviridae, which is available at ictv.global/report/megabirnaviridae.This Profile is dedicated to the memory of our valued colleague Professor Said A. Ghabrial.

Draft Genome Sequence and Transcriptional Analysis of Rosellinia necatrix Infected with a Virulent Mycovirus.

Understanding the molecular mechanisms of pathogenesis is useful in developing effective control methods for fungal diseases. The white root rot fungus Rosellinia necatrix is a soilborne pathogen that causes serious economic losses in various crops, including fruit trees, worldwide. Here, using next-generation sequencing techniques, we first produced a 44-Mb draft genome sequence of R. necatrix strain W97, an isolate from Japan, in which 12,444 protein-coding genes were predicted. To survey differentially expressed genes (DEGs) associated with the pathogenesis of the fungus, the hypovirulent W97 strain infected with Rosellinia necatrix megabirnavirus 1 (RnMBV1) was used for a comprehensive transcriptome analysis. In total, 545 and 615 genes are up- and down-regulated, respectively, in R. necatrix infected with RnMBV1. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses of the DEGs suggested that primary and secondary metabolism would be greatly disturbed in R. necatrix infected with RnMBV1. The genes encoding transcriptional regulators, plant cell wall-degrading enzymes, and toxin production, such as cytochalasin E, were also found in the DEGs. The genetic resources provided in this study will accelerate the discovery of genes associated with pathogenesis and other biological characteristics of R. necatrix, thus contributing to disease control.

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

To reveal mycovirus diversity, we conducted a search of as-yet-unexplored Mediterranean isolates of the phytopathogenic ascomycete Rosellinia necatrix for virus infections. Of seventy-nine, eleven fungal isolates tested RNA virus-positive, with many showing coinfections, indicating a virus incidence of 14%, which is slightly lower than that (approximately 20%) previously reported for extensive surveys of over 1000 Japanese R. necatrix isolates. All viral sequences were fully or partially characterized by Sanger and next-generation sequencing. These sequences appear to represent isolates of various new species spanning at least 6 established or previously proposed families such as Partiti-, Hypo-, Megabirna-, Yado-kari-, Fusagra- and Fusarividae, as well as a newly proposed family, Megatotiviridae. This observation greatly expands the diversity of R. necatrix viruses, because no hypo-, fusagra- or megatotiviruses were previously reported from R. necatrix. The sequence analyses showed a rare horizontal gene transfer event of the 2A-like protease domain between a dsRNA (phlegivirus) and a positive-sense, single-stranded RNA virus (hypovirus). Moreover, many of the newly detected viruses showed the closest relation to viruses reported from fungi other than R. necatrix, such as Fusarium spp., which are sympatric to R. necatrix. These combined results imply horizontal virus transfer between these soil-inhabitant fungi.

Heterodimers as the Structural Unit of the T=1 Capsid of the Fungal Double-Stranded RNA Rosellinia necatrix Quadrivirus 1.

Most double-stranded RNA (dsRNA) viruses are transcribed and replicated in a specialized icosahedral capsid with a T=1 lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids help to organize the viral genome and replicative complex(es). They also act as molecular sieves that isolate the virus genome from host defense mechanisms and allow the passage of nucleotides and viral transcripts. Rosellinia necatrix quadrivirus 1 (RnQV1), the type species of the family Quadriviridae, is a dsRNA fungal virus with a multipartite genome consisting of four monocistronic segments (segments 1 to 4). dsRNA-2 and dsRNA-4 encode two CPs (P2 and P4, respectively), which coassemble into ∼450-Å-diameter capsids. We used three-dimensional cryo-electron microscopy combined with complementary biophysical techniques to determine the structures of RnQV1 virion strains W1075 and W1118. RnQV1 has a quadripartite genome, and the capsid is based on a single-shelled T=1 lattice built of P2-P4 dimers. Whereas the RnQV1-W1118 capsid is built of full-length CP, P2 and P4 of RnQV1-W1075 are cleaved into several polypeptides, maintaining the capsid structural organization. RnQV1 heterodimers have a quaternary organization similar to that of homodimers of reoviruses and other dsRNA mycoviruses. The RnQV1 capsid is the first T=1 capsid with a heterodimer as an asymmetric unit reported to date and follows the architectural principle for dsRNA viruses that a 120-subunit capsid is a conserved assembly that supports dsRNA replication and organization. IMPORTANCE: Given their importance to health, members of the family Reoviridae are the basis of most structural and functional studies and provide much of our knowledge of dsRNA viruses. Analysis of bacterial, protozoal, and fungal dsRNA viruses has improved our understanding of their structure, function, and evolution, as well. Here, we studied a dsRNA virus that infects the fungus Rosellinia necatrix, an ascomycete that is pathogenic to a wide range of plants. Using three-dimensional cryo-electron microscopy and analytical ultracentrifugation analysis, we determined the structure and stoichiometry of Rosellinia necatrix quadrivirus 1 (RnQV1). The RnQV1 capsid is a T=1 capsid with 60 heterodimers as the asymmetric units. The large amount of genetic information used by RnQV1 to construct a simple T=1 capsid is probably related to the numerous virus-host and virus-virus interactions that it must face in its life cycle, which lacks an extracellular phase.

A capsidless ssRNA virus hosted by an unrelated dsRNA virus.

Viruses typically encode the capsid that encases their genome, while satellite viruses do not encode a replicase and depend on a helper virus for their replication(1). Here, we report interplay between two RNA viruses, yado-nushi virus 1 (YnV1) and yado-kari virus 1 (YkV1), in a phytopathogenic fungus, Rosellinia necatrix(2). YkV1 has a close phylogenetic affinity to positive-sense, single-stranded (+)ssRNA viruses such as animal caliciviruses(3), while YnV1 has an undivided double-stranded (ds) RNA genome with a resemblance to fungal totiviruses(4). Virion transfection and infectious full-length cDNA transformation has shown that YkV1 depends on YnV1 for viability, although it probably encodes functional RNA-dependent RNA polymerase (RdRp). Immunological and molecular analyses have revealed trans-encapsidation of not only YkV1 RNA but also RdRp by the capsid protein of the other virus (YnV1), and enhancement of YnV1 accumulation by YkV1. This study demonstrates interplay in which the capsidless (+)ssRNA virus (YkV1), hijacks the capsid protein of the dsRNA virus (YnV1), and replicates as if it were a dsRNA virus.

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

UNLABELLED: RNA silencing acts as a defense mechanism against virus infection in a wide variety of organisms. Here, we investigated inductions of RNA silencing against encapsidated double-stranded RNA (dsRNA) fungal viruses (mycoviruses), including a partitivirus (RnPV1), a quadrivirus (RnQV1), a victorivirus (RnVV1), a mycoreovirus (RnMyRV3), and a megabirnavirus (RnMBV1) in the phytopathogenic fungus Rosellinia necatrix Expression profiling of RNA silencing-related genes revealed that a dicer-like gene, an Argonaute-like gene, and two RNA-dependent RNA polymerase genes were upregulated by RnMyRV3 or RnMBV1 infection but not by other virus infections or by constitutive expression of dsRNA in R. necatrix Massive analysis of viral small RNAs (vsRNAs) from the five mycoviruses showed that 19- to 22-nucleotide (nt) vsRNAs were predominant; however, their ability to form duplexes with 3' overhangs and the 5' nucleotide preferences of vsRNAs differed among the five mycoviruses. The abundances of 19- to 22-nt vsRNAs from RnPV1, RnQV1, RnVV1, RnMyRV3, and RnMBV1 were 6.8%, 1.2%, 0.3%, 13.0%, and 24.9%, respectively. Importantly, the vsRNA abundances and accumulation levels of viral RNA were not always correlated, and the origins of the vsRNAs were distinguishable among the five mycoviruses. These data corroborated diverse interactions between encapsidated dsRNA mycoviruses and RNA silencing. Moreover, a green fluorescent protein (GFP)-based sensor assay in R. necatrix revealed that RnMBV1 infection induced silencing of the target sensor gene (GFP gene and the partial RnMBV1 sequence), suggesting that vsRNAs from RnMBV1 activated the RNA-induced silencing complex. Overall, this study provides insights into RNA silencing against encapsidated dsRNA mycoviruses. IMPORTANCE: Encapsidated dsRNA fungal viruses (mycoviruses) are believed to replicate inside their virions; therefore, there is a question of whether they induce RNA silencing. Here, we investigated inductions of RNA silencing against encapsidated dsRNA mycoviruses (a partitivirus, a quadrivirus, a victorivirus, a mycoreovirus, and a megabirnavirus) in Rosellinia necatrix We revealed upregulation of RNA silencing-related genes in R. necatrix infected with a mycoreovirus or a megabirnavirus but not with other viruses, which was consistent with the relatively high abundances of vsRNAs from the two mycoviruses. We also showed common and different molecular features and origins of the vsRNAs from the five mycoviruses. Furthermore, we demonstrated the activation of RNA-induced silencing complex by mycoviruses in R. necatrix Taken together, our data provide insights into an RNA silencing pathway against encapsidated dsRNA mycoviruses which is differentially induced among encapsidated dsRNA mycoviruses; that is, diverse replication strategies exist among encapsidated dsRNA mycoviruses.

Characterization of a new megabirnavirus that confers hypovirulence with the aid of a co-infecting partitivirus to the host fungus, Rosellinia necatrix.

A new virus termed Rosellinia necatrix megabirnavirus 2 (RnMBV2) was molecularly and biologically characterized. RnMBV2 was originally harbored in isolate W8 of R. necatrix co-infected with the previously reported virus Rosellinia necatrix partitivirus 1 (RnPV1). RnMBV2 has molecular features similar and different from precedent megabirnaviruses, Rosellinia necatrix megabirnavirus 1 (RnMBV1) and Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1). The two genomic segments of RnMBV2 (9.0-kbp dsRNA1 and 8.0-kbp dsRNA2) each possess two open reading frames (ORF1 and 2 on dsRNA1 and ORF3 and 4 on dsRNA2), with a well conserved 5'-long untranslated region (UTR) of 1.7-1.8kb between the segments, and relatively short 3'-UTR. The RnMBV2 dsRNA1-coded capsid protein (CP) and RNA-dependent RNA polymerase (RdRp) show higher sequence identity to those of SsMBV1 than to those of RnMBV1, whereas the RnMBV2 ORF3-coded protein is more closely related to the counterpart of RnMBV1. No significant amino acid sequence similarity was detected among ORF4-coded sequences of the three megabirnaviruses. Virion transfection and co-culturing allowed for single and double infection of mycelial incompatible isolates W37 and W97 by RnMBV2 and/or RnPV1. Their comparative analyses showed RnMBV2 to be able to confer hypovirulence with the aid of a co-infecting RnPV1, while the individual viruses exhibited asymptomatic infections. Interestingly, RnPV1 accumulation appeared to be increased in co-infected fungal strain with two segments of RnMBV2 relative to singly infected fungal strains. Furthermore, the dispensability of RnMBV2 dsRNA2 was demonstrated to be similar to that of the other two megabirnaviruses.

Natural infection of the soil-borne fungus Rosellinia necatrix with novel mycoviruses under greenhouse conditions.

Fungi are an important component of the soil ecosystem. Mycoviruses have numerous potential impacts on soil fungi, including phytopathogenic fungal species. However, the diversity and ecology of mycoviruses in soil fungi is largely unexplored. Our previous work has shown that the soil-borne phytopathogenic fungus Rosellinia necatrix was infected with several novel mycoviruses after growing for 2-3 years in an apple orchard. In this study, we investigated whether natural infection of R. necatrix with mycoviruses occurs under limited conditions. Virus-free R. necatrix isolates were grown in a small bucket containing soil samples for a short time (1.5-4.5 months) under greenhouse conditions. Screening of dsRNA mycoviruses among 365 retrieved isolates showed that four, including 6-31, 6-33, 6-35, and 7-11, harbored virus-like dsRNAs. Molecular characterization of the dsRNAs revealed that three retrieved isolates, 6-31, 6-33, and 6-35 were infected with a novel endornavirus and isolate 7-11 is infected with a novel partitivirus belonging to the genus Alphapartitivirus. These novel mycoviruses had no overt biological impact on R. necatrix. Overall, this study indicates that natural infections of R. necatrix with new mycoviruses can occur under experimental soil conditions.

A novel betapartitivirus RnPV6 from Rosellinia necatrix tolerates host RNA silencing but is interfered by its defective RNAs.

The family Partitiviridae comprises of five genera with bi-segmented dsRNA genomes that accommodate members infecting plants, fungi or protists. All partitiviruses with only a few exceptions cause asymptomatic infections. We report the characterization of a novel betapartitivirus termed Rosellinia necatrix partitivirus 6 (RnPV6) from a field isolate of a plant pathogenic fungus, white root rot fungus. RnPV6 has typical partitivirus features: dsRNA1 and dsRNA2 are 2462 and 2499bps in length encoding RNA-dependent RNA polymerase and capsid protein. Purified particles are spherical with a diameter of 30nm. Taking advantage of infectivity as virions, RnPV6 was introduced into a model filamentous fungal host, chestnut blight fungus to investigate virus/host interactions. Unlike other partitiviruses tested previously, RnPV6 induced profound phenotypic alterations with symptoms characterized by a reduced growth rate and enhanced pigmentation and was tolerant to host RNA silencing. In addition, a variety of defective RNAs derived from dsRNA1 appear after virion transfection. These sub-viral RNAs were shown to interfere with RnPV6 replication, at least for that of cognate segment dsRNA1. Presence of these sub-viral elements resulted in reduced symptom expression by RnPV6, suggesting their nature as defective-interfering RNAs. The features of RnPV6 are similar to but distinct from those of a previously reported alphapartitivirus, Rosellinia necatrix partitivirus 2 that is susceptible to RNA silencing.

Megabirnavirus structure reveals a putative 120-subunit capsid formed by asymmetrical dimers with distinctive large protrusions.

Rosellinia necatrix megabirnavirus 1 (RnMBV1) W779 is a bi-segmented dsRNA virus and a strain of the type species Rosellinia necatrix megabirnavirus 1 of the family Megabirnaviridae. RnMBV1 causes severe reduction of both mycelial growth of Rosellinia necatrix in synthetic medium and fungal virulence to plant hosts, and thus has strong potential for virocontrol (biological control using viruses) of white rot. The structure of RnMBV1 was examined by cryo-electron microscopy and three-dimensional reconstruction at 15.7 Å resolution. The diameter of the RnMBV1 capsid was 520 Å, and the capsid was composed of 60 asymmetrical dimers in the T = 1 (so-called T = 2) lattice that is well conserved among dsRNA viruses. However, RnMBV1 has putatively 120 large protrusions with a width of ∼ 45 Å and a height of ∼ 50 Å on the virus surface, making it distinguishable from the other dsRNA viruses.

Systemic RNA interference is not triggered by locally-induced RNA interference in a plant pathogenic fungus, Rosellinia necatrix.

The white root rot fungus, Rosellinia necatrix, damages a wide range of fruit trees. R. necatrix is known to host a variety of mycoviruses, and several of these have potential as biological control agents. RNA interference (RNAi) is a fungal defense mechanism against viral infection, and it is therefore important to understand the RNAi amplification and transmission systems in R. necatrix for effective use of mycoviruses in disease control. In this study, we describe an intriguing RNAi signal transmission phenomenon in R. necatrix. In R. necatrix transformants with autonomously replicating vectors carrying a hairpin structure to induce RNAi, the gene silencing effect was distributed locally and unevenly, based on the vector distribution. This indicates that R. necatrix has no mechanism to propagate silencing signals systemically, unlike Caenorhabditis elegans and Arabidopsis thaliana. Furthermore, the expression of RNA-dependent RNA polymerase homologs was not upregulated during RNAi induction, suggesting that silencing signals are not amplified at sufficient levels to induce systemic RNAi in R. necatrix. Our results also suggest that, in addition to hairpin-induced RNAi, there is either a 5' transitive RNAi or quelling-like gene silencing system in R. necatrix. This is the first study demonstrating that systemic RNAi is not induced by local RNAi in fungi.

A novel single-stranded RNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix, with similarity to hypo-like viruses.

Here we report a biological and molecular characterization of a novel positive-sense RNA virus isolated from a field isolate (NW10) of a filamentous phytopathogenic fungus, the white root rot fungus that is designated as Rosellinia necatrix fusarivirus 1 (RnFV1). A recently developed technology using zinc ions allowed us to transfer RnFV1 to two mycelially incompatible Rosellinia necatrix strains. A biological comparison of the virus-free and -recipient isogenic fungal strains suggested that RnFV1 infects latently and thus has no potential as a virocontrol agent. The virus has an undivided positive-sense RNA genome of 6286 nucleotides excluding a poly (A) tail. The genome possesses two non-overlapping open reading frames (ORFs): a large ORF1 that encodes polypeptides with RNA replication functions and a smaller ORF2 that encodes polypeptides of unknown function. A lack of coat protein genes was suggested by the failure of virus particles from infected mycelia. No evidence was obtained by Northern analysis or classical 5'-RACE for the presence of subgenomic RNA for the downstream ORF. Sequence similarities were found in amino-acid sequence between RnFV1 putative proteins and counterparts of a previously reported mycovirus, Fusarium graminearum virus 1 (FgV1). Interestingly, several related sequences were detected by BLAST searches of independent transcriptome assembly databases one of which probably represents an entire virus genome. Phylogenetic analysis based on the conserved RNA-dependent RNA polymerase showed that RnFV1, FgV1, and these similar sequences are grouped in a cluster distinct from distantly related hypoviruses. It is proposed that a new taxonomic family termed Fusariviridae be created to include RnFV1 and FgV1.

Functional analysis of a melanin biosynthetic gene using RNAi-mediated gene silencing in Rosellinia necatrix.

Rosellinia necatrix causes white root rot in a wide range of fruit trees and persists for extended periods as pseudosclerotia on root debris. However, the pathogenesis of this disease has yet to be clarified. The functions of endogeneous target genes have not been determined because of the inefficiency in genetic transformation. In this study, the function of a melanin biosynthetic gene was determined to examine its role in morphology and virulence. A polyketide synthase gene (termed as RnPKS1) in the R. necatrix genome is homologous to the 1,8-dihydroxynaphthalene (DHN) melanin biosynthetic gene of Colletotrichum lagenarium. Melanin-deficient strains of R. necatrix were obtained by RNA interference-mediated knockdown of RnPKS1. The virulence of these strains was not significantly reduced compared with the parental melanin-producing strain. However, knockdown strains failed to develop pseudosclerotia and were degraded sooner in soil than the parental strain. Microscopic observations of albino conidiomata produced by knockdown strains revealed that melanization is involved in synnema integrity. These results suggest that melanin is not necessary for R. necatrix pathogenesis but is involved in survival through morphogenesis. This is the first report on the functional analysis of an endogenous target gene in R. necatrix.

Genome rearrangement of a mycovirus Rosellinia necatrix megabirnavirus 1 affecting its ability to attenuate virulence of the host fungus.

Rosellinia necatrix megabirnavirus 1 (RnMBV1) is a bi-segmented double-stranded RNA mycovirus that reduces the virulence of the fungal plant pathogen R. necatrix. We isolated strains of RnMBV1 with genome rearrangements (RnMBV1-RS1) that retained dsRNA1, encoding capsid protein (ORF1) and RNA-dependent RNA polymerase (ORF2), and had a newly emerged segment named dsRNAS1, but with loss of dsRNA2, which contains two ORFs of unknown function. Analyses of two variants of dsRNAS1 revealed that they both originated from dsRNA1 by deletion of ORF1 and partial tandem duplication of ORF2, retaining a much shorter 5' untranslated region (UTR). R. necatrix transfected with RnMBV-RS1 virions showed maintenance of virulence on host plants compared with infection with RnMBV1. This suggests that dsRNAS1 is able to be transcribed and packaged, as well as suggesting that dsRNA2, while dispensable for virus replication, is required to reduce the virulence of R. necatrix.

Biological properties and expression strategy of rosellinia necatrix megabirnavirus 1 analysed in an experimental host, Cryphonectria parasitica.

Rosellinia necatrix megabirnavirus 1 (RnMBV1) with a bipartite dsRNA genome (dsRNA1 and dsRNA2) confers hypovirulence to its natural host, the white root rot fungus, and is thus regarded as a potential virocontrol (biocontrol) agent. Each segment has two large ORFs: ORF1 and partially overlapping ORF2 on dsRNA1 encode the major capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), whilst ORF3 and ORF4 on dsRNA2 encode polypeptides with unknown functions. Here, we report the biological and molecular characterization of this virus in the chestnut blight fungus, Cryphonectria parasitica, a filamentous fungus that has been used as a model for mycovirus research. Transfection with purified RnMBV1 particles into an RNA-silencing-defective strain (Δdcl-2) of C. parasitica and subsequent anastomosis with the WT strain (EP155) resulted in stable persistent infection in both host strains. However, accumulation levels in the two strains were different, being ~20-fold higher in Δdcl-2 than in EP155. Intriguingly, whilst RnMBV1 reduced both virulence and growth rate in Δdcl-2, it attenuated virulence without affecting significantly other traits in EP155. Western blot analysis using antiserum against recombinant proteins encoded by either ORF1 or ORF2 demonstrated the presence of a 250 kDa protein in purified virion preparations, suggesting that RdRp is expressed as a CP fusion product via a -1 frameshift. Antiserum against the ORF3-encoded protein allowed the detection of 150, 30 and 23 kDa polypeptides specifically in RnMBV1-infected mycelia. Some properties of an RnMBV1 mutant with genome rearrangements, which occurred after transfection of Δdcl-2 and EP155, were also presented. This study provides an additional example of C. parasitica serving as a versatile, heterologous fungus for exploring virus-host interactions and virus gene expression strategies.

A mycoreovirus suppresses RNA silencing in the white root rot fungus, Rosellinia necatrix.

RNA silencing is a fundamental antiviral response in eukaryotic organisms. We investigated the counterdefense strategy of a fungal virus (mycovirus) against RNA silencing in the white root rot fungus, Rosellinia necatrix. We generated an R. necatrix strain that constitutively induced RNA silencing of the exogenous green fluorescent protein (GFP) gene, and infected it with each of four unrelated mycoviruses, including a partitivirus, a mycoreovirus, a megabirnavirus, and a quadrivirus. Infection with a mycoreovirus (R. necatrix mycoreovirus 3; RnMyRV3) suppressed RNA silencing of GFP, while the other mycoviruses did not. RnMyRV3 reduced accumulation of GFP-small interfering (si) RNAs and increased accumulation of GFP-double-stranded (ds) RNA; suggesting that the virus interferes with the dicing of dsRNA. Moreover, an agroinfiltration assay in planta revealed that the S10 gene of RnMyRV3 has RNA silencing suppressor activity. These data corroborate the counterdefense strategy of RnMyRV3 against host RNA silencing.

A novel victorivirus from a phytopathogenic fungus, Rosellinia necatrix, is infectious as particles and targeted by RNA silencing.

A 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 ~40 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 (Δdcl-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 Δdcl-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.

Potentiation of mycovirus transmission by zinc compounds via attenuation of heterogenic incompatibility in Rosellinia necatrix.

Heterogenic incompatibility is considered a defense mechanism against deleterious intruders such as mycovirus. Rosellinia necatrix shows strong heterogenic incompatibility. In the heterogenic incompatibility reaction, the approaching hyphae hardly anastomosed, a distinctive barrage line formed, and green fluorescent protein (GFP)-labeled hyphae quickly lost their fluorescence when encountering incompatible hyphae. In this study, transmission of a hypovirulence-conferring mycovirus to strains with different genetic backgrounds was attempted. Various chemical reagents considered to affect the programmed cell death pathway or cell wall modification were examined. Treatment with zinc compounds was shown to aid in transmission of mycoviruses to strains with different genetic backgrounds. In incompatible pairings, treatment with zinc compounds accelerated hyphal anastomosis; moreover, cytosolic GFP was transmitted to the newly joined hyphae. These results suggest that zinc compounds not only increase hyphal anastomosis but also attenuate heterogenic incompatibility.

Viruses of the white root rot fungus, Rosellinia necatrix.

Rosellinia necatrix is a filamentous ascomycete that is pathogenic to a wide range of perennial plants worldwide. An extensive search for double-stranded RNA of a large collection of field isolates led to the detection of a variety of viruses. Since the first identification of a reovirus in this fungus in 2002, several novel viruses have been molecularly characterized that include members of at least five virus families. While some cause phenotypic alterations, many others show latent infections. Viruses attenuating the virulence of a host fungus to its plant hosts attract much attention as agents for virocontrol (biological control using viruses) of the fungus, one of which is currently being tested in experimental fields. Like the Cryphonectria parasitica/viruses, the R. necatrix/viruses have emerged as an amenable system for studying virus/host and virus/virus interactions. Several techniques have recently been developed that enhance the investigation of virus etiology, replication, and symptom induction in this mycovirus/fungal host system.