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Construction of an agroinfectious clone of bean rugose mosaic virus using Gibson Assembly

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Abstract

Construction of agroinfectious viral clones usually requires many steps of cloning and sub-cloning and also a binary vector, which makes the process laborious, time-consuming, and frequently susceptible to some degree of plasmid instability. Nowadays, novel methods have been applied to the assembly of infectious viral clones, and here we have applied isothermal, single-step Gibson Assembly (GA) to construct an agroinfectious clone of Bean rugose mosaic virus (BRMV) using a small binary vector. The procedure has drastically reduced the cloning steps, and BRMV could be recovered from agroinfiltrated common bean twenty days after inoculation, indicating that the infectious clone could spread in the plant tissues and efficiently generate a systemic infection. The virus was also recovered from leaves of common bean and soybean cultivars mechanically inoculated with infectious clone two weeks after inoculation, confirming the efficiency of GA cloning procedure to produce the first BRMV agroinfectious clone to bean and soybean.

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Acknowledgements

We want to acknowledge to the Brazilian Research Council (CNPq) for the financial support. Authors also thank to Dr. John Lindbo and Dr. Massimo Turina for providing the vector pJL89.

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Author notes

    Authors and Affiliations

    1. Universidade Estadual de Maringá, 5790 Av. Colombo, Maringá, PR, Brazil

      Taise Bijora, Danielle K. T. Costa & Eliezer R. Souto

    2. Universidade de Brasília, Brasília, DF, Brazil

      Rosana Blawid & Tatsuya Nagata

    3. Embrapa Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil

      Francisco J. L. Aragão

    4. Department of Cellular Biology, Biological Sciences institute, Universidade de Brasília, Brasília, DF, 70910-900, Brazil

      Tatsuya Nagata

    5. Department of Agronomy, Center of Agricultural Sciences, Universidade Estadual de Maringá, Maringá, PR, 87020-900, Brazil

      Eliezer R. Souto

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    1. Taise Bijora
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    2. Rosana Blawid
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    3. Danielle K. T. Costa
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    4. Francisco J. L. Aragão
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    5. Eliezer R. Souto
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    Contributions

    TB, RB, DKTC, and TN carried out the laboratory experiments; FJLA contributed for the inoculation methodology. ERS firstly planed the research project, collected cultivars of common bean and soybean, and wrote the manuscript with TB. All authors read and approved the final manuscript.

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    Correspondence to Eliezer R. Souto or Tatsuya Nagata.

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    The authors declare that they have no conflict of interest.

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    This article does not contain any studies with human participants or animals performed by any of the authors.

    Additional information

    Edited by Thomas Hohn.

    Eliezer R. Souto and Tatsuya Nagata have equally contributed to this work.

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    Bijora, T., Blawid, R., Costa, D.K.T. et al. Construction of an agroinfectious clone of bean rugose mosaic virus using Gibson Assembly. Virus Genes 53, 495–499 (2017). https://doi.org/10.1007/s11262-017-1446-y

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    • DOI: https://doi.org/10.1007/s11262-017-1446-y

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