Seed transmission of raspberry bushy dwarf virus is blocked in Nicotiana benthamiana plants by preventing virus entry into the embryo from the infected embryo sac and endosperm.

Raspberry bushy dwarf virus (RBDV) is transmitted through seed in infected red raspberry plants after pollination with pollen grains from healthy red raspberry plants. Here, we show that RBDV is not transmitted through seeds in infected Nicotiana benthamiana (Nb) plants after pollination with virus-free Nb pollen grains. Chromogenic in situ hybridization revealed that the virus invades the shoot apical meristem and the ovule, including the embryo sac, of RBDV-infected Nb plants; however, in seeds that developed from infected embryo sacs after fertilization by virus-free sperm cells, RBDV was absent in the embryos and present in the endosperms. When we analyzed seed transmission of RBDV in Nb mutants with mutations in dicer-like enzyme 2 and 4 (NbDCL2&4) or RNA-dependent RNA polymerase 6 (NbRDR6), RBDV was not present in the offspring from seeds with embryos and endosperms that did not express NbDCL2&4 or NbRDR6. These results suggest that seed transmission of RBDV is prevented by evasion of infection by the embryo and that RNA silencing is not essential for preventing seed transmission of RBDV in Nb plants.

Apple Russet Ring and Apple Green Crinkle Diseases: Fulfillment of Koch's Postulates by Virome Analysis, Amplification of Full-Length cDNA of Viral Genomes, in vitro Transcription of Infectious Viral RNAs, and Reproduction of Symptoms on Fruits of Apple Trees Inoculated With Viral RNAs.

Apple russet ring and apple green crinkle are graft-transmitted diseases first reported more than 60 years ago, but at present, no association between a specific virus (variant) and the disease has been clearly demonstrated. In this study, we conducted the following series of experiments to identify the causal viruses (variants) of these apple diseases; (1) comprehensive analysis by next-generation sequencing of all viruses in each apple tree affected with russet ring or green crinkle disease, (2) amplification of full-length genomic cDNA of viruses using primers containing the T3 promoter and the in vitro transcription of infectious viral RNAs, (3) inoculation of viral RNA transcripts to both herbaceous and apple plants, (4) analysis of sequence variants of viruses present in infected plants, (5) back-inoculation of sequence variants of candidate viruses to apple seedlings combined with the virus-induced flowering technology using the apple latent spherical virus vector to reproduce the symptom on the fruit as soon as possible, and (6) reproduction of symptoms on the fruits of apple trees inoculated with sequence variants and the re-isolation of each virus variant from apples showing fruit symptoms. The results showed that one of the sequence variants of the apple chlorotic leaf spot virus causes a characteristic ring-shaped rust on the fruits of infected apple trees and that a sequence variant of the apple stem pitting virus probably causes green crinkle symptoms on an infected apple fruit. Thus, we were able to fulfill Koch's postulates to prove the viral etiology of both the apple russet ring and green crinkle diseases. We also propose an experimental system that can prove whether a virus found in diseased tissues is the pathogen responsible for the diseases when the etiology is undetermined.

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.

ICTV Virus Taxonomy Profile: Hypoviridae.

The Hypoviridae, comprising one genus, Hypovirus, is a family of capsidless viruses with positive-sense, ssRNA genomes of 9.1-12.7 kb that possess either a single large ORF or two ORFs. The ORFs appear to be translated from genomic RNA by non-canonical mechanisms, i.e. internal ribosome entry site-mediated and stop/restart translation. Hypoviruses have been detected in ascomycetous or basidiomycetous filamentous fungi, and are considered to be replicated in host Golgi-derived, lipid vesicles that contain their dsRNA as a replicative form. Some hypoviruses induce hypovirulence to host fungi, while others do not. This is a summary of the current ICTV report on the taxonomy of the Hypoviridae, which is available at www.ictv.global/report/hypoviridae.

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.

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.

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.

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.

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 second quadrivirus strain from the phytopathogenic filamentous fungus Rosellinia necatrix.

We report the biological and molecular characterisation ofa second quadrivirus strain termed Rosellinia necatrix quadrivirus 1 strain W1118 (RnQV1-W1118) from the ascomycete white root rot fungus. Commonalities with the first quadrivirus (RnQV1-W1075) include its quadripartite genome structure, spherical particle morphology, sequence heterogeneity in the extreme terminal end, 72­82%sequence identity between the corresponding proteins, and its ability to cause a latent infection. Distinguishing features include different conserved terminal sequences and the degree of susceptibility of two major capsid proteins to proteolytic degradation, which is thought to occur during virion purification. Identification of this second quadrivirus strain strengthens our earlier proposal that ''Rosellinia necatrix quadrivirus 1'' should be considered the type species of a novel family, ''Quadriviridae''.

Appearance of mycovirus-like double-stranded RNAs in the white root rot fungus, Rosellinia necatrix, in an apple orchard.

In general, mycoviruses are transmitted through hyphal anastomosis between vegetatively compatible strains of the same fungi, and their entire intracellular life cycle within host fungi limits transmission to separate species and even to incompatible strains belonging to the same species. Based on field observations of the white root rot fungus, Rosellinia necatrix, we found two interesting phenomena concerning mycovirus epidemiology. Specifically, apple trees in an orchard were inoculated with one or two R. necatrix strains that belonged to different mycelial compatibility groups (MCGs), strains W563 (virus-free, MCG139) and NW10 (carrying a mycovirus-like double-stranded (ds) RNA element (N10), MCG442). Forty-two sub-isolates of R. necatrix, which were retrieved 2-3 years later, were all genetically identical to W563 or NW10: however, 22 of the sub-isolates contained novel dsRNAs. Six novel dsRNAs (S1-S6) were isolated: S1 was a new victorivirus; S2, S3, and S4 were new partitiviruses; and S5 and S6 were novel viruses that could not be assigned to any known mycovirus family. N10 dsRNA was detected in three W563 sub-isolates. These findings indicated that novel mycoviruses, from an unknown source, were infecting strains W563 and NW10 of R. necatrix in the soil, and that N10 dsRNA was being transmitted between incompatible strains, NW10 to W563.

Characterization of plant virus-encoded gene silencing suppressors.

Agroinfiltration assay using green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c is a powerful method for screening of putative plant virus-encoded gene silencing suppressors. This method allows the investigator to know whether the putative viral suppressor inhibits silencing in a cell (local silencing) and/or spreading of silencing throughout a plant (systemic silencing). Additionally, grafting experiments using transgenic plants expressing the suppressor and the GFP will indicate whether the suppressor blocks systemic silencing steps, which include the production of a silencing signal in a silenced cell, and the cell-to-cell and long-distance movement of a silencing signal throughout a plant. Here, we describe methods and techniques of an agroinfiltration assay and grafting experiments, which were used for the characterization of Apple chlorotic leaf spot virus 50 kDa movement protein as a gene silencing suppressor. This protocol should allow the investigator to characterize putative plant virus-encoded gene silencing suppressors.

Molecular characterization of a new hypovirus infecting a phytopathogenic fungus, Valsa ceratosperma.

A double-stranded (ds) RNA, approximately 9.5kb in size; was identified in the MVC86 isolate of Valsa ceratosperma. Complete sequence of the dsRNA revealed a 9543-bp segment (excluding the 3' poly-A tail) that is predicted to encode a single large protein (P330). P330 has 63%, 49%, and 55% amino acid sequence identities to the proteins encoded by hypoviruses Cryphonectria hypovirus 3 (CHV3), CHV4, and Sclerotinia sclerotiorum hypovirus 1 (SsHV1), respectively. Like polyproteins encoded by CHV3, CHV4, and SsHV1, P330 comprises four conserved domains, including a papain-like protease, a UDP glucose/sterol glucosyltransferase (UGT), an RNA-dependent RNA polymerase (RdRp), and an RNA helicase. These molecular characteristics suggest that this dsRNA represents a new hypovirus that we tentatively designate Valsa ceratosperma hypovirus 1 (VcHV1). Phylogenetic analysis of the RdRp and RNA helicase domains of VcHV1 revealed that VcHV1, CHV3, CHV4, and SsHV1 clustered together into one clade distinct from that of CHV1 and CHV2, indicating the existence of two lineages in the family Hypoviridae. Comparison of biological properties between VcHV1-infected and VcHV1-free isogenic strains did not reveal differences in colony morphology or fungal virulence under laboratory conditions.

A novel colony-print immunoassay reveals differential patterns of distribution and horizontal transmission of four unrelated mycoviruses in Rosellinia necatrix.

A colony-print immunoassay (CPIA) using an anti-dsRNA antibody was developed to visualize the distribution of four unrelated mycoviruses with dsRNA genomes, a partitivirus (RnPV1), mycoreovirus (RnMyRV3), megabirnavirus (RnMBV1), and an unidentified virus (RnQV1), in mycelia of the white root rot fungus, Rosellinia necatrix. CPIA revealed different distribution patterns within single colonies for each virus. Both RnPV1 and RnMBV1 were distributed throughout single colonies, RnMyRV3 was absent from some colony sectors, and RnQV1 exhibited varied accumulation levels between sectors. RnMyRV3 and RnQV1 were transmitted to the recipient virus-free colonies of virus-infected and virus-free colony pairs more slowly than were RnPV1 or RnMBV1. The presence of RnMyRV3 in recipient colonies restricted horizontal transmission of RnPV1 and RnMBV1. These results imply that one or more mechanisms are present in host-virus and virus-virus interactions that restrict the spread of viruses within and between colonies.

Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes.

Apple latent spherical virus (ALSV) vectors were evaluated for virus-induced gene silencing (VIGS) of endogenous genes among a broad range of plant species. ALSV vectors carrying partial sequences of a subunit of magnesium chelatase (SU) and phytoene desaturase (PDS) genes induced highly uniform knockout phenotypes typical of SU and PDS inhibition on model plants such as tobacco and Arabidopsis thaliana, and economically important crops such as tomato, legume, and cucurbit species. The silencing phenotypes persisted throughout plant growth in these plants. In addition, ALSV vectors could be successfully used to silence a meristem gene, proliferating cell nuclear antigen and disease resistant N gene in tobacco and RCY1 gene in A. thaliana. As ALSV infects most host plants symptomlessly and effectively induces stable VIGS for long periods, the ALSV vector is a valuable tool to determine the functions of interested genes among a broad range of plant species.

Inhibition of long-distance movement of RNA silencing signals in Nicotiana benthamiana by Apple chlorotic leaf spot virus 50 kDa movement protein.

Apple chlorotic leaf spot virus 50 kDa movement protein (P50) acts as a suppressor of systemic silencing in Nicotiana benthamiana. Here, we investigate the mode of action of P50 suppressor. An agroinfiltration assay in GFP-expressing N. benthamiana line16c (GFP-plant) showed that P50 could not prevent the short-distance spread of silencing. In grafting experiments, the systemic silencing was inhibited in GFP-plants (scion) grafted on P50-expressing N. benthamiana (P50-plant; rootstock) when GFP silencing was induced in rootstock. In double-grafted plants, GFP-plant (scion)/P50-plant (interstock)/GFP-plant (rootstock), the systemic silencing in scion was inhibited when GFP silencing was induced in rootstock. Analysis of P50 deletion mutants indicated that the N-terminal region (amino acids 1-284) is important for its suppressor activity. In gel mobility shift assay, P50 lacks binding ability with siRNAs. These results indicated that P50 has a unique suppressor activity that specifically inhibits the long-distance movement of silencing signals.

Combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) in the coat protein of apple chlorotic leaf spot virus are crucial for infectivity.

Amino acid sequences of apple chlorotic leaf spot virus (ACLSV) coat protein (CP) were compared between 12 isolates from apple, plum and cherry, and 109 cDNA clones that were amplified directly from infected apple tissues. Phylogenetic analysis based on the amino acid sequences of CP showed that the isolates and cDNA clones were separated into two major clusters in which the combinations of the five amino acids at positions 40, 59, 75, 130 and 184 (Ala(40)-Val(59)-Phe(75)-Ser(130)-Met(184) or Ser(40)-Leu(59)-Tyr(75)-Thr(130)-Leu(184)) were highly conserved within each cluster. Site-directed mutagenesis using an infectious cDNA clone of ACLSV indicated that the combinations of two amino acids (Ala(40) and Phe(75) or Ser(40) and Tyr(75)) are necessary for infectivity to Chenopodium quinoa plants by mechanical inoculation. Moreover, an agroinoculation assay indicated that the substitution of a single amino acid (Ala(40) to Ser(40) or Phe(75) to Tyr(75)) resulted in extreme reduction in the accumulation of viral genomic RNA, double-stranded RNAs and viral proteins (movement protein and CP) in infiltrated tissues, suggesting that the combinations of the two amino acids at positions 40 and 75 are important for effective replication in host plant cells.

Apple chlorotic leaf spot virus 50 kDa movement protein acts as a suppressor of systemic silencing without interfering with local silencing in Nicotiana benthamiana.

Apple chlorotic leaf spot virus (ACLSV) is the type species of the genus Trichovirus and its single-stranded, plus-sense RNA genome encodes a 216 kDa protein (P216) involved in replication, a 50 kDa movement protein (P50) and a 21 kDa coat protein (CP). In this study, it was investigated whether these proteins might have RNA silencing-suppressor activities by Agrobacterium-mediated transient assay in the green fluorescent protein-expressing Nicotiana benthamiana line 16c. The results indicated that none of these proteins could suppress local silencing in infiltrated leaves. However, systemic silencing in upper leaves induced by both single- and double-stranded RNA could be suppressed by P50, but not by a frame-shift mutant of P50, P216 or CP. Moreover, when P50 was expressed separately from where silencing signals were generated in a leaf, systemic silencing in upper leaves was inhibited. Collectively, our data indicate that P50 acts as a suppressor of systemic silencing without interfering with local silencing, probably by inhibiting the movement of silencing signals.