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

Abstract

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.

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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.

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Acknowledgements

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).

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Contributions

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.

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Competing interests

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

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

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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). https://doi.org/10.1038/nbt.2969

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