Recombinant plant dsRNA-binding protein as an effective tool for the isolation of viral replicative form dsRNA and universal detection of RNA viruses

  • Kappei KobayashiEmail author
  • Reiko Tomita
  • Masaru Sakamoto


The isolation of viral replicative form (RF) double-stranded RNA (dsRNA) is a classic technique for plant virus detection when the virus species cannot be predicted from disease symptoms. However, the method has not been very widely used, most likely because dsRNA isolation using CF-11 cellulose is laborious and time-consuming. Here we report an alternative tool, a recombinant plant dsRNA-binding protein, to isolate dsRNA. This tool enables us to isolate viral RF dsRNA in an hour from either extracted nucleic acids or crude detergent extracts. Combining this technique with sequence-non-specific reverse transcription, PCR amplification, cloning, and sequencing, a variety of viruses were efficiently detected using a single set of reagents and procedures.


Detection dsRNA dsRNA-binding protein Replicative form RNA virus 



We thank Tetsuya Mizutani for advice on the experiments, David Baulcombe for PVX and TRV vectors, Yoshikatsu Genda for PVY, Yasuya Iwadate for TSWV-infected material and Kazue Obara for technical assistance. This study was supported by the Iwate Prefectural Government.

Supplementary material

10327_2009_155_MOESM1_ESM.doc (590 kb)
Supplementary material 1 (DOC 590 kb)
10327_2009_155_MOESM2_ESM.doc (35 kb)
Supplementary material 2 (DOC 35 kb)
10327_2009_155_MOESM3_ESM.doc (32 kb)
Supplementary material 3 (DOC 31 kb)


  1. Aramburu J, Navas-Castillo J, Moreno P, Cambra M (1991) Detection of double-stranded RNA by ELISA and dot immunobinding assay using an antiserum to synthetic polynucleotides. J Virol Methods 33:1–11PubMedCrossRefGoogle Scholar
  2. Brigneti G, Martín-Hernández AM, Jin H, Chen J, Baulcombe DC, Baker B, Jones JDG (2004) Virus-induced gene silencing in Solanum species. Plant J 39:264–272PubMedCrossRefGoogle Scholar
  3. Chapman S, Kavanagh T, Baulcombe D (1992) Potato virus X as a vector for gene expression in plants. Plant J 2:549–557PubMedGoogle Scholar
  4. Chaumpluk P, Sasaki Y, Nakajima N, Nagano H, Nakamura I, Suzuki K, Mise K, Inouye N, Okuno T, Furusawa I (1996) Six new subgroup I members of Japanese cucumber mosaic virus as determined by nucleotide sequence analysis on RNA3’s cDNAs. Ann Phytopathol Soc Jpn 62:40–44Google Scholar
  5. Clark MF, Adams AN (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J Gen Virol 34:475–483PubMedCrossRefGoogle Scholar
  6. Dodds JA, Morris TJ, Jordan RL (1984) Plant viral double-stranded RNA. Annu Rev Phytopathol 22:151–168CrossRefGoogle Scholar
  7. Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA (eds) (2005) Virus taxonomy: VIIIth Report of the International Committee on Taxonomy of Viruses. Academic Press, New YorkGoogle Scholar
  8. Hamada H, Tomita R, Iwadate Y, Kobayashi K, Munemura I, Takeuchi S, Hikichi Y, Suzuki K (2007) Cooperative effect of two amino acid mutations in the coat protein of Pepper mild mottle virus overcomes L 3-mediated resistance in Capsicum plants. Virus Genes 34:205–214PubMedCrossRefGoogle Scholar
  9. Hiraguri A, Itoh R, Kondo N, Nomura Y, Aizawa D, Murai Y, Koiwa H, Seki M, Shinozaki K, Fukuhara T (2005) Specific interactions between Dicer-like proteins and HYL1/DRB-family dsRNA-binding proteins in Arabidopsis thaliana. Plant Mol Biol 57:173–188PubMedCrossRefGoogle Scholar
  10. Jelkmann W, Martin RR, Maiss E (1989) Cloning of four plant viruses from small quantities of double-stranded RNA. Phytopathology 79:1250–1253CrossRefGoogle Scholar
  11. Kihara Y, Satho T, Eshita Y, Sakai K, Kotaki A, Takasaki T, Rongsriyam Y, Komalamisra N, Srisawat R, Lapcharoen P, Sumroiphon S, Iwanaga S, Ushijima H, Endoh D, Miyata T, Sakata A, Kashige N, Miake F, Fukushi S, Saijo M, Kurane I, Morikawa S, Mizutani T (2007) Rapid determination of viral RNA sequences in mosquitoes collected in the field. J Virol Methods 146:372–374PubMedCrossRefGoogle Scholar
  12. Lukács N (1994) Detection of virus infection in plants and differentiation between coexisting viruses by monoclonal antibodies to double-stranded RNA. J Virol Methods 47:255–272PubMedCrossRefGoogle Scholar
  13. Mizutani T (2007) Establishment of a system for determination of emerging infectious diseases (RDV method), and its application (in Japanese). Uirusu 57:217–225PubMedCrossRefGoogle Scholar
  14. Mizutani T, Endoh D, Okamoto M, Shirato K, Shimizu H, Arita M, Fukushi S, Saijo M, Sakai K, Lim CK, Ito M, Nerome R, Takasaki T, Ishii K, Suzuki T, Kurane I, Morikawa S, Nishimura H (2007) Rapid genome sequencing of RNA viruses. Emerg Infect Dis 13:322–324PubMedCrossRefGoogle Scholar
  15. Morris TJ, Dodds JA, Hillman B, Jordan RL, Lommel SA, Tamaki SJ (1983) Viral specific dsRNA: diagnostic value for plant virus disease identification. Plant Mol Biol Rep 1:27–30CrossRefGoogle Scholar
  16. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:e63PubMedCrossRefGoogle Scholar
  17. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491PubMedCrossRefGoogle Scholar
  18. Sakai K, Mizutani T, Fukushi S, Saijo M, Endoh D, Kurane I, Takehara K, Morikawa S (2007) An improved procedure for rapid determination of viral RNA sequences of avian RNA viruses. Arch Virol 152:1763–1765PubMedCrossRefGoogle Scholar
  19. Schönborn J, Oberstraß J, Breyel E, Tittgen J, Schumacher J, Lukacs N (1991) Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts. Nucleic Acids Res 19:2993–3000PubMedCrossRefGoogle Scholar
  20. Valverde RA, Nameth ST, Jordan RL (1990) Analysis of double-stranded RNA for plant virus diagnosis. Plant Dis 74:255–258CrossRefGoogle Scholar
  21. Watanabe S, Mizutani T, Sakai K, Kato K, Tohya Y, Fukushi S, Saijo M, Yoshikawa Y, Kurane I, Morikawa S, Akashi H (2008) Ligation-mediated amplification for effective rapid determination of viral RNA sequences (RDV). J Clin Virol 43:56–59PubMedCrossRefGoogle Scholar
  22. Zhang YP, Rowhani A (2000) A strategy for rapid cDNA coning from double-stranded RNA templates isolated from plants infected with RNA viruses by using Taq DNA polymerase. J Virol Methods 84:59–63PubMedCrossRefGoogle Scholar

Copyright information

© The Phytopathological Society of Japan and Springer 2009

Authors and Affiliations

  • Kappei Kobayashi
    • 1
    Email author
  • Reiko Tomita
    • 1
  • Masaru Sakamoto
    • 1
  1. 1.Iwate Biotechnology Research CenterKitakamiJapan

Personalised recommendations