Hypovirulence of Colletotrichum gloesporioides Associated with dsRNA Mycovirus Isolated from a Mango Orchard in Thailand.

The pathogenic fungus Colletotrichum gloeosporioides causes anthracnose disease, which is an important fungal disease affecting the production of numerous crops around the world. The presence of mycoviruses, however, may have an impact on the pathogenicity of the fungal host. Here, we describe a double-stranded RNA (dsRNA) mycovirus, which was isolated from a field strain of C. gloeosporioides, Ssa-44.1. The 2939 bp genome sequence comprises two open reading frames (ORFs) that encode for a putative protein and RNA-dependent RNA polymerase (RdRp). The Ssa-44.1 mycovirus is a member of the unclassified mycovirus family named Colletotrichum gloeosporioides RNA virus 1 strain Ssa-44.1 (CgRV1-Ssa-44.1), which has a phylogenetic similarity to Colletotrichum gleosporioides RNA virus 1 (CgRV1), which was isolated from citrus leaves in China. In C. gloeosporioides, CgRV1-Ssa-44.1 was shown to be linked to hypovirulence. CgRV1-Ssa-44.1 has a low spore transfer efficiency but can successfully spread horizontally to isogenic virus-free isolates. Furthermore, CgRV1-Ssa-44.1 had a strong biological control impact on C. gloeosporioides on mango plants. This study is the first to describe a hypovirulence-associated mycovirus infecting C. gloeosporioides, which has the potential to assist with anthracnose disease biological management.

A Transfectable Fusagravirus from a Japanese Strain of Cryphonectria carpinicola with Spherical Particles.

A novel dsRNA virus (Cryphonectria carpinicola fusagravirus 1, CcFGV1), isolated from a Japanese strain (JS13) of Cryphonectria carpinicola, was thoroughly characterized. The biological comparison of a set of isogenic CcFGV1-infected and -free (JS13VF) strains indicated asymptomatic infection by CcFGV1. The sequence analysis showed that the virus has a two open reading frame (ORF) genome of 9.6 kbp with the RNA-directed RNA polymerase domain encoded by ORF2. The N-terminal sequencing and peptide mass fingerprinting showed an N-terminally processed or degraded product (150 kDa) of the 5'-proximal ORF1-encoded protein (1462 amino acids) to make up the CcFGV1 spherical particles of ~40 nm in diameter. Interestingly, a portion of CcFGV1 dsRNA co-fractionated with a host protein of 70 kDa. The purified CcFGV1 particles were used to transfect protoplasts of JS13VF as well as the standard strain of an experimental model filamentous fungal host Cryphonectria parasitica. CcFGV1 was confirmed to be associated with asymptomatic infection of both fungi. RNA silencing was shown to target the virus in C. parasitica, resulting in reduced CcFGV1 accumulation by comparing the CcFGV1 content between RNA silencing-competent and -deficient strains. These results indicate the transfectability of spherical particles of a fusagravirus associated with asymptomatic infection.

Establishment of Neurospora crassa as a model organism for fungal virology.

The filamentous fungus Neurospora crassa is used as a model organism for genetics, developmental biology and molecular biology. Remarkably, it is not known to host or to be susceptible to infection with any viruses. Here, we identify diverse RNA viruses in N. crassa and other Neurospora species, and show that N. crassa supports the replication of these viruses as well as some viruses from other fungi. Several encapsidated double-stranded RNA viruses and capsid-less positive-sense single-stranded RNA viruses can be experimentally introduced into N. crassa protoplasts or spheroplasts. This allowed us to examine viral replication and RNAi-mediated antiviral responses in this organism. We show that viral infection upregulates the transcription of RNAi components, and that Dicer proteins (DCL-1, DCL-2) and an Argonaute (QDE-2) participate in suppression of viral replication. Our study thus establishes N. crassa as a model system for the study of host-virus interactions.

Investigation of Host Range of and Host Defense against a Mitochondrially Replicating Mitovirus.

Mitoviruses (genus Mitovirus, family Narnaviridae) are mitochondrially replicating viruses that have the simplest positive-sense RNA genomes of 2.2 to 4.4 kb with a single open reading frame (ORF) encoding an RNA-dependent RNA polymerase. Cryphonectria parasitica mitovirus 1 (CpMV1) from U.S. strain NB631 of the chestnut blight fungus, Cryphonectria parasitica, was the first virus identified as a mitochondrially replicating virus. Despite subsequent discovery of many other mitoviruses from diverse fungi, no great advances in understanding mitovirus biology have emerged, partly because of the lack of inoculation methods. Here we developed a protoplast fusion-based protocol for horizontal transmission of CpMV1 that entailed fusion of recipient and donor protoplasts, hyphal anastomosis, and single-conidium isolation. This method allowed expansion of the host range to many other C. parasitica strains. Species within and outside the family Cryphonectriaceae, Cryphonectria radicalis and Valsa ceratosperma, also supported the replication of CpMV1 at a level comparable to that in the natural host. No stable maintenance of CpMV1 was observed in Helminthosporium victoriae PCR-based haplotyping of virus-infected fungal strains confirmed the recipient mitochondrial genetic background. Phenotypic comparison between CpMV1-free and -infected isogenic strains revealed no overt effects of the virus. Taking advantage of the infectivity to the standard strain C. parasitica EP155, accumulation levels were compared among antiviral RNA silencing-proficient and -deficient strains in the EP155 background. Comparable accumulation levels were observed among these strains, suggesting the avoidance of antiviral RNA silencing by CpMV1, which is consistent with its mitochondrial replication. Collectively, the results of study provide a foundation to further explore the biology of mitoviruses.IMPORTANCE Capsidless mitoviruses, which are ubiquitously detected in filamentous fungi, have the simplest RNA genomes of 2.2 to 4.4 kb, encoding only RNA-dependent RNA polymerase. Despite their simple genomes, detailed biological characterization of mitoviruses has been hampered by their mitochondrial location within the cell, posing challenges to their experimental introduction and study. Here we developed a protoplast fusion-based protocol for horizontal transfer of the prototype mitovirus, Cryphonectria parasitica mitovirus 1 (CpMV1), which was isolated from strain NB631 of the chestnut blight fungus (Cryphonectria parasitica), a model filamentous fungus for studying virus-host interactions. The host range of CpMV1 has been expanded to many different strains of C. parasitica and different fungal species within and outside the Cryphonectriaceae. Comparison of CpMV1 accumulation among various RNA silencing-deficient and -competent strains showed clearly that the virus was unaffected by RNA silencing. This study provides a solid foundation for further exploration of mitovirus-host interactions.

Mycoreovirus genome rearrangements associated with RNA silencing deficiency.

Mycoreovirus 1 (MyRV1) has 11 double-stranded RNA genome segments (S1 to S11) and confers hypovirulence to the chestnut blight fungus, Cryphonectria parasitica. MyRV1 genome rearrangements are frequently generated by a multifunctional protein, p29, encoded by a positive-strand RNA virus, Cryphonectria hypovirus 1. One of its functional roles is RNA silencing suppression. Here, we explored a possible link between MyRV1 genome rearrangements and the host RNA silencing pathway using wild-type (WT) and mutant strains of both MyRV1 and the host fungus. Host strains included deletion mutants of RNA silencing components such as dicer-like (dcl) and argonaute-like (agl) genes, while virus strains included an S4 internal deletion mutant MyRV1/S4ss. Consequently, intragenic rearrangements with nearly complete duplication of the three largest segments, i.e. S1, S2 and S3, were observed even more frequently in the RNA silencing-deficient strains Δdcl2 and Δagl2 infected with MyRV1/S4ss, but not with any other viral/host strain combinations. An interesting difference was noted between genome rearrangement events in the two host strains, i.e. generation of the rearrangement required prolonged culture for Δagl2 in comparison with Δdcl2. These results suggest a role for RNA silencing that suppresses genome rearrangements of a dsRNA virus.

The chestnut blight fungus for studies on virus/host and virus/virus interactions: from a natural to a model host.

The chestnut blight fungus, Cryphonectria parasitica, is an important plant pathogenic ascomycete. The fungus hosts a wide range of viruses and now has been established as a model filamentous fungus for studying virus/host and virus/virus interactions. This is based on the development of methods for artificial virus introduction and elimination, host genome manipulability, available host genome sequence with annotations, host mutant strains, and molecular tools. Molecular tools include sub-cellular distribution markers, gene expression reporters, and vectors with regulatable promoters that have been long available for unicellular organisms, cultured cells, individuals of animals and plants, and certain filamentous fungi. A comparison with other filamentous fungi such as Neurospora crassa has been made to establish clear advantages and disadvantages of C. parasitica as a virus host. In addition, a few recent studies on RNA silencing vs. viruses in this fungus are introduced.

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.

Mycoreovirus genome alterations: similarities to and differences from rearrangements reported for other reoviruses.

The family Reoviridae is one of the largest virus families with genomes composed of 9-12 double-stranded RNA segments. It includes members infecting organisms from protists to humans. It is well known that reovirus genomes are prone to various types of genome alterations including intragenic rearrangement and reassortment under laboratory and natural conditions. Recently distinct genetic alterations were reported for members of the genus Mycoreovirus, Mycoreovirus 1 (MyRV1), and MyRV3 with 11 (S1-S11) and 12 genome segments (S1-S12), respectively. While MyRV3 S8 is lost during subculturing of infected host fungal strains, MyRV1 rearrangements undergo alterations spontaneously and inducibly. The inducible MyRV1 rearrangements are different from any other previous examples of reovirus rearrangements in their dependence on an unrelated virus factor, a multifunctional protein, p29, encoded by a distinct virus Cryphonectria parasitica hypovirus 1 (CHV1). A total of 5 MyRV1 variants with genome rearranged segments (S1-S3, S6 and S10) are generated in the background of a single viral strain in the presence of CHV1 p29 supplied either transgenically or by coinfection. MyRV1 S4 and S10 are rearranged, albeit very infrequently, in a CHV1 p29 independent fashion. A variant of MyRV1 with substantial deletions in both S4 and S10, generated through a combined reassortment and rearrangement approach, shows comparable replication levels to the wild-type MyRV1. In vivo and in vitro interactions of CHV1 p29 and MyRV1 VP9 are implicated in the induction of MyRV1 rearrangements. However, the mechanism underlying p29-mediated rearrangements remains largely unknown. MyRV1 S4 rearrangements spontaneously occurred independently of CHV1 p29. In the absence of reverse genetics systems for mycoreoviruses, molecular and biological characterization of these MyRV1 and MyRV3 variants contribute to functional analyses of the protein products encoded by those rearranged segments.

Mycoreovirus 1 S4-coded protein is dispensable for viral replication but necessary for efficient vertical transmission and normal symptom induction.

Rearrangements of two segments, S6 and S10, of Mycoreovirus 1 (MyRV1), a member of the family Reoviridae, were previously shown to be induced at a high rate by the multifunctional protein p29 encoded by a distinct ssRNA virus, the prototype hypovirus CHV1-EP713 (Sun and Suzuki, RNA 14, 2557-2571, 2008). Here we report the occurrence of rearrangements of MyRV1 S4, albeit at a very low frequency, in the absence of CHV1 p29, resulting in internal 80-90% deletions of the open reading frame (ORF) in S4. Comparative analyses of fungal strains infected by wild-type MyRV1 and its variants carrying rearrangements of S4, S4 plus S10 and S10 indicated that S4-encoded VP4, like VP10, is non-essential for virus replication but required for efficient vertical transmission and symptom expression caused by MyRV1. This is the first example of a reovirus variant that carries deletions of over 75% of the ORFs in two genome segments and is still replication-competent.

Cytological and electrophoretic karyotyping of the chestnut blight fungus Cryphonectria parasitica.

The karyotypes of nine strains including three transformants of the chestnut blight fungus Cryphonectria parasitica were analyzed by pulsed-field gel electrophoresis (PFGE) and cytology using a fluorescence microscope. Cytology of the mitotic metaphase showed n=9 for both standard strain EP155 and field strain GH2 infected by Cryphonectria hypovirus 3. Chromosomes were morphologically characterized by size, heterochromatic segment, and constriction. PFGE resolved 5 or 6 chromosomal DNA bands ranging from 3.3Mbp to 9.7Mbp, but accurate determination of the chromosome number was hampered by clumping of some bands. Banding profiles in PFGE were similar among the strains except for GH2, in which a chromosome translocation was detected by Southern blot analysis. By integrating the data from cytology and PFGE, the genome size of C. parasitica was estimated to be ca. 50Mbp. This is the first report of a cytological karyotype in the order Diaporthales.

A host factor involved in hypovirus symptom expression in the chestnut blight fungus, Cryphonectria parasitica.

The prototype hypovirus CHV1-EP713 causes virulence attenuation and severe suppression of asexual sporulation and pigmentation in its host, the chestnut blight fungus, Cryphonectria parasitica. We identified a factor associated with symptom induction in C. parasitica using a transformation of C. parasitica strain EP155 with a full-length cDNA clone from a mild mutant virus strain, Cys(72). This was accomplished by using mutagenesis of the transformant fungal strain TCys(72)-1 by random integration of plasmid pHygR, conferring hygromycin resistance. The mutant, namA (after nami-gata, meaning wave shaped), showed an irregular fungal morphology with reduced conidiation and pigmentation while retaining similar levels of virulence and virus accumulation relative to TCys(72)-1- or Cys(72)-infected strain EP155. However, the colony morphology of virus-cured namA (VC-namA) was indistinguishable from those of EP155 and virus-cured TCys(72)-1 [VC-TCys(72)-1]. The phenotypic difference between VC-namA and VC-TCys(72)-1 was found only when these strains infected with the wild type or certain mutant CHV1-EP713 strains but not when infected with Mycoreovirus 1. Sequence analysis of inverse-PCR-amplified genomic DNA fragments and cDNA identified the insertion site of the mutagenic plasmid in exon 8 of the nam-1 gene. NAM-1, comprising 1,257 amino acids, shows sequence similarities to counterparts from other filamentous fungi and possesses the CorA domain that is conserved in a class of Mg(2+) transporters from prokaryotes and eukaryotes. Complementation assays using the wild-type and mutant alleles and targeted disruption of nam-1 showed that nam-1 with an extension of the pHygR-derived sequence contributed to the altered phenotype in the namA mutant. The molecular mechanism underlying virus-specific fungal symptom modulation in VC-namA is discussed.