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Establishment of an agroinoculation system for broad bean wilt virus 2

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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.

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We thank Kazue Obara for technical assistance, and Yasuya Iwadate and Kazumichi Fujiwara for collecting the gentian sample. This study was supported by the Iwate Prefectural Government.

Conflict of interest

The authors declare that they have no conflict of interest.

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Corresponding author

Correspondence to Ken-Taro Sekine.

Additional information

The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB746938 (BBWV-2-Ty RNA1) and AB746939 (BBWV-2-Ty RNA2).

Electronic supplementary material

Below is the link to the electronic supplementary material.


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)


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)


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)

Supplementary material 4 Primers used in this study (PPTX 191 kb)

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Atsumi, G., Tomita, R., Kobayashi, K. et al. Establishment of an agroinoculation system for broad bean wilt virus 2. Arch Virol 158, 1549–1554 (2013).

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