Molecular characterization of dsRNA segments 2 and 5 and electron microscopy of a novel reovirus from a hypovirulent isolate, W370, of the plant pathogen Rosellinia necatrix

Chuan Zhao Wei1, Hideki Osaki1, Toru Iwanami1,{dagger}, Naoyuki Matsumoto2 and Yoshihiro Ohtsu1

1 National Institute of Fruit Tree Science, Fujimoto, Tsukuba 305-8605, Japan
2 National Institute for Agro-Environmental Sciences, Kan-nondai, Tsukuba 305-8604, Japan

Correspondence
Chuan Zhao Wei
chenwei{at}vesta.ocn.ne.jp


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A hypovirulent isolate, W370, of the white root rot fungus Rosellinia necatrix has previously been shown to harbour 12 dsRNA segments. In this study, complete nucleotide sequences of segments 2 and 5 of W370 dsRNAs were determined. The nucleotide sequence of genome segment 2 was 3773 bases long with a single long open reading frame (ORF) encoding 1226 amino acid residues with a predicted molecular mass of approximately 138·5 kDa. The nucleotide sequence of segment 5 was 2089 bases long with a single long ORF, whose deduced polypeptide contained 646 amino acid residues with a predicted molecular mass of about 72 kDa. Comparative analysis showed that the deduced protein sequence of segment 2 had significant homology with the putative VP2 of Colorado tick fever virus (CTFV) and European Eyach virus (EYAV) in the genus Coltivirus, but the deduced protein sequence of segment 5 had no similarity with other virus proteins. Double-shelled spherical particles approximately 80 nm in diameter associated with W370 dsRNAs were observed in a preparation from the mycelial tissue of isolate W370. The results demonstrated that the virus associated with W370 dsRNAs is a novel reovirus of the family Reoviridae. The virus was named Rosellinia anti-rot virus (RArV).

The GenBank accession numbers of the sequences reported in this paper are AB098022 (segment 2) and AB098023 (segment 5).

{dagger}Present address: National Agricultural Research Center for Kyushu Okinawa Region, Suya 2421, Nishigoshi, Kumamoto 861-1192, Japan.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
White root rot, also known as Rosellinia (Dematophora) root rot, is a destructive disease of about 170 species of plants in 63 genera (Sztejnberg, 1994). The disease has a wide geographical distribution in all temperate regions of the world (Sztejnberg, 1994). In Japan, it is one of the most serious diseases of fruit trees and large amounts of fungicide have been used for its control in Japanese pear and apple orchards. During a search for hypovirulent isolates of the white root rot fungus Rosellinia necatrix Prillieux to exploit for biocontrol of the disease, a hypovirulent isolate, W370, was found (Arakawa et al., 2001, 2002). Isolate W370 was found to contain 12 dsRNA species and its hypovirulence was ascribed to the presence of these dsRNAs (Arakawa et al., 2001, 2002).

The 12 dsRNA species were designated segments (S) 1–12, on the basis of their electrophoretic mobility (Osaki et al., 2002). Previous sequence analysis of the eight smaller segments has suggested that W370 dsRNA segments might have originated from a member of the virus family Reoviridae (Osaki et al., 2002). In this paper we report the complete nucleotide sequences of W370 dsRNA genome segments 2 and 5. Double-shelled spherical particles of approximately 80 nm in diameter associated with W370 dsRNAs were observed in a preparation from the mycelial tissue of isolate W370. These results show that isolate W370 of R. necatrix harbours a novel reovirus member of the family Reoviridae. Since it has potential for use as a biocontrol agent of white root rot disease, we named it Rosellinia anti-rot virus (RArV).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Fungal isolates and culture conditions.
Isolate W370 of R. necatrix was a dsRNA-containing hypovirulent strain. An isogenic dsRNA-free isolate derived from isolate W370 and a dsRNA-free isolate W380 of R. necatrix were used as control materials for electron microscopy (Arakawa et al., 2001, 2002). They were cultured and maintained on oatmeal agar. For purification of dsRNAs and virus particles, the isolates were grown in 50 ml of oatmeal extract broth (15 g Quaker oatmeal in 1000 ml H2O) at 25 °C in the dark for 7 days, then transferred into 1000 ml of fresh oatmeal extract broth and incubated for 7 days at 25 °C in the dark.

Extraction and purification of dsRNAs.
Total dsRNA was prepared as described by Morris & Dodds (1979), with minor modifications (Osaki et al., 2002). The dsRNA was concentrated by ethanol precipitation and further purified using RNeasy Plant Mini Kits (Qiagen). The RNaid kit (BIO101) was used to isolate and purify dsRNA segments from polyacrylamide gels.

cDNA synthesis and cloning.
One cDNA library was synthesized, starting with 3 µg of purified total dsRNA. First-strand synthesis for the library was primed with 0·037 µg pd(N)6 random hexamer. A total volume of 20 µl containing primers and dsRNA mixture in water was denatured by boiling for 10 min. The TimeSaver cDNA synthesis kit (Amersham Pharmacia) was used for cDNA synthesis. Double-stranded cDNA was passed through a Sizesep 400 spun column to remove cDNA products with a length under 400 bp. The purified cDNA was ligated into the EcoRI site of pUC118, after addition of an EcoRI/NotI adaptor and transformed into Escherichia coli DH5{alpha} (Toyobo).

General recombinant DNA methods were as described in Sambrook et al. (1989). Recombinant plasmids were screened from ampicillin-resistant white colonies using the QIAprep Spin Miniprep Kit (Qiagen).

Nothern blotting.
Northern blot hybridization analysis was performed as described by Valverde et al. (1990), with minor modifications. Prior to electroblotting, dsRNA in polyacrylamide gels was denatured with 50 % (v/v) DMSO and 1 M glyoxal in 1x TBE at 50 °C for 1 h. Gels were electroblotted on to nylon membranes in the presence of 1x TBE at 100 mA for 2 h. Membranes were baked at 120 °C for 2 h and used for hybridization. Digoxigenin (Dig)-labelling and detection were carried out following the protocol of the DIG DNA labelling and detection kit (Boehringer Mannheim).

RT-PCR.
Plasmids containing cDNA inserts of 1·5 kb and longer were sequenced. PCR primers were designed from the terminal sequences of inserts and used for RT-PCR on isolated segments to confirm the identity of the template. RT-PCR was carried out using the RNA LA PCR kit (AMV) (Version 1.1) (TaKaRa) and 20 µM of each primer was used. Reverse transcription was carried out at 42 °C for 60 min. PCR amplification was carried out using 35 cycles of denaturation at 94 °C for 0·5 min, annealing at 45 °C for 0·5 min and extension at 72 °C for 3 min, with a final extension step at 72 °C for 7 min.

The distal ends of S2 and S5 were amplified by the 5' RACE (rapid amplification of cDNA ends) approach (Frohman, 1990) using a 5' RACE kit (Invitrogen). Segments 2 and 5 isolated from polyacrylamide gels were used as template dsRNA. The RT-PCR products and 5' RACE products were cloned into the pCR2.1-TOPO vector using the TOPO TA cloning kit (Invitrogen).

Sequence analysis.
DNA sequences were analysed using GENETYX-WIN software (Software Development Co.). RNA secondary structures in both termini of the dsRNA segments were predicted using the program DNASIS Version 2.1 (Hitachi Software Engineering). Comparison of sequences with those available from nucleic acid and protein databases was performed using the NCBI BLAST 2.0 program (http://www3.ncbi.nlm.gov/BLAST). Multiple sequence alignment was performed using CLUSTAL W (Thompson et al., 1994).

The Pfam program (http://www.sanger.ac.uk/Pfam/search.shtml) was used to search for previously described protein family domains. The Motif program (http://www.motif.genome.ad.jp) was used to analyse the theoretical protein sequences for the presence of known functional amino acid motifs.

Virus particle purification and electron microscopy.
Extraction of particle-containing fractions from the mycelia was performed with phosphate buffer (Kimura & Black, 1971; Omura et al., 1982), with minor modifications. W370 mycelia (100 g) were frozen in liquid nitrogen and ground to a fine powder. Potassium phosphate buffer (PB; 0·1 M, pH 7·2, 400 ml) was added to the ground mycelia. The mixture was stirred for 1 h, then passed through four layers of finely woven cotton cloth. Triton X-100 was added to a final concentration of 2 %. The mixture was stirred for 60 min, then centrifuged for 15 min at 3000 g. Polyethylene glycol 6000 and NaCl were added to the supernatant fluid to final concentrations of 6 % (w/v) and 0·3 M, respectively. The mixture was stirred for 40 min, then centrifuged for 15 min at 6000 g.

The resulting pellets were resuspended in 9 ml 0·1 M PB, pH 7·0. Carbon tetrachloride was added to a final concentration of 10 % and vortexed for 1 min. After centrifugation at 3000 g for 15 min, the supernatants were layered on to 1 ml sucrose cushions (20 % sucrose, w/v, in 0·1 M PB, pH 7·0) and centrifuged at 96 000 g for 2 h. The pellets were resuspended in 0·01 M PB, pH 7·0, containing 0·01 M MgCl2 (PB-Mg) (MgCl2 was added just before use) for 1 h and centrifuged for 15 min at 3000 g. The supernatant was layered on to 10–40 % (w/v) linear sucrose gradients in PB-Mg and centrifuged for 80 min at 62 800 g in a Hitachi SRP-28SA1 rotor. Two bands, which corresponded to inner capsids and intact particles, respectively, were observed and collected separately. Appropriate fractions were centrifuged for 2 h at 96 000 g in a Hitachi RP-65T-320 rotor and the final pellets were resuspended in PB-Mg.

Particle preparations were stained with either 2 % phosphotungstic acid (PTA), pH 7·0, or 2 % aqueous uranyl acetate and then examined with a JEM-1200 EX electron microscope.

Isolate W380 and the isogenic dsRNA-free isolate from W370 were also treated in the same way.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sequence determination of S2 and S5
dsRNA isolated from the hypovirulent isolate of the white root rot fungus R. necatrix W370 was separated by electrophoresis into 12 discrete sizes (S1–S12) (Fig. 1). In our previous report (Osaki et al., 2002) we were unable to separate segments 4 and 5.



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Fig. 1. Electrophoresis of dsRNAs extracted from R. necatrix strain W370 on a 5 % polyacrylamide gel. Lane 1, 12 dsRNA genome segments of rice dwarf virus as a dsRNA marker; lane 2, dsRNA preparation from mycelia of W370. The gels were stained with ethidium bromide and the nucleic acids visualized by UV.

 
The S2 and S5 specificity of five cDNA clones with inserts longer than 1·5 kb were characterized by Northern blot analysis and preliminary sequence analysis. Primers (18- to 20-mers) were designed from the terminal sequences of the inserts and used for RT-PCR on isolated segments 2 and 5 to confirm the identity of the template. In all experiments, eight specific RT-PCR products were amplified by five sets of primers. On comparison of these sequences, all PCR products were divided into just two groups. Nothern blot hybridization analysis confirmed that one corresponded to S2 and the other to S5.

Using the sequences of these RT-PCR products, the cDNA sequences of the entire dsRNA segments 2 and 5 could be completed, with the exception of the terminal sequences. The remainder of the S2 and S5 sequences was then determined by sequencing the 5' RACE products, using specific primers derived from the RT-PCR products or nested primers from the truncated 5' RACE products. The distal 3' end of S5 was determined according to the protocol for 5' RACE of GC-rich cDNA suggested by the manufacturer, since the dCTP-tailing method resulted in truncated products.

Sequences analysis of segments 2 and 5
The complete nucleotide sequences of S2 and S5 were 3773 and 2089 bases long, with GC contents of 50 % and 49·5 %, respectively. Sequence comparisons of the two segments with those available from nucleic acid databases showed no significant homology with reported virus sequences at the nucleotide level.

The extreme 5' and 3' ends of the sense strand of both segments had the sequence 5'-ACAAUUU...UGCAGAC-3'. The same terminal sequence has also been identified in eight other previously analysed segments (S4, S6–12) (Osaki et al., 2002). Thus, as commonly found for members of the family Reoviridae, all segments were found to have conserved sequences located at the termini. Such conserved motifs are detected in many viruses possessing multisegmented genomes and have been suggested to play an important role in transcription, replication and packaging of RNA, as well as in virus maturation (Attoui et al., 1997; Anzola et al., 1987; Xu et al., 1989). Segment-specific panhandle structures, formed by inverted terminal repeats, were found in the two segments (data not shown).

A single long open reading frame (ORF) was present in S2. The deduced polypeptide (designated VP2) contained 1226 amino acid residues (nt 63–3740) with a predicted molecular mass of about 138·5 kDa. A single long ORF was also present in S5. The deduced polypeptide (designated VP5) contained 646 amino acid residues (nt 46–1983) with a predicted molecular mass of about 72 kDa.

The BLAST 2.0 program was used to search for protein sequence homologies. The deduced VP2 protein sequence showed homology with the putative VP2 of Colorado tick fever virus (CTFV; 23 % identity, 39 % similarity) and the putative VP2 of European Eyach virus (EYAV; 24 % identity, 39 % similarity) (Attoui et al., 2000, 2002). Analysis of amino acid sequences using the NCBI BLAST program showed that the deduced VP5 protein sequence had no similarity to other viral proteins.

A search for functional motifs in the deduced protein sequences encoded by the ORFs of S2 and S5 was conducted. The VP2 sequence was found to contain a methyltransferase family motif (LATKIAFLMNPLAYERNRHVKAAVLFDFIR) at aa 510–539, which is also conserved in VP2 of CTFV and EYAV (Fig. 2). Therefore, these putative proteins are proposed to act as methyltransferases (Attoui et al., 2000, 2002). No known functional amino acid motifs or previously described protein domains were found in the sequence of S5 using either the Motif or the Pfam programs.




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Fig. 2. Alignment of the putative VP2 protein sequence from the ORF of segment 2 of R. necatrix W370 dsRNA virus (RAr-VP2) with VP2 of Colorado tick fever virus (CTF-VP2) and European Eyach virus (EYA-VP2) (Attoui et al., 2000, 2002). Amino acids identical among the three viruses are indicated with an asterisk; chemically similar amino acids are indicated with one or two dots.

 
Electron microscopy
The virus particles appeared to be tightly bound to cellular material, since only a few virus particles of approximately 80 nm in diameter were observed in the crude preparation of hypovirulent isolate W370 after cellular debris had been removed by low speed centrifugation (10 000 g, 10 min) (Fig. 3A). On the other hand, no such particles were observed in the preparation from mycelial tissue from the dsRNA-free strain that originated from the hypovirulent isolate W370 or from the healthy isolate W380 of R. necatrix. Double-shelled spherical particles approximately 80 nm in diameter were observed in the partially purified preparations from the mycelial tissue of hypovirulent strain W370 containing dsRNAs (Fig. 3B). Round particles 60–65 nm in diameter were also observed in the preparation from the mycelial tissue of strain W370 containing dsRNA that had been partially purified by centrifugation through sucrose cushions (Fig. 3C). From observations of partially degraded virus particles and virus particles variably penetrated by stain, it was concluded that the 60–65 nm particles represented the virion inner capsid core with B spikes (Milne & Lovisolo, 1977) and that the 80 nm particles represented intact virions with the typical double shells (Fig. 3B, C). This conclusion was confirmed by dsRNA extraction from different fractions of a sucrose density gradient containing the two particles of 80 nm and 60–65 nm in diameter (Fig. 3D, E); the two different particles contained the same 12 genome segments of dsRNA with the same electrophoretic profile as the dsRNA segments extracted from the mycelial tissue of R. necatrix W370 (data not shown). (These particles were disrupted with 2 % PTA, pH 7·0; data not shown.)



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Fig. 3. Rosellinia anti-rot virus (RArV) particles from mycelia of W370 of R. necatrix. (A) Intact virus particles from the crude preparation. (B) An intact virus particle from the partially purified preparation. (C) The spiked core particles from the partially purified preparation (arrows indicate B spikes). (D) A purified intact virus particle. (E) A purified spiked core particle (arrow indicates B spike). Viral preparations were stained with uranyl acetate and are shown at the same magnification. Bar, 100 nm.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This study reports the first observation of viral particles containing W370 dsRNAs in preparations from the mycelial tissue of isolate W370 of R. necatrix. RArV shares many properties with members of the family Reoviridae, such as double-shelled particles (Mertens et al., 2000). Intact particles of RArV were found to be similar in size to those of CTFV and EYAV (80 nm in diameter) (Attoui et al., 2002), but the inner capsids of RArV (60–65 nm) were slightly larger than those of CTFV and EYAV (50 nm). The RArV particles had a B-spiked core and showed a resemblance to Fijivirus particles (Milne & Lovisolo, 1977); however, similar surface projections have not been observed with CTFV or EYAV particles, in the genus Coltivirus. Whereas reoviruses such as Coltivirus and Fijivirus have been characterized from invertebrate, vertebrate and plant hosts, none has yet been characterized from a fungal host. The relative size and the number of segments of W370 dsRNAs were similar to those of the dsRNA molecules in C-18 of Cryphonectria parasitica, but the size of the RArV particles (80 nm) was distinct from those found in C. parasitica (Enebak et al., 1994).

Previous sequence analysis of the eight smaller segments (S4, S6–12) suggested that W370 dsRNA segments might have originated from a member of the family Reoviridae. Conserved terminal sequences at both 5' and 3' ends were found in all eight segments and the deduced amino acid sequences of W370 dsRNAs showed partial similarities to several proteins of viruses in the family Reoviridae (Osaki et al., 2002.). Combined with the results of the sequence analysis of these eight smaller segments, the lack of any significant homology in the majority of gene products supports the suggestion that W370 dsRNAs represent a virus from a member of a distinct new genus of the family Reoviridae (Mertens et al., 2000). However, the sequences of segments 1 and 3 have yet to be determined.

Hypovirulent isolate W370 contained RArV particles associated with 12 dsRNA segments, but the same particles were not found in an isogenic dsRNA-free isolate from W370 obtained by hyphal tip isolation. Similarly, viral particles and dsRNAs were not detected from a healthy isolate, W380, of R. necatrix; these results suggest that RArV acts as a hypovirulence factor to the host R. necatrix. We may thus be able to use the virus to attenuate fungal virulence to control white root rot by transfection of the virus into the virulent host R. necatrix directly or by as yet unidentified potential vectors.


   ACKNOWLEDGEMENTS
 
This research was supported by the programme for Promotion of Basic Research Activities for Innovative Biosciences through the Bio-oriented Technology Research Advancement Institution (BRAIN). We thank Dr Omura, National Agricultural Research Center, Tsukuba, Japan, for providing the purified RDV and for helpful discussions of results prior to publication.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 15 January 2003; accepted 7 May 2003.