Hideki Osaki

Nucleotide Sequences of Double-Stranded RNA Segments from a Hypovirulent Strain of the White Root Rot Fungus Rosellinia necatrix: Possibility of the First Member of the Reoviridae from Fungus

Twelve double-stranded (ds) RNA segments were detected from a hypovirulent strain W370 of the white root rot fungus Rosellinia necatrix. The estimated molecular weights ranged from 0.41×106 to 2.95×106. Full length cDNA clones for eight segments were obtained. Northern blot analysis suggested that each segment was genetically unique. The nucleotide sequences of eight full length dsRNA segments were determined. One long open reading frame was found in each segment. Conserved sequences at the 5′-end (5′-ACAAUUU-3′) and at the 3′-end (5′-UGCAGAC-3′) were identified in all eight segments. Segment-specific panhandle structures, formed by inverted terminal repeats, were also found in all segments. Comparative analyses of the predicted translational products of eight dsRNA segments showed that the deduced amino acid sequence partially matched those of the Reoviridae family members: Colorado tick fever virus, Nilaparvata lugens reovirus, and rice black streaked dwarf virus. The results suggested that W370 dsRNA is derived from a new member of the family Reoviridae detected in fungus.

Detection of a double-stranded RNA virus from a strain of the violet root rot fungus Helicobasidium mompa Tanaka.

Three double-stranded (ds) RNA species (ca. 1.30, 1.27 and 1.23×106) were isolated by CF-11 cellulose chromatography from a strain of the violet root rot fungus Helicobasidium mompa recovered from apple roots. Purified virion preparations contained isometric particles about 25nm in diameter, and also the same three species of dsRNA isolated from total extracts by CF-11 cellulose chromatography. The molecular mass of the coat protein was about 67K when estimated by SDS-PAGE. The largest dsRNA (referred to as dsRNA1) contains a single, long open reading frame of 1794 nucleotides that encodes a putative polypeptide containing 598 amino acid residues with a molecular mass of 69.9K. This polypeptide contains amino acid sequence motifs conserved in putative RNA-dependent RNA polymerases of RNA viruses. Phylogenetic analysis revealed similarities to RNA-dependent RNA polymerases from Atkinsonella hypoxylon 2H virus, a member of the family Partitiviridae.

Cloning and characterization of a Totivirus double-stranded RNA from the plant pathogenic fungus, Helicobasidium mompa Tanaka

Virus-like particles (VLPs, named HmTV1-17), about 40 nm in diameter were found in the violet root rot fungus Helicobasidium mompa Tanaka strain No. 17, which had been isolated from an apple tree. Purified preparations of HmTV1-17 contained two species of double-stranded RNA (dsRNA), designated 17L and 17S. cDNAs were constructed from HmTV1-17 genomic dsRNAs purified using CF-11 cellulose column chromatography. The sequences of 17L and 17S cDNA comprised 5207 and 2096 bp, respectively. Although 17S has no large open reading flame (ORF) on either strand, 17L has two large overlapping ORFs. The 5′ located ORF1 encodes the coat protein (CP, 788 amino acids), whereas the gene product of ORF2, which is in the −1 frame relative to ORF1, shows the typical features of a RNA dependent RNA polymerase (RDRP, 845 amino acids). Phylogenetic analysis based on RDRP showed that HmTV1-17 is closely related to Sphaeropsis sapinea SsRV1, a member of the genus Totivirus from filamentous fungus S. sapinea.

Characterization of double-stranded RNA elements in the violet root rot fungus Helicobasidium mompa

Double-stranded (ds) RNA of various types was detected by electrophoresis in 23 of 25 isolates of Helicobasidium mompa. These dsRNAs varied in size from ca. 2 kbp to more than 10 kbp. dsRNAs from an isolate V1 had two distinct nucleotide sequences for putative RNA-dependent RNA polymerase (RDRP). Their complete sequences revealed that V1 dsRNA1 was 2247 bp in length, with a single ORF that encoded a 706-amino acid residue polypeptide with a predicted molecular mass of 82.6 kDa, and that V1 dsRNA3 was 1776 bp in length, with a single ORF that encoded a 538-amino acid residue polypeptide with a predicted molecular mass of 62.6 kDa. RDRP-conserved motifs were identified in both predicted amino acid sequences. Phylogenetic analysis indicated that V1 dsRNA1 was most closely related to Fusarium poae virus 1, while V1 dsRNA3 was most closely related to Helicobasidium mompa 70 virus. These results indicate coinfection of isolate V1 by two distinct partitiviruses.