Skip to main content

Thank you for visiting 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.

Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew


Sequence-specific nucleases have been applied to engineer targeted modifications in polyploid genomes1, but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator–like effector nuclease (TALEN)2,3 and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (refs. 4,5) technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW-RESISTANCE LOCUS (MLO) proteins6. Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations7. We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew. We further use CRISPR-Cas9 technology to generate transgenic wheat plants that carry mutations in the TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks caused by TALENs. Our findings provide a methodological framework to improve polyploid crops.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Targeted knockout of TaMLO genes using the TALEN and CRISPR-Cas9 systems.
Figure 2: Loss of TaMLO function confers resistance of bread wheat to powdery mildew disease.
Figure 3: NHEJ-mediated knock-in of a GFP reporter gene at a TaMLO site in wheat protoplasts.


  1. Kim, H. & Kim, J.S. A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15, 321–334 (2014).

    Article  CAS  Google Scholar 

  2. Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 (2009).

    Article  CAS  Google Scholar 

  3. Moscou, M.J. & Bogdanove, A.J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).

    Article  CAS  Google Scholar 

  4. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  5. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  Google Scholar 

  6. Elliott, C. et al. Functional conservation of wheat and rice Mlo orthologs in defense modulation to the powdery mildew fungus. Mol. Plant Microbe Interact. 15, 1069–1077 (2002).

    Article  CAS  Google Scholar 

  7. Várallyay, É., Giczey, G. & Burgyán, J. Virus-induced gene silencing of Mlo genes induces powdery mildew resistance in Triticum aestivum. Arch. Virol. 157, 1345–1350 (2012).

    Article  Google Scholar 

  8. Slade, A.J. et al. A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat. Biotechnol. 23, 75–81 (2005).

    Article  CAS  Google Scholar 

  9. Dvorřák, J. in Genetics and Genomics of the Triticeae Vol. 7. (eds. Muehlbauer, G.J. & Feuillet, C.) 685–711 (Springer US, 2009).

  10. Bibikova, M., Beumer, K., Trautman, J.K. & Carroll, D. Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003).

    Article  CAS  Google Scholar 

  11. Symington, L.S. & Gautier, J. Double-strand break end resection and repair pathway choice. Annu. Rev. Genet. 45, 247–271 (2011).

    Article  CAS  Google Scholar 

  12. Voytas, D.F. & Gao, C. Precision genome engineering and agriculture: opportunities and regulatory challenges. PLoS Biol. 12, e1001877 (2014).

    Article  Google Scholar 

  13. Zhang, F. et al. High frequency targeted mutagenesis in Arabidopsis thaliana using zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 107, 12028–12033 (2010).

    Article  CAS  Google Scholar 

  14. Feng, Z. et al. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc. Natl. Acad. Sci. USA 111, 4632–4637 (2014).

    Article  CAS  Google Scholar 

  15. Christian, M., Qi, Y., Zhang, Y. & Voytas, D.F. Targeted mutagenesis of Arabidopsis thaliana using engineered TAL effector nucleases. G3 (Bethesda) 3, 1697–1705 (2013).

    Article  Google Scholar 

  16. Li, T. et al. High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat. Biotechnol. 30, 390–392 (2012).

    Article  CAS  Google Scholar 

  17. Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31, 686–688 (2013).

    Article  CAS  Google Scholar 

  18. Büschges, R. et al. The barley Mlo gene: A novel control element of plant pathogen resistance. Cell 88, 695–705 (1997).

    Article  Google Scholar 

  19. Piffanelli, P. et al. A barley cultivation-associated polymorphism conveys resistance to powdery mildew. Nature 430, 887–891 (2004).

    Article  CAS  Google Scholar 

  20. Consonni, C. et al. Conserved requirement for a plant host cell protein in powdery mildew pathogenesis. Nat. Genet. 38, 716–720 (2006).

    Article  CAS  Google Scholar 

  21. Bai, Y. et al. Naturally occurring broad-spectrum powdery mildew resistance in a central american tomato accession is caused by loss of mlo function. Mol. Plant Microbe Interact. 21, 30–39 (2008).

    Article  CAS  Google Scholar 

  22. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).

    Article  CAS  Google Scholar 

  23. Shan, Q. et al. Rapid and efficient gene modification in rice and brachypodium using TALENs. Mol. Plant 6, 1365–1368 (2013).

    Article  CAS  Google Scholar 

  24. Christensen, A. & Quail, P. Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 5, 213–218 (1996).

    Article  CAS  Google Scholar 

  25. Feng, Z. et al. Efficient genome editing in plants using a CRISPR/Cas system. Cell Res. 23, 1229–1232 (2013).

    Article  CAS  Google Scholar 

  26. Xie, K. & Yang, Y. RNA-guided genome editing in plants using a CRISPR-Cas system. Mol. Plant 6, 1975–1983 (2013).

    Article  CAS  Google Scholar 

  27. McDonald, B.A. & Linde, C. Pathogen population genetics, evolutionary potential, and durable resistance. Annu. Re. Phytopathol. 40, 349–379 (2002).

    Article  CAS  Google Scholar 

  28. Zhang, Y. et al. Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiol. 161, 20–27 (2013).

    Article  CAS  Google Scholar 

  29. Guerineau, F., Lucy, A. & Mullineaux, P. Effect of two consensus sequences preceding the translation initiator codon on gene expression in plant protoplasts. Plant Mol. Biol. 18, 815–818 (1992).

    Article  CAS  Google Scholar 

  30. Rasco-Gaunt, S. et al. Procedures allowing the transformation of a range of European elite wheat (Triticum aestivum L.) varieties via particle bombardment. J. Exp. Bot. 52, 865–874 (2001).

    Article  CAS  Google Scholar 

  31. Hein, I. et al. Virus-induced gene silencing-based functional characterization of genes associated with powdery mildew resistance in barley. Plant Physiol. 138, 2155–2164 (2005).

    Article  CAS  Google Scholar 

Download references


We thank D. Wang for critical reading of the manuscript and providing the E22 and B13 strains, Q. Shen for providing E09 strain, and T. Li for technical support in flow cytometry (all three are at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences). This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB11030500) and the National Basic Research Program of China (973 Program, 2011CB100702) to J.-L.Q. and a grant to C.G. from the National Natural Science Foundation of China (31271795).

Author information

Authors and Affiliations



J.-L.Q., C.G. and Y.W. designed the experiments; Y.W., X.C., Q.S., Y.Z. and J.L. performed the experiments; and J.-L.Q., C.G. and Y.W. wrote the manuscript.

Corresponding authors

Correspondence to Caixia Gao or Jin-Long Qiu.

Ethics declarations

Competing interests

The authors have filed a patent application (Chinese patent application number 201410027631.2) based on the results reported in this paper.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–6 (PDF 17919 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wang, Y., Cheng, X., Shan, Q. et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32, 947–951 (2014).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

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