Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Conferring resistance to geminiviruses with the CRISPR–Cas prokaryotic immune system

Nature Plants volume 1, Article number: 15145 (2015) Cite this article

Subjects

Abstract

To reduce crop losses due to geminivirus infection, we targeted the bean yellow dwarf virus (BeYDV) genome for destruction with the CRISPR–Cas (clustered, regularly interspaced short palindromic repeats–CRISPR-associated proteins) system. Transient assays using BeYDV-based replicons revealed that CRISPR–Cas reagents introduced mutations within the viral genome and reduced virus copy number. Transgenic plants expressing CRISPR–Cas reagents and challenged with BeYDV had reduced virus load and symptoms, thereby demonstrating a novel strategy for engineering resistance to geminiviruses.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Testing Cas9-sgRNA activity at targets within the BeYDV.
Figure 2: Restricting BeYDV infection with the CRISPR–Cas system.

Similar content being viewed by others

References

  1. Mansoor, S., Zafar, Y. & Briddon, R. W. Geminivirus disease complexes: the threat is spreading. Trends Plant Sci. 11, 209–212 (2006).

    Article  CAS  Google Scholar 

  2. Hanley-Bowdoin, L., Bejarano, E. R., Robertson, D. & Mansoor, S. Geminiviruses: masters at redirecting and reprogramming plant processes. Nature Rev. Microbiol. 11, 777–788 (2013).

    Article  CAS  Google Scholar 

  3. Pilartz, M. & Jeske, H. Abutilon mosaic geminivirus double-stranded DNA is packed into minichromosomes. Virology 189, 800–802 (1992).

    Article  CAS  Google Scholar 

  4. Aragao, F. J. L. & Faria, J. C. First transgenic geminivirus-resistant plant in the field. Nature Biotechnol. 27, 1086–1088 (2009).

    Article  CAS  Google Scholar 

  5. Yang, C.-F. et al. Generation of marker-free transgenic plants concurrently resistant to a DNA geminivirus and a RNA tospovirus. Sci. Rep. 4, 5717 (2014).

    Article  Google Scholar 

  6. Westra, E. R., Buckling, A. & Fineran, P. C. CRISPR-Cas systems: beyond adaptive immunity. Nature Rev. Microbiol. 12, 317–326 (2014).

    Article  CAS  Google Scholar 

  7. Halley-Stott, R. P., Tanzer, F., Martin, D. P. & Rybicki, E. P. The complete nucleotide sequence of a mild strain of Bean yellow dwarf virus. Arch. Virol. 152, 1237–1240 (2007).

    Article  CAS  Google Scholar 

  8. Li, J.-F. et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature Biotechnol. 31, 688–691 (2013).

    Article  CAS  Google Scholar 

  9. Stenger, D. C., Revington, G. N., Stevenson, M. C. & Bisaro, D. M. Replicational release of geminivirus genomes from tandemly repeated copies: evidence for rolling-circle replication of a plant viral DNA. Proc. Natl Acad. Sci. USA 88, 8029–8033 (1991).

    Article  CAS  Google Scholar 

  10. Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    Article  CAS  Google Scholar 

  11. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    Article  CAS  Google Scholar 

  12. Kearse, M. et al. Geneious Basic: An integrated and extendable 531 desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647–1649 (2012).

    Article  Google Scholar 

  13. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  Google Scholar 

  14. Baltes, N. J., Gil-Humanes, J., Cermak, T., Atkins, P. A. & Voytas, D. F. DNA replicons for plant genome engineering. Plant Cell 26, 151–163 (2014).

    Article  CAS  Google Scholar 

  15. Liu, D. et al. Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR. PLoS ONE 7, e46451 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K. Leffler for the help in generating the figures. We thank J. Gil for technical assistance with the qPCR experiment. This work was supported by a grant from the National Science Foundation (IOS-1339209).

Author information

Author notes

  1. Nicholas J. Baltes and Aaron W. Hummel: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, 55455, Minnesota, USA

    Nicholas J. Baltes, Aaron W. Hummel, Eva Konecna & Daniel F. Voytas

  2. Center for Genome Engineering, University of Minnesota, Minneapolis, 55455, Minnesota, USA

    Nicholas J. Baltes, Aaron W. Hummel, Eva Konecna & Daniel F. Voytas

  3. Department of Plant Developmental Genetics, Institute of Biophysics ASCR, v.v.i., Kralovopolska street 135, Brno, 612 00, Czech Republic

    Radim Cegan

  4. Department of Molecular Genetics, Center for Applied Plant Sciences, and Ohio State Biochemistry Program, The Ohio State University, Columbus, 43210, Ohio, USA

    Aaron N. Bruns & David M. Bisaro

Authors
  1. Nicholas J. Baltes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aaron W. Hummel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eva Konecna
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Radim Cegan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Aaron N. Bruns
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. David M. Bisaro
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Daniel F. Voytas
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.J.B. and A.W.H. designed the experiments, with input from D.F.V. N.J.B, A.W.H. and E.K. generated constructs. N.J.B., A.W.H. and E.K. performed the transient assays. E.K. provided technical support. R.C. analysed the NGS data. A.N.B. and D.M.B. helped design the infection experiments; E.K. and A.B. performed the experiments. N.J.B., A.W.H. and D.F.V. wrote the paper. D.F.V. supervised the entire project. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Daniel F. Voytas.

Ethics declarations

Competing interests

N.J.B., A.W.H. and D.F.V. are inventors on a patent application (WO2015048707A2) for the technology described in this work.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Baltes, N., Hummel, A., Konecna, E. et al. Conferring resistance to geminiviruses with the CRISPR–Cas prokaryotic immune system. Nature Plants 1, 15145 (2015). https://doi.org/10.1038/nplants.2015.145

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nplants.2015.145

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology