Molecular characterization of a single-negative-stranded RNA virus from the rice blast fungus Magnaporthe oryzae isolate NJ39.

A novel negative-sense single-stranded RNA mycovirus, designated as "Magnaporthe oryzae mymonavirus 1" (MoMNV1), was identified in the rice blast fungus Magnaporthe oryzae isolate NJ39. MoMNV1 has a single genomic RNA segment consisting of 10,515 nucleotides, which contains six open reading frames. The largest open reading frame contains 5837 bases and encodes an RNA replicase. The six open reading frames have no overlap and are arranged linearly on the genome, but the spacing of the genes is small, with a maximum of 315 bases and a minimum of 80 bases. Genome comparison and phylogenetic analysis indicated that MoMNV1 is a new member of the genus Penicillimonavirus of the family Mymonaviridae.

A novel narnavirus from the plant-pathogenic fungus Magnaporthe oryzae.

A novel mycovirus with the proposed name "Magnaporthe oryzae narnavirus virus 1" (MoNV1), was described in the rice blast fungus Magnaporthe oryzae. The virus has a single-stranded (+ss) RNA genome of 2452 nucleotides, contains a single open reading frame (ORF) predicted to encode an RNA-dependent RNA polymerase (RDRP), and is closely related to some viruses of the genus Narnavirus, family Narnaviridae, including Aspergillus fumigatus narnavirus 1 (AfNV1), Neofusicoccum parvum narnavirus 2 (NpNV2) and Alternaria tenuissima narnavirus 1 (AtNV2). Genome sequence comparisons and phylogenetic analysis suggested that MoNV1 is a new member of the genus Narnavirus. The RDRPs of MoNV1 and some closely related narnaviruses do not contain a typical metal-binding "GDD" motif and catalytic site. Further studies are needed to investigate the replication mechanism of these viruses.

Characterization of a Novel Ourmia-Like Mycovirus Infecting Magnaporthe oryzae and Implications for Viral Diversity and Evolution.

Here, the molecular characterization of a novel mycovirus that was isolated from a phytopathogenic fungus Magnaporthe oryzae and designed as Magnaporthe oryzae ourmia-like virus 4 (MOLV4) is reported. MOLV4 has a genome that is 2497 bp long and possesses a single open reading frame (ORF), which encodes the product RNA-dependent RNA polymerase (RdRp). Sequence similarities were found between the MOLV4 encoded RdRp and the counterparts of a few previously reported ourmia-like mycoviruses. Virus-curing and biological comparison indicate that the virus has no or mild effects on the morphology and mycelium growth rate of the host fungus. Phylogenetic analysis using the RdRp aa sequences was performed. The results show that MOLV4 is clustered with the ourmia-like mycoviruses, forming a clade closely related to ourmiaviruses but distinct from narnaviruses. In addition, database searches revealed that several MOLV4-related sequences are present in the transcriptome shotgun assembly (TSA) library, expressed sequence tag database (ESTdb), whole-genome shotgun (WGS) library, and genomic survey sequences (GSS) libraries of a few other species of eukaryote organisms. Our results show that MOLV4, together with other similar ourmia-like mycoviruses, might represent a virus clade that links the plant ourmiaviruses and fungal narnaviruses and has a wide range of hosts.

Characterization of the Papain-Like Protease p29 of the Hypovirus CHV1-CN280 in Its Natural Host Fungus Cryphonectria parasitica and Nonhost Fungus Magnaporthe oryzae.

Cryphonectria hypovirus 1 strain CN280 (CHV1-CN280) was isolated from North China and exhibited typical hypovirulence-associated traits. We previously reported that CHV1-CN280 was more aggressive and had a higher horizontal transmission ability between Cryphonectria parasitica isolates belonging to different vegetative compatibility groups than two other CHV1 hypoviruses (namely, CHV1-EP713 and CHV1-Euro7), thus displaying greater potential for biological control of chestnut blight. The genome sequence of CHV1-CN280 shared approximately 70% identity with three other hypoviruses (CHV1-EP713, CHV1-Euro7, and CHV1-EP721). The coding region for p29, a papain-like protease encoded by CHV1-CN280 hypovirus, displayed an average of only approximately 60% amino acid identity among them, while the identity between the other three CHV1 isolates was higher than 89%. Protease p29 acted as a virus-encoded determinant responsible for altering fungal host phenotypes in other CHV1 isolates. In this study, the impacts of CHV1-CN280 p29 expression in virus-free C. parasitica were investigated. CHV1-CN280 p29 expression in C. parasitica resulted in significantly reduced sporulation, pigmentation, extracellular laccase activities, and pathogenicity, which is consistent with previous investigations. Subsequently, the potential of CHV1-CN280 p29 as a viral determinant responsible for suppression of host phenotypes in other phytopathogenic fungi such as Magnaporthe oryzae, the causal agent of rice blast disease, was discussed. However, heterologous expression of p29 in M. oryzae induced the opposite effect on sporulation, extracellular laccase activities, and pathogenicity; had no significant effect on pigmentation and mycelial growth; and contributed to extracellular peroxidase activities, suggesting that CHV1-CN280 p29 may disturb a unique regulatory pathway in C. parasitica, rather than a basic regulatory pathway conserved in diverse range of fungi. Alternatively, CHV1-CN280 p29-mediated modulation of fungal phenotypes may be facilitated by the specific interaction between p29 and a special fungal-host component, which exists only with C. parasitica but not M. oryzae.

Chrysoviruses in Magnaporthe oryzae.

Magnaporthe oryzae, the fungus that causes rice blast, is the most destructive pathogen of rice worldwide. A number of M. oryzae mycoviruses have been identified. These include Magnaporthe oryzae. viruses 1, 2, and 3 (MoV1, MoV2, and MoV3) belonging to the genus, Victorivirus, in the family, Totiviridae; Magnaporthe oryzae. partitivirus 1 (MoPV1) in the family, Partitiviridae; Magnaporthe oryzae. chrysovirus 1 strains A and B (MoCV1-A and MoCV1-B) belonging to cluster II of the family, Chrysoviridae; a mycovirus related to plant viruses of the family, Tombusviridae (Magnaporthe oryzae. virus A); and a (+)ssRNA mycovirus closely related to the ourmia-like viruses (Magnaporthe oryzae. ourmia-like virus 1). Among these, MoCV1-A and MoCV1-B were the first reported mycoviruses that cause hypovirulence traits in their host fungus, such as impaired growth, altered colony morphology, and reduced pigmentation. Recently we reported that, although MoCV1-A infection generally confers hypovirulence to fungi, it is also a driving force behind the development of physiological diversity, including pathogenic races. Another example of modulated pathogenicity caused by mycovirus infection is that of Alternaria alternata chrysovirus 1 (AaCV1), which is closely related to MoCV1-A. AaCV1 exhibits two contrasting effects: Impaired growth of the host fungus while rendering the host hypervirulent to the plant, through increased production of the host-specific AK-toxin. It is inferred that these mycoviruses might be epigenetic factors that cause changes in the pathogenicity of phytopathogenic fungi.

Molecular characterization of a novel ssRNA ourmia-like virus from the rice blast fungus Magnaporthe oryzae.

In this study we characterize a novel positive and single stranded RNA (ssRNA) mycovirus isolated from the rice field isolate of Magnaporthe oryzae Guy11. The ssRNA contains a single open reading frame (ORF) of 2,373 nucleotides in length and encodes an RNA-dependent RNA polymerase (RdRp) closely related to ourmiaviruses (plant viruses) and ourmia-like mycoviruses. Accordingly, we name this virus Magnaporthe oryzae ourmia-like virus 1 (MOLV1). Although phylogenetic analysis suggests that MOLV1 is closely related to ourmia and ourmia-like viruses, it has some features never reported before within the Ourmiavirus genus. 3' RLM-RACE (RNA ligase-mediated rapid amplification of cDNA ends) and extension poly(A) tests (ePAT) suggest that the MOLV1 genome contains a poly(A) tail whereas the three cytosine and the three guanine residues present in 5' and 3' untranslated regions (UTRs) of ourmia viruses are not observed in the MOLV1 sequence. The discovery of this novel viral genome supports the hypothesis that plant pathogenic fungi may have acquired this type of viruses from their host plants.

Complete nucleotide sequence of Magnaporthe oryzae partitivirus 1.

A novel double-stranded RNA (dsRNA) mycovirus, designated Magnaporthe oryzae partitivirus 1 (MoPV1), was isolated from a strain of the plant pathogenic fungus Magnaporthe oryzae. The MoPV1 genome has two dsRNA genome segments. The larger segment (1763 bp) has a single open reading frame (ORF) with a conserved RNA-dependent RNA polymerase (RdRp) domain. The smaller segment (1491 bp) contains a single ORF encoding a putative coat protein (CP). Homology searches and phylogenetic analysis indicated that MoPV1 is a new member of the genus Gammapartitivirus. This is the first report of a mycovirus of the family Partitiviridae identified in Magnaporthe oryzae.

Detection of Magnaporthe oryzae chrysovirus 1 in Japan and establishment of a rapid, sensitive and direct diagnostic method based on reverse transcription loop-mediated isothermal amplification.

Magnaporthe oryzae chrysovirus 1 (MoCV1) is a mycovirus with a dsRNA genome that infects the rice blast fungus Magnaporthe oryzae and impairs its growth. To date, MoCV1 has only been found in Vietnamese isolates of M. oryzae, and the distribution of this virus in M. oryzae isolates from other parts of the world remains unknown. In this study, using a one-step reverse transcription PCR (RT-PCR) assay, we detected a MoCV1-related virus in M. oryzae in Japan (named MoCV1-AK) whose sequence shares considerable similarity with that of the MoCV1 Vietnamese isolate. To establish a system for a comprehensive survey of MoCV1 infection in the field, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for direct detection of the virus. The sensitivity of the RT-LAMP assay was at least as high as that of the one-step RT-PCR assay. In addition, we detected MoCV1-AK in M. oryzae-infected oatmeal agar plates and lesions on rice leaves using the RT-LAMP assay without dsRNA extraction, by simple sampling with a toothpick. Preliminary screening of MoCV1 in Japanese M. oryzae isolates indicated that MoCV1 is currently distributed in rice fields in Japan. Our results provide a first example of the application of RT-LAMP for the detection of mycoviruses, which will accelerate surveys for mycovirus infection.

A novel single-stranded RNA virus isolated from the rice-pathogenic fungus Magnaporthe oryzae with similarity to members of the family Tombusviridae.

Here, we report a novel virus isolated from rice blast fungus, Magnaporthe oryzae, an important plant pathogen. This virus has an RNA genome of 3246 nucleotides. Its genome possesses two in-frame open reading frames (ORFs). The smaller ORF1 encodes a protein with significant similarity to a protein encoded by the ssRNA mycovirus Diaporthe ambigua RNA virus 1 (DaRV1). The larger ORF2 encodes a protein with similarity to RNA-dependent RNA polymerases (RdRp) of DaRV1 and other plant viruses of the family Tombusviridae. In silico analysis and comparisons with DaRV1 genome expression suggest that ORF2 is translated via a readthrough mechanism together with ORF1. Based upon results of this study, this virus, for which the provisional name Magnaporthe oryzae virus A (MoVA) is proposed, belongs to a new virus species. Furthermore, MoVA along with DaRV1 belong to a new taxon of mycoviruses that are evolutionarily related to plant viruses belonging to the family Tombusviridae.

Genomic organization of a novel victorivirus from the rice blast fungus Magnaporthe oryzae.

Two double-stranded RNA (dsRNA) mycoviruses were found in isolate QSP5 of the rice blast fungus Magnaporthe oryzae. Sequence analysis of the two dsRNA mycoviruses revealed that one is closely related to Magnaporthe oryzae virus 2 (MoV2), and the other one is related to Magnaporthe oryzae chrysovirus 1-A (MoCV1-A). Therefore, they were named Magnaporthe oryzae virus 3 (MoV3) and Magnaporthe oryzae chrysovirus 1-C (MoCV1-C), respectively. In this paper, the molecular and structural characteristics of MoV3 were analyzed in detail. The full genome sequence (5181 bp) of MoV3 was obtained by cDNA cloning. Sequence analysis indicated that MoV3 has two overlapping open reading frames (ORF1 and ORF2). The 5'-proximal ORF1 encodes a putative coat protein (CP) with a molecular weight of 80,939 Da; the 3'-proximal ORF2 encodes a putative RNA-dependent RNA polymerase (RdRp) with a molecular weight of 90,506 Da. The stop codon of ORF1 overlaps the start codon of ORF2, with the tetranucleotide sequence AUGA, which is characteristic of members of the genus Victorivirus of the family Totiviridae. Phylogenetic analysis of RdRp and CP further supported the view that MoV3, a novel mycovirus, belongs to the genus Victorivirus of the family Totiviridae.

Molecular genetics and Biochemical analyses of mycoviruses in rice blast fungus, Magnaporthe oryzae.

We have found a novel mycovirus, MoCV1 in the rice blast fungus, Magnaporthe oryzae. MoCV1 has five dRNA segments as genome, and belong to Chrysoviridae tentatively. Using micro-spin column method or one-step reverse-transcription PCR (RT-PCR) assay, we detected a MoCV1-related virus from M. oryzae in Japan, whose sequence shares considerable identity with that of the MoCV1 Vietnamese isolate. To establish a system for comprehensive survey of MoCV1 infection in the field, we developed a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for direct detection of the virus. In this review, we introduce our current knowledges of MoCV1 properties for biochemical and molecular genetic aspects and also describe its negative effects to host fungus, which imply potentiality to utilize MoCV1 as bio-controller. Heterologous gene-expression system in yeast is employed to investigate biological activities or functions of mycoviral proteins in fungal host cells. MoCV1-A infection caused hypovirulence to the host fungus, unexpectedly, also resulted in the change of pathogenic races in several differential rice lines, namely S (compatible) to R (incompatible) reaction or R to S. The cause of epigenetic alteration is also discussed.

Heterologous expression of a gene of Magnaporthe oryzae chrysovirus 1 strain A disrupts growth of the human pathogenic fungus Cryptococcus neoformans.

Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) is the causal agent of growth repression and attenuated virulence (hypovirulence) of the rice blast fungus, M. oryzae. We have previously reported that heterologous expression of MoCV1-A ORF4 in Saccharomyces cerevisiae results in growth defects, a large central vacuole and other cytological changes. In this study, the effects of open reading frame (ORF) 4 expression in Cryptococcus neoformans, a human pathogenic fungus responsible for severe opportunistic infection, were investigated. Cells expressing the ORF4 gene in C. neoformans showed remarkably enlarged vacuoles, nuclear diffusion and a reduced growth rate. In addition, expression of ORF4 apparently suppressed formation of the capsule that surrounds the entire cell wall, which is one of the most important components of expression of virulence. After 5-fluoroorotic acid treatment of ORF4-expressing cells to remove the plasmid carrying the ORF4 gene, the resultant plasmid-free cells recovered normal morphology and growth, indicating that heterologous expression of the MoCV1-A ORF4 gene induces negative effects in C. neoformans. These data suggest that the ORF4 product is a candidate for a pharmaceutical protein to control disease caused by C. neoformans.

A dsRNA mycovirus, Magnaporthe oryzae chrysovirus 1-B, suppresses vegetative growth and development of the rice blast fungus.

A double-stranded RNA (dsRNA) mycovirus was found in isolate S-0412-II 2a of the rice blast fungus Magnaporthe oryzae. Sequence analysis of the five dsRNA segments (dsRNA1 through dsRNA5) revealed that this mycovirus is closely related to Magnaporthe oryzae chrysovirus 1-A (MoCV1-A), tentatively classified as a member of the Chrysoviridae; therefore, it was named Magnaporthe oryzae chrysovirus 1-B (MoCV1-B). Virus particles were spherical and composed of the ORF1, ORF3 and ORF4 proteins. MoCV1-B-infected isolate S-0412-II 2a showed a more severe impaired phenotype than the MoCV1-A-infected isolate. In a virus-cured isolate, normal growth was restored, implied that MoCV1-B could be involved in this observed phenotype. An unanticipated result was the occurrence of a fungal isolate lacking dsRNA5. The nonessential dsRNA5 had higher sequence identity (96%) with dsRNA5 of MoCV1-A than with the other dsRNA segments (71-79%), indicating that dsRNA5 could be a portable genomic element between MoCV1-A and MoCV1-B.

Characterization of Magnaporthe oryzae chrysovirus 1 structural proteins and their expression in Saccharomyces cerevisiae.

Magnaporthe oryzae chrysovirus 1 (MoCV1), which is associated with an impaired growth phenotype of its host fungus, harbors four major proteins: P130 (130 kDa), P70 (70 kDa), P65 (65 kDa), and P58 (58 kDa). N-terminal sequence analysis of each protein revealed that P130 was encoded by double-stranded RNA1 (dsRNA1) (open reading frame 1 [ORF1] 1,127 amino acids [aa]), P70 by dsRNA4 (ORF4; 812 aa), and P58 by dsRNA3 (ORF3; 799 aa), although the molecular masses of P58 and P70 were significantly smaller than those deduced for ORF3 and ORF4, respectively. P65 was a degraded form of P70. Full-size proteins of ORF3 (84 kDa) and ORF4 (85 kDa) were produced in Escherichia coli. Antisera against these recombinant proteins detected full-size proteins encoded by ORF3 and ORF4 in mycelia cultured for 9, 15, and 28 days, and the antisera also detected smaller degraded proteins, namely, P58, P70, and P65, in mycelia cultured for 28 days. These full-size proteins and P58 and P70 were also components of viral particles, indicating that MoCV1 particles might have at least two forms during vegetative growth of the host fungus. Expression of the ORF4 protein in Saccharomyces cerevisiae resulted in cytological changes, with a large central vacuole associated with these growth defects. MoCV1 has five dsRNA segments, as do two Fusarium graminearum viruses (FgV-ch9 and FgV2), and forms a separate clade with FgV-ch9, FgV2, Aspergillus mycovirus 1816 (AsV1816), and Agaricus bisporus virus 1 (AbV1) in the Chrysoviridae family on the basis of their RdRp protein sequences.

Mycoviruses related to chrysovirus affect vegetative growth in the rice blast fungus Magnaporthe oryzae.

Mycoviruses causing impaired growth and abnormal pigmentation of the host were found in the rice blast fungus, Magnaporthe oryzae. Four dsRNAs, dsRNA 1 (3554 bp), dsRNA 2 (3250 bp), dsRNA 3 (307 bp) and dsRNA 4 (3043 bp), were detected in isolate S-0412-II 1a of M. oryzae. By picking up single conidia of S-0412-II 1a, cured strains of the fungus were isolated that had completely lost the mycovirus. The cured strains had normal mycelial growth and pigmentation, suggesting that this mycovirus modulates host traits. The buoyant densities of isometric virus particles (∼35 nm diameter) containing these dsRNAs in CsCl ranged from 1.37 to 1.40 g cm⁻³. The single ORF (3384 nt) of dsRNA 1 encoded a gene product highly homologous to the viral RNA-dependent RNA polymerase of members of the family Chrysoviridae. It is noteworthy that mycovirus S-0412-II 1a was detected not only in host cells but also in culture supernatant. Furthermore, abnormal aggregation of mycelia was observed after adding the mycovirus-containing culture supernatant to an uninfected strain of M. oryzae and mycoviral dsRNAs were detectable from the aggregated mycelia. This novel dsRNA mycovirus was named Magnaporthe oryzae chrysovirus 1.

Significantly low level of small RNA accumulation derived from an encapsidated mycovirus with dsRNA genome.

The role of RNA silencing as an antiviral defence has been well elucidated in plants and invertebrates, but not in filamentous fungi. We have previously determined the complete genome sequence of Magnaporthe oryzae virus 2 (MoV2), a dsRNA virus that infects the rice blast fungus Magnaporthe oryzae. In this study, we detected small interfering RNAs (siRNAs) from both positive- and negative-strand MoV2 viral RNA, suggesting that the RNA silencing machinery in M. oryzae functions against the mycovirus. Cloning and characterisation of MoV2 siRNAs indicated that, in MoV2, the ratio of virus-derived siRNAs to total small RNA is significantly lower than that in either plant viruses or Cryphonectria hypovirus 1 (CHV1), another mycovirus. Nevertheless, any MoV2-encoded proteins did not exhibit RNA silencing suppressor activity in both the plant and fungal systems. Our study suggests the existence of a novel viral strategy employed to evade host RNA silencing.

The nucleotide sequence and genome organization of Magnaporthe oryzae virus 1.

Magnaporthe oryzae virus 1 (MoV1) found in Magnaporthe oryzae, the pathogenic fungus responsible for rice blast, is a small icosahedral virus with a nonsegmented double-stranded RNA genome. The viral genome has two open reading frames (ORF 1 and 2). The deduced amino acid sequences of both ORF 1 and ORF 2 show a significant similarity to those of capsid protein and RdRp, respectively, of members of the family Totiviridae. Both a comparison of genome organization and phylogenic analysis have indicated that MoV1 is closely related to some of the totiviruses that infect filamentous fungi. These results suggest that MoV1 belongs to the family Totiviridae.