Faculty Profiles - SUZUKI Nobuhiro

Papers - SUZUKI Nobuhiro

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

Juan Manuel Arjona-Lopez, Paul Telengech, Atif Jamal, Sakae Hisano, Hideki Kondo, Mery Dafny Yelin, Isabel Arjona-Girona, Satoko Kanematsu, Carlos José Lopez-Herrera, Nobuhiro Suzuki

Environmental Microbiology 20 ( 4 ) 1464 - 1483 2018.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing Ltd

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.

DOI： 10.1111/1462-2920.14065

Erratum: A fungal Argonaute interferes with RNA interference (Nucleic acids research (2018) 46 5 (2495-2508))

Quyet Nguyen, Akihide Iritani, Shuhei Ohkita, Ba V. Vu, Kana Yokoya, Ai Matsubara, Ken-Ichi Ikeda, Nobuhiro Suzuki, Hitoshi Nakayashiki

Nucleic acids research 46 ( 5 ) 2698 2018.3

First evidence for internal ribosomal entry sites in diverse fungal virus genomes Reviewed

Sotaro Chiba, Atif Jamal, Nobuhiro Suzuki

mBio 9 ( 2 ) 2018.3

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology

In contrast to well-established internal ribosomal entry site (IRES)-mediated translational initiation in animals and plants, no IRESs were established in fungal viral or cellular RNAs. To identify IRES elements in mycoviruses, we developed a luciferase-based dual-reporter detection system in Cryphonectria parasitica, a model filamentous fungus for virus-host interactions. A bicistronic construct entails a codon-optimized Renilla and firefly luciferase (ORluc and OFluc, respectively) gene, between which potential IRES sequences can be inserted. In this system, ORluc serves as an internal control, while OFluc represents IRES activity. Virus sequences in the 5= untranslated regions (UTRs) of the genomes of diverse positive-sense singlestranded RNA and double-stranded RNA (dsRNA) viruses were analyzed. The results show relatively high IRES activities for Cryphonectria hypovirus 1 (CHV1) and CHV2 and faint but measurable activity for CHV3. The weak IRES signal of CHV3 may be explained by its monocistronic nature, differing from the bicistronic nature of CHV1 and CHV2. This would allow these three hypoviruses to have similar rates of translation of replication-associated protein per viral mRNA molecule. The importance of 24 5=-proximal codons of CHV1 as well as the 5= UTR for IRES function was confirmed. Furthermore, victoriviruses and chrysoviruses tested IRES positive, whereas mycoreoviruses, partitiviruses, and quadriviruses showed similar Fluc activities as the negative controls. Overall, this study represents the first development of an IRES identification system in filamentous fungi based on the codon-optimized dual-luciferase assay and provides evidence for IRESs in filamentous fungi. IMPORTANCE Cap-independent, internal ribosomal entry site (IRES)-mediated translational initiation is often used by virus mRNAs and infrequently by cellular mRNAs in animals and plants. However, no IRESs have been established in fungal virus RNAs or cellular RNAs in filamentous fungi. Here, we report the development of a dualluciferase assay system and measurement of the IRES activities of fungal RNA viruses in a model filamentous fungal host, Cryphonectria parasitica. Viruses identified as IRES positive include hypoviruses (positive-sense RNA viruses, members of the expanded Picornavirus supergroup), totiviruses (nonsegmented dsRNA viruses), and chrysoviruses (tetrasegmented dsRNA viruses). No IRES activities were observed in the 5= untranslated regions of mycoreoviruses (11-segmented dsRNA viruses), quadriviruses (tetrasegmented dsRNA viruses), or partitiviruses (bisegmented dsRNA viruses). This study provides the first evidence for IRES activities in diverse RNA viruses in filamentous fungi and is a first step toward identifying trans-acting host factors and cis-regulatory viral RNA elements.

DOI： 10.1128/mBio.02350-17

A fungal Argonaute interferes with RNA interference. Reviewed International journal

Nguyen Q, Iritani A, Ohkita S, Vu BV, Yokoya K, Matsubara A, Ikeda KI, Suzuki N, Nakayashiki H

Nucleic acids research 46 ( 5 ) 2495 - 2508 2018.3

Language：English Publishing type：Research paper (scientific journal)

Small RNA (sRNA)-mediated gene silencing phenomena, exemplified by RNA interference (RNAi), require a unique class of proteins called Argonautes (AGOs). An AGO protein typically forms a protein-sRNA complex that contributes to gene silencing using the loaded sRNA as a specificity determinant. Here, we show that MoAGO2, one of the three AGO genes in the fungus Pyricularia oryzae (Magnaporthe oryzae) interferes with RNAi. Gene knockout (KO) studies revealed that MoAGO1 and MoAGO3 additively or redundantly played roles in hairpin RNA- and retrotransposon (MAGGY)-triggered RNAi while, surprisingly, the KO mutants of MoAGO2 (Δmoago2) showed elevated levels of gene silencing. Consistently, transcript levels of MAGGY and mycoviruses were drastically reduced in Δmoago2, supporting the idea that MoAGO2 impeded RNAi against the parasitic elements. Deep sequencing analysis revealed that repeat- and mycovirus-derived small interfering RNAs were mainly associated with MoAGO2 and MoAGO3, and their populations were very similar based on their size distribution patterns and positional base preference. Site-directed mutagenesis studies indicated that sRNA binding but not slicer activity of MoAGO2 was essential for the ability to diminish the efficacy of RNAi. Overall, these results suggest a possible interplay between distinct sRNA-mediated gene regulation pathways through a competition for sRNA.

DOI： 10.1093/nar/gkx1301

A neo-virus lifestyle exhibited by a (+)ssRNA virus hosted in an unrelated dsRNA virus: Taxonomic and evolutionary considerations Reviewed

Sakae Hisano, Rui Zhang, Md. Iqbal Faruk, Hideki Kondo, Nobuhiro Suzuki

Virus Research 244 75 - 83 2018.1

Language：English Publisher：Elsevier B.V.

Recent studies illustrate that fungi as virus hosts provides a unique platform for hunting viruses and exploring virus/virus and virus/host interactions. Such studies have revealed a number of as-yet-unreported viruses and virus/virus interactions. Among them is a unique intimate relationship between a (+)ssRNA virus, yado-kari virus (YkV1) and an unrelated dsRNA virus, yado-nushi virus (YnV1). YkV1 dsRNA, a replicated form of YkV1, and RNA-dependent RNA polymerase, are trans-encapsidated by the capsid protein of YnV1. While YnV1 can complete its replication cycle, YkV1 relies on YnV1 for its viability. We previously proposed a model in which YkV1 diverts YnV1 capsids as the replication sites. YkV1 is neither satellite virus nor satellite RNA, because YkV1 appears to encode functional RdRp and enhances YnV1 accumulation. This represents a unique mutualistic virus/virus interplay and similar relations in other virus/host fungus systems are detectable. We propose to establish the family Yadokariviridae that accommodates YkV1 and recently discovered viruses phylogenetically related to YkV1. This article overviews what is known and unknown about the YkV1/YnV1 interactions. Also discussed are the YnV1 Phytoreo_S7 and YkV1 2A-like domains that may have been captured via horizontal transfer during the course of evolution and are conserved across extant diverse RNA viruses. Lastly, evolutionary scenarios are envisioned for YkV1 and YnV1.

DOI： 10.1016/j.virusres.2017.11.006

ICTV virus taxonomy profile: Partitiviridae Reviewed

Eeva J. Vainio, Sotaro Chiba, Said A. Ghabrial, Edgar Maiss, Marilyn Roossinck, Sead Sabanadzovic, Nobuhiro Suzuki, Jiatao Xie, Max Nibert, Elliot J. Lefkowitz, Andrew J. Davison, Stuart G. Siddell, Donald B. Smith, Richard J. Orton, Peter Simmonds, ICTV Report Consortium

Journal of General Virology 99 ( 1 ) 17 - 18 2018.1

Language：English Publishing type：Research paper (scientific journal) Publisher：Microbiology Society

The Partitiviridae is a family of small, isometric, non-enveloped viruses with bisegmented double-stranded (ds) RNA genomes of 3-4.8 kbp. The two genome segments are individually encapsidated. The family has five genera, with characteristic hosts for members of each genus: either plants or fungi for genera Alphapartitivirus and Betapartitivirus, fungi for genus Gammapartitivirus, plants for genus Deltapartitivirus and protozoa for genus Cryspovirus. Partitiviruses are transmitted intracellularly via seeds (plants), oocysts (protozoa) or hyphal anastomosis, cell division and sporogenesis (fungi)
there are no known natural vectors. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Partitiviridae, which is available at www.ictv.global/report/partitiviridae.

DOI： 10.1099/jgv.0.000985

Viruses of Plant-Interacting Fungi Reviewed

Bradley I. Hillman, Aulia Annisa, Nobuhiro Suzuki

Advances in Virus Research 100 99 - 116 2018.1

Language：English Publishing type：Part of collection (book) Publisher：Academic Press Inc.

Plant-associated fungi are infected by viruses at the incidence rates from a few % to over 90%. Multiple viruses often coinfect fungal hosts, and occasionally alter their phenotypes, but most of the infections are asymptomatic. Phenotypic alterations are grouped into two types: harmful or beneficial to the host fungi. Harmful interactions between viruses and hosts include hypovirulence and/or debilitation that are documented in a number of phytopathogenic fungi, exemplified by the chestnut blight, white root rot, and rapeseed rot fungi. Beneficial interactions are observed in a limited number of plant endophytic and pathogenic fungi where heat tolerance and virulence are enhanced, respectively. Coinfections of fungi provided a platform for discoveries of interesting virus/virus interactions that include synergistic, as in the case for those in plants, and unique antagonistic and mutualistic interactions between unrelated RNA viruses. Also discussed here are coinfection-induced genome rearrangements and frequently observed coinfections by the simplest positive-strand RNA virus, the mitoviruses.

DOI： 10.1016/bs.aivir.2017.10.003

Acquisition of functions on the outer capsid surface during evolution of double-stranded RNA fungal viruses Reviewed

Carlos P. Mata, Daniel Luque, Josue Gomez-Blanco, Javier M. Rodriguez, Jose M. Gonzalez, Nobuhiro Suzuki, Said A. Ghabrial, Jose L. Carrascosa, Benes L. Truss, Jose R. Caston

PLOS PATHOGENS 13 ( 12 ) e1006755 2017.12

Language：English Publishing type：Research paper (scientific journal) Publisher：PUBLIC LIBRARY SCIENCE

Unlike their counterparts in bacterial and higher eukaryotic hosts, most fungal viruses are transmitted intracellularly and lack an extracellular phase. Here we determined the cryo-EM structure at 3.7 angstrom resolution of Rosellinia necatrix quadrivirus 1 (RnQV1), a fungal double-stranded (ds) RNA virus. RnQV1, the type species of the family Quadriviridae, has a multipartite genome consisting of four monocistronic segments. Whereas most dsRNA virus capsids are based on dimers of a single protein, the similar to 450-angstrom-diameter, T = 1 RnQV1 capsid is built of P2 and P4 protein heterodimers, each with more than 1000 residues. Despite a lack of sequence similarity between the two proteins, they have a similar a-helical domain, the structural signature shared with the lineage of the dsRNA bluetongue virus-like viruses. Domain insertions in P2 and P4 preferential sites provide additional functions at the capsid outer surface, probably related to enzyme activity. The P2 insertion has a fold similar to that of gelsolin and profilin, two actin-binding proteins with a function in cytoskeleton metabolism, whereas the P4 insertion suggests protease activity involved in cleavage of the P2 383-residue C-terminal region, absent in the mature viral particle. Our results indicate that the intimate virus-fungus partnership has altered the capsid genome-protective and/or receptor-binding functions. Fungal virus evolution has tended to allocate enzyme activities to the virus capsid outer surface.

DOI： 10.1371/journal.ppat.1006755

A possible occurrence of genome reassortment among bipartite rhabdoviruses Reviewed

Hideki Kondo, Keisuke Hirota, Kazuyuki Maruyama, Ida Bagus An'dika, Nobuhiro Suzuki

VIROLOGY 508 18 - 25 2017.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Orchid fleck virus (OFV) represents a rhabdovirus with a unique bipartite genome. OFV genetic diversity at the whole genome level has not been described. Using the partial genome sequence of RNA1, we have determined that several OFV isolates derived from orchids in Japan belong to two genetically distant subgroups: subgroup I, the members of which are distributed worldwide but previously not known in Asia, and subgroup II, which is commonly distributed in Japan. However, complete genome sequence analysis of a novel Japanese subgroup I isolate revealed that although its RNA1 sequence differs considerably from those of subgroup II isolates, its RNA2 sequence is almost identical to them. Based on phylogenetic and recombination analyses, the genome reassortment events were predicted to occur between OFV subgroups including other unseen strains. Our data show that genome reassortment contributes to the genetic diversities of the bipartite rhabdoviruses and its occurrence may be geographically constrained.

DOI： 10.1016/j.virol.2017.04.027

Roles of superoxide anion and hydrogen peroxide during replication of two unrelated plant RNA viruses in Nicotiana benthamiana Reviewed

Kiwamu Hyodo, Nobuhiro Suzuki, Kazuyuki Mise, Tetsuro Okuno

Plant Signaling and Behavior 12 ( 6 ) e1338223 2017.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Taylor and Francis Inc.

Reactive oxygen species (ROS), including superoxide anion (O2 −), hydrogen peroxide (H2O2), and hydroxyl radical, act as signaling molecules to transduce biotic and abiotic stimuli into stress adaptations in plants. A respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) is responsible for O2 − production to inhibit pathogen infection during plant innate immunity. RBOH-derived O2 − can be immediately converted into H2O2 by the action of superoxide dismutase. Interestingly, we recently showed that red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, hijacks the host's ROS-generating machinery during infection. An RCNMV replication protein associates with NbRBOHB and triggers intracellular ROS bursts. These bursts are required for robust viral RNA replication. However, what types of ROS are required for viral replication is currently unknown. Here, we found that RCNMV replication was sensitive to an O2 − scavenger but insensitive to an H2O2 scavenger. Interestingly, replication of another plant (+)RNA virus, brome mosaic virus, was sensitive to both types of scavengers. These results indicate a virus-specific pattern requirement of O2 − and H2O2 for (+)RNA virus replication and suggest a conserved nature of the roles of ROS in (+)RNA virus replication.

DOI： 10.1080/15592324.2017.1338223

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

Daniel Luque, Carlos P. Mata, Fernando Gonzalez-Camacho, Jose M. Gonzalez, Josue Gomez-Blanco, Carlos Alfonso, German Rivas, Wendy M. Havens, Satoko Kanematsu, Nobuhiro Suzuki, Said A. Ghabrial, Benes L. Trus, Jose R. Caston

JOURNAL OF VIROLOGY 90 ( 24 ) 11220 - 11230 2016.12

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

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 similar to 450-angstrom-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.

DOI： 10.1128/JVI.01013-16

Reprint of “Sequence and phylogenetic analyses of novel totivirus-like double-stranded RNAs from field-collected powdery mildew fungi” Reviewed

Hideki Kondo, Sakae Hisano, Sotaro Chiba, Kazuyuki Maruyama, Ida Bagus Andika, Kazuhiro Toyoda, Fumihiro Fujimori, Nobuhiro Suzuki

Virus Research 219 39 - 50 2016.7

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier B.V.

The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (&lt
44%) and with other known totiviruses (&lt
59%). Nine of these identified sequences (RPaTV1a, 1b and 2–8) resembled the genome of the prototype totivirus, Saccharomyces cerevisiae virus-L-A (ScV-L-A) in that they contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA dependent RNA polymerase (RdRp), while one sequence (RPaTV9) showed similarity to another totivirus, Ustilago maydis virus H1 (UmV-H1) that encodes a single polyprotein (CP-RdRp fusion). Similar to yeast totiviruses, each ScV-L-A-like RPaTV contains a –1 ribosomal frameshift site downstream of a predicted pseudoknot structure in the overlapping region of these ORFs, suggesting that the RdRp is translated as a CP-RdRp fusion. Moreover, several ScV-L-A-like sequences were also found by searches of the transcriptome shotgun assembly (TSA) libraries from rust fungi, plants and insects. Phylogenetic analyses show that nine ScV-L-A-like RPaTVs along with ScV-L-A-like sequences derived from TSA libraries are clustered with most established members of the genus Totivirus, while one RPaTV forms a new distinct clade with UmV-H1, possibly establishing an additional genus in the family. Taken together, our results indicate the presence of diverse, novel totiviruses in the powdery mildew fungus populations infecting red clover plants in the field.

DOI： 10.1016/j.virusres.2016.05.011

The world of diverse viruses in the kingdom Fungi Foreword Reviewed

Nobuhiro Suzuki

VIRUS RESEARCH 219 1 - 1 2016.7

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

Atsuko Sasaki, Hitoshi Nakamura, Nobuhiro Suzuki, Satoko Kanernatsu

VIRUS RESEARCH 219 73 - 82 2016.7

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

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 1.7-1.8 kb 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. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2015.12.009

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

Sotaro Chiba, Yu-Hsin Lin, Hideki Kondo, Satoko Kanematsu, Nobuhiro Suzuki

VIRUS RESEARCH 219 62 - 72 2016.7

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

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 2499 bps in length encoding RNA dependent RNA polymerase and capsid protein. Purified particles are spherical with a diameter of 30 nm. 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. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2015.10.017

The victorivirus Helminthosporium victoriae virus 1905 is the primary cause of disease/hypovirulence in its natural host and a heterologous host (Reprinted from Virus Res., vol 213, pg 238-245, 2016) Reviewed

Jiatao Xie, Wendy M. Havens, Yu-Hsin Lin, Nobuhiro Suzuki, Said A. Ghabrial

VIRUS RESEARCH 219 100 - 107 2016.7

Language：English Publisher：ELSEVIER SCIENCE BV

A transmissible disease of the plant pathogenic fungus Helminthosporium victoriae, the causal agent of Victoria blight of oats, was reported more than 50 years ago. Diseased, but not normal, isolates, of H. victoriae contain two distinct viruses designated according to their sedimentation values as victorivirus Helminthosporium victoriae virus 190S (HvV190S) and chrysovirus Helminthosporium victoriae 145S (HvV145S). Although a viral etiology of the disease was previously proposed, conclusive evidence was lacking. Here we present unequivocal evidence based on transfecting virus-free H. victoriae protoplasts with purified virus particles showing that HvV190S is essential for disease development. Furthermore, we show an expansion of the host range of HvV190S to include Cryphonectria parasitica and we also show similarity in a subset of phenotypic traits between HvV190S-infected RNA silencing deficient mutant (Delta dcl-2) of C. parasitica and a strain of H. victoriae. In virulence assays on detached American chestnut branches and Red Delicious apple fruits, HvV190S-infected C parasitica strain Delta dcl-2 was markedly less virulent than wild type and virus-free Delta dcl-2 C parasitica strains. Furthermore, the hypovirulent HvV190S-infected C parasitica Delta dcl-2 strain exhibited strong antifungal activity in dual culture with the plant pathogenic fungus Sclerotinia sclerotiorum. No such inhibitory activity was observed in comparable dual cultures with wild type and virus-free Delta dcl-2 C. parasitica strains. The discovery that infection with HvV190S induced a hypovirulent phenotype in a heterologous plant pathogenic host is very significant since it might be possible to convert other economically important plant pathogenic fungi to hypovirulence using HvV190S. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2016.05.010

Sequence and phylogenetic analyses of novel totivirus-like double-stranded RNAs from field-collected powdery mildew fungi Reviewed

Hideki Kondo, Sakae Hisano, Sotaro Chiba, Kazuyuki Maruyama, Ida Bagus Andika, Kazuhiro Toyoda, Fumihiro Fujimori, Nobuhiro Suzuki

VIRUS RESEARCH 213 353 - 364 2016.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (<44%) and with other known totiviruses (<59%). Nine of these identified sequences (RPaTV1a, 1b and 2-8) resembled the genome of the prototype totivirus, Saccharomyces cerevisiae virus-L-A (ScV-L-A) in that they contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA dependent RNA polymerase (RdRp), while one sequence (RPaTV9) showed similarity to another totivirus, Ustilago maydis virus H1 (UmV-H1) that encodes a single polyprotein (CP-RdRp fusion). Similar to yeast totiviruses, each ScV-L-A-like RPaTV contains a-1 ribosomal frameshift site downstream of a predicted pseudoknot structure in the overlapping region of these ORFs, suggesting that the RdRp is translated as a CP-RdRp fusion. Moreover, several ScV-L-A-like sequences were also found by searches of the transcriptome shotgun assembly (TSA) libraries from rust fungi, plants and insects. Phylogenetic analyses show that nine ScV-L-A-like RPaTVs along with ScV-L-A-like sequences derived from TSA libraries are clustered with most established members of the genus Totivirus, while one RPaTV forms a new distinct Glade with UmV-H1, possibly establishing an additional genus in the family. Taken together, our results indicate the presence of diverse, novel totiviruses in the powdery mildew fungus populations infecting red clover plants in the field. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2015.11.015

The victorivirus Helminthosporium victoriae virus 190S is the primary cause of disease/hypovirulence in its natural host and a heterologous host Reviewed

Jiatao Xie, Wendy M. Havens, Yu-Hsin Lin, Nobuhiro Suzuki, Said A. Ghabrial

VIRUS RESEARCH 213 238 - 245 2016.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

A transmissible disease of the plant pathogenic fungus Helminthosporium victoriae, the causal agent of Victoria blight of oats, was reported more than 50 years ago. Diseased, but not normal, isolates, of H. victoriae contain two distinct viruses designated according to their sedimentation values as victorivirus Helminthosporium victoriae virus 190S (HvV190S) and chrysovirus Helminthosporium victoriae 145S (HvV145S). Although a viral etiology of the disease was previously proposed, conclusive evidence was lacking. Here we present unequivocal evidence based on transfecting virus-free H. victoriae protoplasts with purified virus particles showing that HvV190S is essential for disease development. Furthermore, we show an expansion of the host range of HvV190S to include Cryphonectria parasitica and we also show similarity in a subset of phenotypic traits between HvV190S-infected RNA silencing deficient mutant (Delta dcl-2) of C parasitica and a strain of H. victoriae. In virulence assays on detached American chestnut branches and Red Delicious apple fruits, HvV190S-infected C parasitica strain Delta dcl-2 was markedly less virulent than wild type and virus-free Delta dcl-2 C parasitica strains. Furthermore, the hypovirulent HvV190S-infected C parasitica Delta dcl-2 strain exhibited strong antifungal activity in dual culture with the plant pathogenic fungus Sclerotinia sclerotiorum. No such inhibitory activity was observed in-comparable dual cultures with wild type and virus-free Delta dcl-2 C. parasitica strains. The discovery that infection with HvV190S induced a hypovirulent phenotype in a heterologous plant pathogenic host is very significant since it might be possible to convert other economically important plant pathogenic fungi to hypovirulence using HvV190S. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2015.12.011

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

Naoyuki Miyazaki, Lakha Salaipeth, Satoko Kanematsu, Kenji Iwasaki, Nobuhiro Suzuki

JOURNAL OF GENERAL VIROLOGY 96 ( 8 ) 2435 - 2441 2015.8

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC GENERAL MICROBIOLOGY

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 angstrom resolution. The diameter of the RnMBV1 capsid was 520 angstrom, 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 similar to 45 angstrom and a height of similar to 50 angstrom on the virus surface, making it distinguishable from the other dsRNA viruses.

DOI： 10.1099/vir.0.000182

Different Dicer-like protein components required for intracellular and systemic antiviral silencing in Arabidopsis thaliana Reviewed

Ida Bagus Andika, Kazuyuki Maruyama, Liying Sun, Hideki Kondo, Tetsuo Tamada, Nobuhiro Suzuki

PLANT SIGNALING & BEHAVIOR 10 ( 8 ) e1039214 2015.8

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS INC

Eukaryotes employ RNA silencing as an innate defense system against invading viruses. Dicer proteins play the most crucial role in initiating this antiviral pathway as they recognize and process incoming viral nucleic acids into small interfering RNAs. Generally, 2 successive infection stages constitute viral infection in plants. First, the virus multiplies in initially infected cells or organs after viral transmission and then the virus subsequently spreads systemically through the vasculature to distal plant tissues or organs. Thus, antiviral silencing in plants must cope with both local and systemic invasion of viruses. In a recent study using 2 sets of different experiments, we clearly demonstrated the differential requirement for Dicer-like 4 (DCL4) and DCL2 proteins in the inhibition of intracellular and systemic infection by potato virus X in Arabidopsis thaliana. Taken together with the results of other studies, here we further discuss the functional specificity of DCL proteins in the antiviral silencing pathway.

DOI： 10.1080/15592324.2015.1039214

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