Hajime Yaegashi

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.

Characterization of virus-induced gene silencing in tobacco plants infected with apple latent spherical virus

SummaryApple latent spherical virus (ALSV) expressing green fluorescent protein (GFP-ALSV) was used for analysis of virus-induced gene silencing (VIGS) in tobacco plants expressing GFP (GFP-tobacco). In GFP-tobacco inoculated with GFP-ALSV, small dark spots appeared on inoculated leaves at 5 days post-inoculation (dpi), then expanded, and finally covered the whole area of the leaves after 12 dpi. Most of the fluorescence of upper leaves above the 12th true leaf disappeared at 21 dpi. Thus, GFP-ALSV infection efficiently triggered VIGS of a transgene (GFP gene) in tobacco plants. Analysis of GFP-silenced leaves showed that viral RNAs and proteins accumulated in all leaves where most GFP mRNA had been degraded. The siRNAs derived from ALSV-RNAs were not detected in samples from which siRNA of GFP mRNA could be easily detected. Direct tissue blot analysis showed that the spread of GFP-ALSV always preceded the induction of VIGS in infected leaves of GFP-tobacco. GFP leaf patch tests using Nicotiana benthamiana line 16c showed that Vp20, one of the three capsid proteins, is a silencing suppressor which interferes with systemic silencing.

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.

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, 72–82%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’’.

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.

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

ABSTRACT 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.

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.

Apple necrotic mosaic virus, a novel ilarvirus from mosaic-diseased apple trees in Japan and China

The causal agent of apple mosaic disease has been previously thought to be solely caused by apple mosaic virus (ApMV). In this study, we report that a novel ilarvirus is also associated with apple mosaic disease. Next-generation sequencing analysis of an apple tree showing mosaic symptoms revealed that the tree was infected with three apple latent viruses (apple stem pitting virus, apple stem grooving virus, and apple chlorotic leaf spot virus) and a novel ilarvirus (given the name apple necrotic mosaic virus (ApNMV)) that is closely related to Prunus necrotic ringspot virus (PNRSV) and ApMV. The genome of ApNMV consists of RNA1 (3378 nt), RNA2 (2767 nt), and RNA3 (1956 nt). A phylogenetic analysis based on the coat protein amino acid sequences indicated that the novel virus belongs to the same subgroup 3 of the genus Ilarvirus as PNRSV and ApMV. The presence of mosaic leaves, which tend to be unevenly distributed in diseased apple trees, was correlated with the internal distribution of ApNMV. RT-PCR detection of mosaic-diseased apple trees in Japan indicated that ApNMV was detected in apple trees introduced from China, whereas ApMV was detected from cultivated apple trees in domestic orchards. Consistent with these findings, a survey of mosaic-diseased apple trees in major apple-producing provinces in China revealed that the majority of apple trees showing mosaic symptoms in China are infected with ApNMV.