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.

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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.

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

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.

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.

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.