Archives of Virology

, Volume 158, Issue 7, pp 1549–1554 | Cite as

Establishment of an agroinoculation system for broad bean wilt virus 2

  • Go Atsumi
  • Reiko Tomita
  • Kappei Kobayashi
  • Ken-Taro Sekine
Brief Report

Abstract

We determined the complete nucleotide sequence of a broad bean wilt virus 2 (BBWV-2) isolate from gentian in Japan. The full-length RNA1 and RNA2 sequences, excluding poly(A) tails, were 5955 and 3600 nucleotides long, respectively. Analysis indicated that, in contrast to other BBWV-2 isolates, the 5’ end of both RNA1 and RNA2 starts with a GUU sequence. We successfully inoculated Nicotiana benthamiana with BBWV-2 by infiltrating a mixed suspension of two Agrobacterium tumefaciens clones carrying binary vectors with the full-length RNA1 and RNA2 sequences. This is the first report on the efficient, easy and high-throughput use of agroinoculation for generating BBWV-2 infections.

Supplementary material

705_2013_1625_MOESM1_ESM.pptx (56 kb)
Supplementary material 1 Maps of primers used in this study. (a) Primers for the amplification of cDNA fragments from RNA1 and RNA2 for cloning and sequencing. (b) Primers for 5’ and 3’RACE analysis. (c) Primers for amplification and cloning of full-length sequences of BBWV-2 RNA1 and RNA2 combined with the A. thaliana Actin 2 promoter. The amplified fragments were cloned into the pBpless-Tx vector (c-G). CapAt, polyadenylation signal sequence from cauliflower mosaic virus; aadA, aminoglycoside adenylyltranferase gene, which confers spectinomycin resistance (SpecR); Prrn, promoter for rRNA of tobacco chloroplast. The sequences of primers are shown in Supplementary Table 1. (PPTX 56 kb)
705_2013_1625_MOESM2_ESM.pptx (102 kb)
Supplementary material 2 Phylogenetic tree of BBWV-2 inferred based on the partial sequences of the small CP gene. Unrooted maximum-likelihood trees were inferred from nucleotide sequence alignment of partial sequences of SCP, including BBWV-2-Ty, in addition to the sequences in the reports of Ferrer and colleagues using the same model and parameters (General Time Reversible + Gamma distribution).(PPTX 102 kb)
705_2013_1625_MOESM3_ESM.pptx (3.9 mb)
Supplementary material 3 Symptoms observed in plants inoculated with BBWV-2-Ty. BBWV-2-Ty was sap-inoculated into S. oleracea (a), V. faba (b), C. quinoa (c) and P. sativum (d). Photographs were taken at 7 (a), 13 (b), 6 and 11 (c; inoculated and upper leaf, respectively) and 13 (d) days after inoculation. (PPTX 3980 kb)
705_2013_1625_MOESM4_ESM.pptx (191 kb)
Supplementary material 4 Primers used in this study (PPTX 191 kb)

References

  1. 1.
    Atsumi G, Nakahara KS, Wada TS, Choi SH, Masuta C, Uyeda I (2012) Heterologous expression of viral suppressors of RNA silencing complements virulence of the HC-Pro mutant of clover yellow vein virus in pea. Arch Virol 157:1019–1028PubMedCrossRefGoogle Scholar
  2. 2.
    Ferrer RM, Ferriol I, Moreno P, Guerri J, Rubio L (2011) Genetic variation and evolutionary analysis of broad bean wilt virus 2. Arch Virol 156:1445–1450PubMedCrossRefGoogle Scholar
  3. 3.
    Grimsley N, Hohn B, Hohn T, Walden R (1986) “Agroinfection,” an alternative route for viral infection of plants by using the Ti plasmid. PNAS, USA 83:3282–3286CrossRefGoogle Scholar
  4. 4.
    Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  5. 5.
    Kobayashi YO, Nakano M, Kashiwazaki S, Naito T, Mikoshiba Y, Shiota A, Kameya-Iwaki M, Honda Y (1999) Sequence analysis of RNA-2 of different isolates of broad bean wilt virus confirms the existence of two distinct species. Arch Virol 144:1429–1438PubMedCrossRefGoogle Scholar
  6. 6.
    Koh LH, Cooper JI, Wong SM (2001) Complete sequences and phylogenetic analyses of a Singapore isolate of broad bean wilt fabavirus. Arch Virol 146:135–147PubMedCrossRefGoogle Scholar
  7. 7.
    Kuroda T, Okumura A, Takeda I, Miura Y, Suzuki K (2000) Nucleotide sequence and synthesis of infectious RNA from cloned cDNA of broad bean wilt virus 2 RNA 2. Arch Virol 145:787–793PubMedCrossRefGoogle Scholar
  8. 8.
    Lee U, Hong JS, Choi JK, Kim KC, Kim YS, Curtis IS, Nam HG, Lim PO (2000) Broad bean wilt virus Causes Necrotic Symptoms and Generates Defective RNAs in Capsicum annuum. Phytopathology 90:1390–1395PubMedCrossRefGoogle Scholar
  9. 9.
    Leiser RM, Ziegler-Graff V, Reutenauer A, Herrbach E, Lemaire O, Guilley H, Richards K, Jonard G (1992) Agroinfection as an alternative to insects for infecting plants with beet western yellows luteovirus. PNAS, USA 89:9136–9140CrossRefGoogle Scholar
  10. 10.
    Mori M, Mise K, Kobayashi K, Okuno T, Furusawa I (1991) Infectivity of plasmids containing brome mosaic virus cDNA linked to the cauliflower mosaic virus 35S RNA promoter. J Gen Virol 72(Pt 2):243–246PubMedCrossRefGoogle Scholar
  11. 11.
    Nakamura S, Iwai T, Honkura R (1998) Complete nucleotide sequence and genome organization of broad bean wilt virus 2. Ann Phytopathol Soc Jpn 64:565–568CrossRefGoogle Scholar
  12. 12.
    Qi Y, Zhou X, Li D (2000) Complete nucleotide sequence and infectious cDNA clone of the RNA1 of a Chinese isolate of broad bean wilt virus 2. Virus Genes 20:201–207PubMedCrossRefGoogle Scholar
  13. 13.
    Qi Y, Zhou X, Xue C, Li D (2000) Nucleotide sequence of RNA2 and polyprotein processing sites of a Chinese isolate of broad bean wilt virus 2. Prog Nat Sci 10:680–686Google Scholar
  14. 14.
    Sanfacon H, Wellink J, Le Gall O, Karasev A, van der Vlugt R, Wetzel T (2009) Secoviridae: a proposed family of plant viruses within the order Picornavirales that combines the families Sequiviridae and Comoviridae, the unassigned genera Cheravirus and Sadwavirus, and the proposed genus Torradovirus. Arch Virol 154:899–907PubMedCrossRefGoogle Scholar
  15. 15.
    Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739PubMedCrossRefGoogle Scholar
  16. 16.
    Wülfing C, Plückthun A (1993) A versatile and highly repressive Escherichia coli expression system based on invertible promoters: expression of a gene encoding a toxic product. Gene 136:199–203PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2013

Authors and Affiliations

  • Go Atsumi
    • 1
  • Reiko Tomita
    • 1
  • Kappei Kobayashi
    • 2
  • Ken-Taro Sekine
    • 1
  1. 1.Iwate Biotechnology Research CenterKitakamiJapan
  2. 2.Faculty of AgricultureEhime UniversityMatsuyamaJapan

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