Discrimination of four soybean dwarf virus strains by dot-blot hybridization with specific probes

Journal of Virological Methods 133 (2006) 219–222

Short communication

Discrimination of four soybean dwarf virus strains by dot-blot hybridization with specific probes Noriko Yamagishi a , Hidetaka Terauchi a,1 , Ken-ichiro Honda b , Seiji Kanematsu a , Soh Hidaka a,∗ a

National Agricultural Research Center for Tohoku Region, 4 Akahira, Shimo-kuriyagawa, Morioka, Iwate 020-0198, Japan b National Institute of Vegetable and Tea Science, Ano-cho, Age-gun, Mie 514-2392, Japan Received 6 July 2005; received in revised form 27 October 2005; accepted 27 October 2005 Available online 2 December 2005

Abstract Soybean dwarf virus (SbDV) is divided into four strains (YS, YP, DS, and DP) on the basis of host symptoms in infected soybean plants and on aphid vector specificity. To detect and discriminate each strain of SbDV by dot-blot hybridization, probes Y, D, S, and P were prepared. Probes Y and D, covering most of the 3 -noncoding region of the viral genome containing the sequence of small subgenomic RNA, could discriminate strains in accord with the host symptoms. Probes S and P were derived from the 5 -half of open reading frame 5 encoding the N-terminal half of the readthrough domain which is closely related to the aphid vector specificity of each strain. Thus, the four SbDV strains could be discriminated by the combination of these probes. This method, based on a procedure specific to the SbDV sequence, is a good alternative for routine examination of infected plants in soybean breeding programs for evaluation of resistance to SbDV and for assessment of the distribution of each strain in epidemiological studies. © 2005 Elsevier B.V. All rights reserved. Keywords: Dot-blot hybridization; DNA probe; Discrimination; SbDV; Strain

Soybean dwarf virus (SbDV) is a member of the family Luteoviridae. It causes serious yield losses in soybean production, especially in the northern part of Japan (Tamada, 1975). SbDV isolates are classified into four strains (YS, YP, DS, and DP) on the basis of host symptoms in infected soybean plants (Glycine max (L.) Merr.) and aphid vector specificity (Terauchi et al., 2001). Strains YS and YP cause yellowing in soybean, whereas strains DS and DP cause dwarfing (Tamada, 1973, 1975; Damsteegt et al., 1990; Mikoshiba et al., 1991; Honda et al., 1999). Strains YS and DS are transmitted by the aphid vector Aulacorthum solani (Kaltenbach), and strains YP and DP are transmitted by both Acyrthosiphon pisum (Harris) and Nearctaphis bakeri (Cowen) (Tamada, 1975; Mikoshiba et al., 1991; Honda et al., 1999). Each strain of SbDV has a unique distribution pattern in northern Japan (Mikoshiba et al., 1995), and the clear discrim∗

Corresponding author. Tel.: +81 19 643 3524; fax: +81 19 643 3524. E-mail address: [email protected] (S. Hidaka). 1 Present address: Invitrogen Japan, K.K. Haneda Laboratories, 2-4-3 Showajima, Ohta-ku, Tokyo 143-0004, Japan. 0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.10.028

ination of the four strains is important in assessing their distribution and determining which aphid vectors are present in the field. Discrimination of SbDV in infected plants has been based routinely on an enzyme-linked immunosorbent assay (ELISA) using specific monoclonal antibodies (MAbs) (Mikoshiba et al., 1994). These MAbs can discriminate between the different types of aphid transmissibility (strains YS and YP or strains DS and DP). Other methods are required to discriminate the four strains clearly. Another approach for detecting luteoviruses is based on RNA-sequence-specific procedures, such as hybridization with specific nucleic acid probes (Herrbach et al., 1991; Smith et al., 1993; Lemaire et al., 1995; Figueira et al., 1997; Singh, 1999) or reverse transcription-polymerase chain reaction (RT-PCR) with specific primer pairs. (Figueira et al., 1997; Singh, 1999). The complete nucleotide sequences of the genomic RNAs of representative isolates of the four strains, M93-1 (YS), M94-1 (YP), HS97-8 (DS) and M96-1 (DP) were determined (Terauchi et al., 2001). The genomic RNA of these strains consists of five open reading frames (ORFs). ORFs 1 and 2 encode the replicationrelated proteins; ORFs 3, 4, and 5 encode the coat protein, a

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N. Yamagishi et al. / Journal of Virological Methods 133 (2006) 219–222

Fig. 1. Schematic representation of the SbDV genome and localization of the probes used for dot-blot hybridization. Probes Y and P were derived from strain YP (M94-1: DDBJ accession no. AB038148), probe S from strain YS (M93-1: DDBJ accession no. AB038147), and probes D from strain DS (HS97-8: DDBJ accession no. AB038149), respectively. CP indicates coat protein. RTD indicates readthrough domain. Gray indicates N-terminal half of readthrough domain (N-RTD).

putative movement protein, and a readthrough domain (RTD), as in the case of SbDV Tas-1 reported by Rathjen et al. (1994) (Fig. 1). Comparison of the complete nucleotide sequences of these genomic RNAs of the four strains showed that sequence identity was highest between strains with the same symptoms – yellowing (strains YS and YP) and dwarfing (strains DS and DP) – except in the 5 -half of ORF 5, which encodes the Nterminal half of the readthrough domain (N-RTD). This region was related closely to the aphid vector specificity of SbDV, and the nucleotide sequences were very similar between strains YS and DS, and between strains YP and DP (Terauchi et al., 2001, 2003). These features in the sequence will facilitate the identification of the strains at the genome level. It was also reported that small subgenomic RNA (S-sgRNA) was constantly present in SbDV-infected leaves throughout infection, and accumulated in large amounts compared with the genomic RNA and the large subgenomic RNA (Yamagishi et al., 2003). Therefore, the S-sgRNA was expected to be a sensitive indicator of SbDV infection. From these results, a method of dot-blot hybridization was developed using symptom-specific probes (Y, D) and aphidspecific probes (S, P) for the discrimination of the four strains of SbDV. Several advantages of this method are discussed. SbDV was maintained in white clover (Y strains: M93-1, M94-1) and red clover (D strains: HS97-8, M96-1). To transmit each strain to soybean or broad bean by aphids, the method described by Terauchi et al. (2001) was used with some modifications. Briefly, aphids were allowed to acquire the virus from the infected clover at 15 ◦ C for 7 days, and then placed on healthy soybean or broad bean seedlings for inoculation under the same conditions. Inoculated plants were sprayed with insecticide before transfer into a greenhouse maintained at 25 ◦ C. Three weeks after inoculation, newly emerged leaves of each plant were tested for discrimination of the virus by dot-blot hybridization using specific probes. For the dot-blot hybridization, leaves were used of soybeans or broad beans infected with field isolates obtained from Hokkaido (HS99-5) and Iwate pre-

fectures (IK00-1, IK00-2, II00-1, II00-2, IT00-1, and IT00-2) (Terauchi et al., 2003) which had been stored at −80 ◦ C. Infected leaves (about 80 mg) were pulverized in a Micro Smash MS-100 bead beater (TOMY, Tokyo, Japan), and total RNA was extracted by using the acid guanidium–phenol–chloroform method (Chomczynski and Sacchi, 1987). Extracted total RNA was resuspended in 50 ␮l of RNase-free water. For RNA denaturation, 1 volume (50 ␮l) of RNA was combined with 3 volumes (150 ␮l) of solution containing 10× SSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7.0) and 18.6% formaldehyde, and incubated at 65 ◦ C for 15 min, followed by cooling on ice as described by Smith et al. (1993). Full-length cDNA clones of strains YS (M93-1), YP (M94-1), and DS (HS97-8) were constructed by assembling 1–3 kb cDNA segments which had been prepared for sequencing (Terauchi et al., 2001). Using these clones as templates, four probes were synthesized by PCR. Primers were designed for the amplification of specific regions on the SbDV genome as shown in Fig. 1 and Table 1. For dot-blot hybridization, Hybond-N+ membrane (Amersham Biosciences, NJ, USA) was treated with 20× SSC before use, and wells of Immunodot AE-6190 dot blotting apparatus (ATTO, Tokyo, Japan) were washed with 10× SSC before sample application. For discrimination of the strains, denatured RNA samples were divided into four aliquots for hybridization with each of probes Y, D, S, and P, and were applied to the dotblotting apparatus according to the instruction manual. After sample application, wells were washed with 10× SSC, then the membranes were treated with 20× SSC (20 min) and baked in an oven. Labelling of the probe, hybridization, washing and signal detection were carried out according to the instruction manual of AlkPhos Direct Labeling and Detection System (Amersham Biosciences), except that hybridization and washing were carried out at 60 ◦ C. For discrimination of the four strains of SbDV, probes Y, D, S, and P (Fig. 1) were prepared. Probes Y and D which were

Table 1 Primers used for probe preparation by PCR Probe

Corresponding position in the genome

Forward primer

Reverse primer

Y D S P

5502–5823 of the YP(M94-1) 5408–5689 of the DS(HS97-8) 3874–4399 of the YS(M93-1) 3751–4399 of the YP(M94-1)

5 -GCTTCTGGTGATTACACTGC-3

5 -GCCACCTTAACAACAAAGAGG-3

5 -CTCACAGAACTCTGTTGAGGC-3 5 -ATCAACGCTTATCAGTGCCC-3

5 -GACGCTAATATGACCTGGTC-3

N. Yamagishi et al. / Journal of Virological Methods 133 (2006) 219–222

Fig. 2. Discrimination of four strains of SbDV by dot-blot hybridization using specific probes. Total RNA was extracted from broadbeans infected with strains YS (M93-1), YP (M94-1), DS (HS97-8), and DP (M96-1: DDBJ accession no. AB038150), respectively. H indicates healthy plant.

designed to avoid cross-hybridization between strains Y and D, can discriminate strains in agreement with host symptoms. These probes covered the 3 -terminal region of the genome with 66–67% similarity between strains Y and D, but 93–95% similarity between strains S and P. Therefore, probe Y detected strains YS and YP, and probe D detected strains DS and DP (Fig. 2). Probes Y and D cover the 3 -noncoding region containing the sequence of S-sgRNA (Yamagishi et al., 2003). As S-sgRNA can be detected even in senescent infected leaves, when other viral-specific RNAs are hardly visible by Northern blot analysis (results not shown), S-sgRNA was expected to be the most sensitive target for the detection of SbDV from a small amount of infected leaves. Further, probes S and P, covering almost entire 5 half of ORF 5 (78–79% similarity between strains S and P, in contrast to 88–98% between strains Y and D), could discriminate the aphid vector specificity of each strain. Thus, probe S detected strains YS and DS, and probe P detected strains YP and DP (Fig. 2). None of the healthy plants gave any background signals, irrespective of the probe used (Fig. 2).

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To investigate the effectiveness of this method, RNA extracted from infected leaves of the seven field isolates was tested (Fig. 3). Six isolates hybridized strongly with probe Y. Of these, four that hybridized with probe S were identified as strain YS, and two isolates that hybridized with probe P were strain YP. The remaining isolate hybridized with probes D and S, and was classified as strain DS. Thus, Fig. 3 shows that classification of the seven SbDV field isolates by this dot-blot hybridization method agreed well with the results based on their host symptoms and aphid vector specificity (Terauchi et al., 2003). One of the advantages of dot-blot hybridization assays over serological assays is that detection can be carried out without the need for specific antibodies, which are sometimes difficult to produce. Polyclonal and monoclonal antibodies have been used routinely in ELISA for detection of SbDV (Tamada, 1975; Mikoshiba et al., 1994). However, these antibodies are not sufficiently specific to discriminate the four strains effectively. In some instances, an isolate of strain Y identified by biological assay did not react with Y-specific MAb in ELISA, for an unknown reason, and RT-PCR was necessary to confirm the strain (Honda, unpublished results). RT-PCR is a useful method for detecting viral RNA sequences, and is the most sensitive (Figueira et al., 1997; Singh, 1999). Four specific primers based on ORF 5 designed for discrimination of the strains by RT-PCR could be used to classify many field isolates successfully, but the amplified DNA of several isolates could not be detected due to a change in the nucleotide sequence within the primer-specific region (Terauchi, unpublished results). In dot-blot hybridization, the intensity of the dot signal is largely unaffected by minor changes in the sequence of the target region, and the signal can be identified easily on exposed film. The flexibility of hybridization to specific sequences can help resolve ambiguities in RT-PCR or ELISA assays. This method is able to discriminate the four SbDV strains effectively without laborious steps, as in the case of RT-PCR in dealing with a large number of samples. Therefore, the dot-blot hybridization assay is a very suitable method for routine analysis of SbDV strains, although these methods should complement each other for more reliable discrimination of the virus.

Fig. 3. Discrimination of seven field isolates of SbDV by dot-blot hybridization using specific probes. Seven isolates of SbDV (IK00-1, IK00-2, II00-1, II00-2, IT00-1, IT00-2, and HS99-5) which had been classified based on host symptoms and aphid vector specificity were tested. Total RNA was extracted from soybeans infected with IK00-1, II00-2, and HS99-5 or from broadbeans infected with IK00-2, II00-1, IT00-1, and IT00-2. H indicates healthy plant.

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