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

Journal name:
Nature Biotechnology
Volume:
32,
Pages:
947–951
Year published:
DOI:
doi:10.1038/nbt.2969
Received
Accepted
Published online

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.

At a glance

Figures

  1. Targeted knockout of TaMLO genes using the TALEN and CRISPR-Cas9 systems.
    Figure 1: Targeted knockout of TaMLO genes using the TALEN and CRISPR-Cas9 systems.

    (a) Sites within a conserved region of exon 2 of wheat TaMLO homoeologs targeted by the TALEN and CRISPR-Cas9 systems. The TALEN-targeted sequences in MLO-A1, MLO-B1 and MLO-D1 are underlined, and the AvaII restriction site in the spacer is blue. Of the three SNPs highlighted in red, two are in the spacer region and one lies near the far right of the TALEN binding site. The CRISPR-Cas9 targeted sequence in MLO-A1 is indicated in the box, and the protospacer-adjacent motif (PAM) sequence is highlighted in green. (b) Outcome of PCR-RE assay to detect TALEN-induced mutations in 15 representative T0 transgenic wheat plants. Mutations were identified in TaMLO genes amplified with gene-specific primers from independent seedlings. Lanes T0-1 to T0-15 show PCR fragments amplified from the transgenic wheat plants digested with AvaII. Lanes labeled WT show PCR fragments amplified from a wild-type control plant with or without AvaII digestion. The bands marked by red arrowheads are caused by TALEN-induced mutations. (c) TALEN-induced mutant TaMLO alleles identified by sequencing 15 representative transgenic wheat plants. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. (d) Outcome of T7E1 assay to detect CRISPR-induced mutations in 15 representative T0 transgenic wheat plants. Red arrowheads indicate the fragments digested by T7E1. (e) Mutations in the TaMLO-A1 site that were induced by sgMLO-A1.

  2. Loss of TaMLO function confers resistance of bread wheat to powdery mildew disease.
    Figure 2: Loss of TaMLO function confers resistance of bread wheat to powdery mildew disease.

    (a) Percentage of microcolonies formed from the total number of germinated spores of Blumeria graminis f. sp. tritici (Bgt) inoculated on the leaves of wild-type (WT) and various tamlo mutants. At least 2,000 germinated spores per genotype per experiment were examined 72 h after inoculation with virulent Bgt isolate E09. Values are the mean ± s.d. of four independent experiments. **P < 0.01 (t-test). (b) Micrographs of microcolony formation of Bgt on the surfaces of leaves of the indicated genotypes 3 d postinoculation. Powdery mildew spores and colonies were stained with Coomassie blue. Scale bars, 200 μm. (c) Macroscopic infection phenotypes of representative leaves of WT and the indicated mlo mutants 7 d after inoculation of detached leaves with Bgt. Scale bar, 1 cm. (d) Disease symptoms of wild-type (WT) and tamlo-aabbdd mutant plants. The photograph was taken 7 d after inoculation in planta. Scale bars, 2 cm.

  3. NHEJ-mediated knock-in of a GFP reporter gene at a TaMLO site in wheat protoplasts.
    Figure 3: NHEJ-mediated knock-in of a GFP reporter gene at a TaMLO site in wheat protoplasts.

    (a) Structure of the GFP donor plasmid and the anticipated outcome of a GFP knock-in event. A cauliflower mosaic virus (CaMV) 35S terminator lies downstream of the GFP coding sequence. The cassette is flanked by two T-MLO sites, which generate a linear structure by recombination with the co-transformed T-MLO plasmid. The locations and names of the primers used for PCR analysis of knock-in events are shown. (b) Measurement of GFP knock-in efficiency in wheat protoplasts by flow cytometry. Three fields of protoplasts are shown. Protoplasts were transformed with the following DNA constructs (from left to right): (i) T-MLO plus GFP donor plasmids; (ii) GFP donor plasmid alone; (iii) positive control with GFP expression driven by the maize Ubiquitin 1 promoter. Flow cytometry was used to quantify the percentage of GFP-expressing protoplasts. Scale bars, 100 μm. (c) Sequencing of 5′ and 3′ junctions confirm NHEJ-mediated knock-in events. The 5′ junction sequences were PCR-amplified with primers F1 and R1, and the 3′ junctions with primers F2 and R2. T-MLO sites are underlined. Inherent SNPs in the T-MLO site are highlighted in red. Sequences from the coding region of GFP and the CaMV 35S terminator are highlighted in green. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating nucleotide deletion and insertion, respectively.

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Author information

  1. These authors contributed equally to this work.

    • Yanpeng Wang &
    • Xi Cheng

Affiliations

  1. State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

    • Yanpeng Wang,
    • Qiwei Shan,
    • Yi Zhang,
    • Jinxing Liu &
    • Caixia Gao
  2. State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

    • Xi Cheng &
    • Jin-Long Qiu

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.

Competing financial 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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