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Genome-edited powdery mildew resistance in wheat without growth penalties

Abstract

Disruption of susceptibility (S) genes in crops is an attractive breeding strategy for conferring disease resistance1,2. However, S genes are implicated in many essential biological functions and deletion of these genes typically results in undesired pleiotropic effects1. Loss-of-function mutations in one such S gene, Mildew resistance locus O (MLO), confers durable and broad-spectrum resistance to powdery mildew in various plant species2,3. However, mlo-associated resistance is also accompanied by growth penalties and yield losses3,4, thereby limiting its widespread use in agriculture. Here we describe Tamlo-R32, a mutant with a 304-kilobase pair targeted deletion in the MLO-B1 locus of wheat that retains crop growth and yields while conferring robust powdery mildew resistance. We show that this deletion results in an altered local chromatin landscape, leading to the ectopic activation of Tonoplast monosaccharide transporter 3 (TaTMT3B), and that this activation alleviates growth and yield penalties associated with MLO disruption. Notably, the function of TMT3 is conserved in other plant species such as Arabidopsis thaliana. Moreover, precision genome editing facilitates the rapid introduction of this mlo resistance allele (Tamlo-R32) into elite wheat varieties. This work demonstrates the ability to stack genetic changes to rescue growth defects caused by recessive alleles, which is critical for developing high-yielding crop varieties with robust and durable disease resistance.

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Fig. 1: Tamlo-R32 wheat exhibits immunity to powdery mildew without growth and yield penalties.
Fig. 2: Chromosomal rearrangement in Tamlo-R32 leads to activation of TaTMT3B in leaves.
Fig. 3: Increased TMT3 expression rescues growth phenotypes in mlo mutants of wheat and Arabidopsis.
Fig. 4: Introduction of the Tamlo-R32 allele into elite wheat varieties.

Data availability

The sequencing data obtained in this study have been deposited in the Genome Sequence Archive (GSA) database in the BIG Data Center (https://ngdc.cncb.ac.cn/) under accession number PRJCA005687. Chinese Spring wheat reference genome RefSeq v1.1 is available on IWGSC (http://www.wheatgenome.org/). Transcriptome data from different wheat tissues are from ref. 29Source data are provided with this paper.

References

  1. van Schie, C. C. & Takken, F. L. Susceptibility genes 101: how to be a good host. Annu. Rev. Phytopathol. 52, 551–581 (2014).

    PubMed  Google Scholar 

  2. Schulze-Lefert, P. & Vogel, J. Closing the ranks to attack by powdery mildew. Trends Plant Sci. 5, 343–348 (2000).

    CAS  PubMed  ADS  Google Scholar 

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

    PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  5. van Esse, H. P., Reuber, T. L. & van der Does, D. Genetic modification to improve disease resistance in crops. New Phytol. 225, 70–86 (2020).

    PubMed  Google Scholar 

  6. Li, W., Deng, Y., Ning, Y., He, Z. & Wang, G.-L. Exploiting broad-spectrum disease resistance in crops: from molecular dissection to breeding. Annu. Rev. Plant Biol. 71, 575–603 (2020).

    CAS  PubMed  Google Scholar 

  7. Dangl, J. L., Horvath, D. M. & Staskawicz, B. J. Pivoting the plant immune system from dissection to deployment. Science 341, 746–751 (2013).

    CAS  PubMed  ADS  Google Scholar 

  8. Deng, Y. et al. Epigenetic regulation of antagonistic receptors confers rice blast resistance with yield balance. Science 355, 962–965 (2017).

    CAS  ADS  PubMed  Google Scholar 

  9. Saintenac, C. et al. Wheat receptor-kinase-like protein Stb6 controls gene-for-gene resistance to fungal pathogen Zymoseptoria tritici. Nat. Genet. 50, 368–374 (2018).

    CAS  PubMed  Google Scholar 

  10. Jones, J. D. & Dangl, J. L. The plant immune system. Nature 444, 323–329 (2006).

    CAS  PubMed  ADS  Google Scholar 

  11. Dangl, J. L. & Jones, J. D. Plant pathogens and integrated defence responses to infection. Nature 411, 826–833 (2001).

    CAS  PubMed  ADS  Google Scholar 

  12. Dodds, P. N. & Rathjen, J. P. Plant immunity: towards an integrated view of plant–pathogen interactions. Nat. Rev. Genet. 11, 539–548 (2010).

    CAS  PubMed  Google Scholar 

  13. Oliva, R. et al. Broad-spectrum resistance to bacterial blight in rice using genome editing. Nat. Biotechnol. 37, 1344–1350 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Lapin, D. & Van den Ackerveken, G. Susceptibility to plant disease: more than a failure of host immunity. Trends Plant Sci. 18, 546–554 (2013).

    CAS  PubMed  Google Scholar 

  15. Vogel, J. P., Raab, T. K., Schiff, C. & Somerville, S. C. PMR6, a pectate lyase–like gene required for powdery mildew susceptibility in Arabidopsis. Plant Cell 14, 2095–2106 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Eckardt, N. A. Plant disease susceptibility genes? Plant Cell 14, 1983–1986 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Kim, M. C. et al. Calmodulin interacts with MLO protein to regulate defence against mildew in barley. Nature 416, 447–451 (2002).

    CAS  PubMed  ADS  Google Scholar 

  18. Devoto, A. et al. Topology, subcellular localization, and sequence diversity of the Mlo family in plants. J. Biol. Chem. 274, 34993–35004 (1999).

    CAS  PubMed  Google Scholar 

  19. Kusch, S. & Panstruga, R. mlo-based resistance: an apparently universal "weapon" to defeat powdery mildew disease. Mol. Plant Microbe Interact. 30, 179–189 (2017).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  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).

    CAS  PubMed  Google Scholar 

  22. Humphry, M., Consonni, C. & Panstruga, R. mlo-based powdery mildew immunity: silver bullet or simply non-host resistance? Mol. Plant Pathol. 7, 605–610 (2006).

    PubMed  Google Scholar 

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

    CAS  PubMed  ADS  Google Scholar 

  24. Acevedo-Garcia, J., Kusch, S. & Panstruga, R. Magical mystery tour: MLO proteins in plant immunity and beyond. New Phytol. 204, 273–281 (2014).

    CAS  PubMed  Google Scholar 

  25. Appiano, M. et al. Monocot and dicot MLO powdery mildew susceptibility factors are functionally conserved in spite of the evolution of class-specific molecular features. BMC Plant Biol. 15, 257 (2015).

    PubMed  PubMed Central  Google Scholar 

  26. Singh, R. P. et al. Disease impact on wheat yield potential and prospects of genetic control. Annu. Rev. Phytopathol. 54, 303–322 (2016).

    CAS  PubMed  Google Scholar 

  27. Acevedo-Garcia, J. et al. mlo-based powdery mildew resistance in hexaploid bread wheat generated by a non-transgenic TILLING approach. Plant Biotechnol. J. 15, 367–378 (2017).

    CAS  PubMed  Google Scholar 

  28. Bogdanove, A. J. & Voytas, D. F. TAL effectors: customizable proteins for DNA targeting. Science 333, 1843–1846 (2011).

    CAS  PubMed  ADS  Google Scholar 

  29. Ramírez-González, R. H. et al. The transcriptional landscape of polyploid wheat. Science 361, eaar6089 (2018).

    PubMed  Google Scholar 

  30. Wormit, A. et al. Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport. Plant Cell 18, 3476–3490 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Bieluszewski, T., Xiao, J., Yang, Y. & Wagner, D. PRC2 activity, recruitment, and silencing: a comparative perspective. Trends Plant Sci. 21, S1360–S1385 (2021).

    Google Scholar 

  32. Kajimura, T., Mizuno, N. & Takumi, S. Utility of leaf senescence-associated gene homologs as developmental markers in common wheat. Plant Physiol. Biochem. 48, 851–859 (2010).

    CAS  PubMed  Google Scholar 

  33. Consonni, C. et al. Tryptophan-derived metabolites are required for antifungal defense in the Arabidopsis mlo2 mutant. Plant Physiol. 152, 1544–1561 (2010).

    CAS  PubMed  Google Scholar 

  34. Gao, C. Genome engineering for crop improvement and future agriculture. Cell 184, 1621–1635 (2021).

    CAS  PubMed  Google Scholar 

  35. Zhang, Y. et al. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat. Commun.7, 12617–12624 (2016).

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  36. Liang, Z. et al. Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat. Commun.8, 14261–14265 (2017).

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  37. Jansen, M., Jarosch, B. & Schaffrath, U. The barley mutant emr1 exhibits restored resistance against Magnaporthe oryzae in the hypersusceptible mlo-genetic background. Planta 225, 1381–1391 (2007).

    CAS  PubMed  Google Scholar 

  38. Li, S. et al. MYB75 phosphorylation by MPK4 is required for light-induced anthocyanin accumulation in Arabidopsis. Plant Cell 28, 2866–2883 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  40. Liang, Z. et al. Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins. Nat. Protoc. 13, 413–430 (2018).

    CAS  PubMed  Google Scholar 

  41. Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    CAS  PubMed  Google Scholar 

  42. Wang, Z. et al. Genetic and physical mapping of powdery mildew resistance gene MlHLT in Chinese wheat landrace Hulutou. Theor. Appl. Genet. 128, 365–373 (2015).

    CAS  PubMed  Google Scholar 

  43. Shen, Q. H. et al. Nuclear activity of MLA immune receptors links isolate-specific and basal disease-resistance responses. Science 315, 1098–1103 (2007).

    CAS  PubMed  ADS  Google Scholar 

  44. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Danecek, P. et al. Twelve years of SAMtools and BCFtools. Gigascience 10, giab008 (2021).

    PubMed  PubMed Central  Google Scholar 

  46. Thorvaldsdóttir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 14, 178–192 (2013).

    PubMed  Google Scholar 

  47. Kim, D., Paggi, J. M., Park, C., Bennett, C. & Salzberg, S. L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 37, 907–915 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    CAS  PubMed  Google Scholar 

  49. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    CAS  PubMed  Google Scholar 

  50. Weber, B., Jamge, S. & Stam, M. 3C in maize and Arabidopsis. Methods Mol. Biol. 1675, 247–270 (2018).

    CAS  PubMed  Google Scholar 

  51. Krijger, P. H., Geeven, G., Bianchi, V., Hilvering, C. R., & de Laat, W. 4C-seq from beginning to end: a detailed protocol for sample preparation and data analysis. Methods 170, 17–32. (2020).

    CAS  PubMed  Google Scholar 

  52. Ricci, W. A. et al. Widespread long-range cis-regulatory elements in the maize genome. Nat. Plants 5, 1237–1249 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Rao, S. S. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665–1680 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Kim, D. & Sung, S. Vernalization-triggered intragenic chromatin-loop formation by long noncoding RNAs. Dev. Cell 40, 302–312 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Paolacci, A. R., Tanzarella, O. A., Porceddu, E. & Ciaffi, M. Identification and validation of reference genes for quantitative RT–PCR normalization in wheat. BMC Mol. Biol. 10, 11 (2009).

    PubMed  PubMed Central  Google Scholar 

  56. Bajic, M., Maher, K. A. & Deal, R. B. Identification of open chromatin regions in plant genomes using ATAC-seq. Methods Mol. Biol. 1675, 183–201 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Zhang, Y. et al. Model-based analysis of ChIP-seq (MACS). Genome Biol. 9, R137 (2008).

    PubMed  PubMed Central  Google Scholar 

  58. Ramírez, F., Dündar, F., Diehl, S., Grüning, B. A. & Manke, T. deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res. 42, W187–W191 (2014).

    PubMed  PubMed Central  Google Scholar 

  59. Kaya-Okur, H. S. et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat. Commun. 10, 1930 (2019).

    PubMed  PubMed Central  ADS  Google Scholar 

  60. Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884–i890 (2018).

    PubMed  PubMed Central  Google Scholar 

  61. Xiao, J. et al. Cis- and trans-determinants of epigenetic silencing by Polycomb Repressive Complex 2 in Arabidopsis. Nat. Genet. 49, 1546–1552 (2017).

    CAS  PubMed  Google Scholar 

  62. Gou, J. Y. et al. Wheat stripe rust resistance protein WKS1 reduces the ability of the thylakoid-associated ascorbate peroxidase to detoxify reactive oxygen species. Plant Cell 27, 1755–1770 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Qin, D. et al. Characterization and fine mapping of a novel barley Stage Green-Revertible Albino gene (HvSGRA) by bulked segregant analysis based on ssr assay and specific length amplified fragment sequencing. BMC Genomics 16, 838–851 (2015).

    PubMed  PubMed Central  Google Scholar 

  64. Ni, Z. et al. Altered circadian rhythms regulate growth vigour in hybrids and allopolyploids. Nature 457, 327–331 (2009).

    CAS  PubMed  ADS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA24020101 to J.-L.Q., XDA24020102 to C.G., XDA24010204 to J.X., XDA24020310 to Y.W., XDPB16 to J.-L.Q.), the National Natural Science Foundation of China (31788103 to C.G., 32001891 to S.L., 31970529 to J.X. and 31971370 to K.C.) and the Key Research Program of Frontier Sciences (QYZDY-SSW-SMC030 to C.G.) and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2020000003 to Y.W.).

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Authors and Affiliations

Authors

Contributions

C.G. and J.-L.Q. conceived and conceptualized the study. C.G. and J.-L.Q. designed the experiments. S.L., D.L. and Y.Z. performed most of the experiments. S.L., Y.W. and J.X. prepared the figures. S.L. and Y.Z. performed the powdery mildew infection experiment. D.L., B. Li, Y. Lei, J.L. and K.C carried out genome-editing experiments and mutant identification. M.D. conducted the 4C and 3C experiments. L.Z. and J.X. conducted the CUT&Tag, ATAC-seq and bioinformatics analyses. B. Lv, and Y. Liang performed the marker-assisted selection (MAS) and powdery mildew microscopic analyses. S.L. and Y.W. characterized the phenotypes of mutant plants. Y.C. and Z.L. carried out traditional breeding and field trials. J.-L.Q., C.G., S.L. and J.X wrote the manuscript. All authors commented on the results and contributed to the manuscript.

Corresponding authors

Correspondence to Jun Xiao, Jin-Long Qiu or Caixia Gao.

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C.G., J.-L.Q., S.L. and Y.W. have filed patent applications based on the work published here.

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Nature thanks Wendy Harwood, Nian Wang and the other, anonymous, reviewers for their contribution to the peer review of this work. Peer review reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Genotyping by agarose gel electrophoresis and Sanger sequencing.

a, Detection of the transgene in the Tamlo-R32 mutants by PCR using five independent primer sets. Plasmid DNA of the TALEN vector was used as a positive control. b, Schematic diagram of the structure of the wheat TaMLO1 gene. Green rectangles and solid black lines represent exons and introns, respectively. The conserved TALEN target site within TaMLO1 is indicated by the red vertical line. Black arrows denote the positions and orientations of the three pairs of gene-specific primers (F1/R1, F2/R2, F3/R3) for amplifying TaMLO-A1, TaMLO-B1 and TaMLO-D1, respectively. c, Agarose gel electrophoresis of TaMLO1 amplicons from genomic DNA of the BW wild-type (upper) and Tamlo-R32 mutant (lower) using gene-specific primers. d, Agarose gel electrophoresis of the PCR products amplified by the primer pairs F2/R2 (upper) and F4/R4 (lower) from genomic DNA of the wild type and Tamlo-R32 mutants. The positions and orientations of the F4/R4 primer pairs are denoted by black arrows in Fig. 1g. e, DNA sequence of the edited sites in Tamlo-R32. Blue letters indicate inserted sequences. Red letters indicate the original sequence. Black vertical lines indicate the target site. The black dotted line indicates the deleted region. For a, c, d, experiments were repeated 3 times with the same results.

Extended Data Fig. 2 The Tamlo-R32 mutant displays no yield penalties when grown in the field.

a–d, The agronomic traits, including grain yield per plant (a), thousand kernel weight (b), tiller number per plant (c) and grain number per spike (d) were evaluated in field conditions in two wheat-growing areas in the North China Plain, Beijing and Zhaoxian in Hebei Province in 2019 and 2020. For box plots, the box limits indicate the twenty-fifth and seventy-fifth percentiles, the whiskers indicate the full range of the data, and the center line indicates the median. Individual data points are plotted. n represents the sample size. Statistical significance was determined by two-tailed Mann-Whitney tests or two-tailed Student’s t-tests. P values are indicated

Source data

Extended Data Fig. 3 Macroscopic powdery mildew infection phenotypes of F2 plants from various crosses.

a, b, Representative detached leaves of the F2 generations of Tamlo-R32×Tamlo-aaBBdd (a) and Tamlo-R32×Tamlo-aabbdd (b) are shown seven days after inoculation of Bgt isolate E09. Red triangles indicate resistant plants. Scale bar, 1 cm.

Extended Data Fig. 4 Expression patterns and chromatin landscapes of genes around the large deletion.

a, Expression of TaTMT3B and 11 other nearby genes in different tissues; data are from a previous publication30. Genes within the deleted region are indicated. b, c, Amino acid sequence alignment between homeologs on the A, B and D genomes of downregulated genes in the deletion region. d, Expression levels of TaTMT3B in different tissues of Tamlo-R32 and WT plants measured by quantitative RT-PCR. Results are normalized to TaPAGE gene. n.d., not detected. Data are means ± s.d., of three independent RNA preparations from biological replicates. e, Chromatin accessibility, and histone modification profiles in the TaTMT3B-MLO-B1 region in leaf tissue of Chinese Spring wheat. The Integrative Genomics Viewer (IGV) views show the various chromatin status profiles near TaTMT3B. The y-axis represents signal enrichment computed from reads at each position normalized to the total number of reads (RPKM). The dark shading indicates regions with either repressive (H3K27me3) or active (H3K4me3, H3K36me3, H3K27ac) histone modifications (a-f). f, Schematic illustration of a possible model for regulation of the activation of TaTMT3B expression in Tamlo-R32

Source data

Extended Data Fig. 5 Expression levels of twenty genes around the large deletion measured by quantitative RT-PCR.

a, Schematic diagram of the gene distribution around the large B-genome deletion identified in Tamlo-R32. b, Expression levels of twenty genes of the B genome were measured by quantitative RT-PCR in both wild-type Bobwhite and Tamlo-R32 mutant leaves. Results are normalized to TaPARG gene and the expression level of gene in wild-type BW plants was set at one except TaTMT3B. n.d., not detected. Data are means ± s.d. of three independent RNA preparations from biological replicates

Source data

Extended Data Fig. 6 Wheat and Arabidopsis mlo mutants overexpressing TMT3 maintain powdery mildew resistance.

a, Targeted knockout of TaTMT3B by CRISPR-Cas9 in the Tamlo-R32 background. Blue letters indicate TaTMT3B sgRNA. The PAM sequence is highlighted in red. The numbers on the right show the type of mutation and how many nucleotides are involved, with “-” indicating deletion of the given number of nucleotides. b, Expression levels of TaTMT3 in TaTMT3B-overexpressors in the Tamlo-aabbdd mutant background, assessed by quantitative RT-PCR. The primers were designed to detect complete TaTMT3 transcripts. The results are normalized to TaACTIN, and expression of the gene in KN199 (wild type) is set at one. Data are means ± s.d., of three independent RNA preparations from biological replicates. c, Macroscopic infection phenotypes of representative detached leaves of the indicated wheat plants seven days after inoculation with Bgt isolate E09. Scale bar, 1 cm. d, Micrographs of microcolony formation by Bgt on wheat leaves of the indicated genotypes three days postinoculation. Powdery mildew spores and colonies were stained with Coomassie blue. Scale bars, 100 μm. e, Percentages of microcolonies formed from the total number of germinated spores of Bgt on the leaves of the indicated wheat plants. f, Expression levels of AtTMT3 in TMT3-overexpressors in the Atmlo2/6/12 background measured by quantitative RT-PCR. The primers were designed to detect both transgenic and endogenous AtTMT3 transcripts. The results are normalized to Arabidopsis AtACTIN8, and the expression level of the gene in WT was set at one. Data are means ± s.d. of three independent RNA preparations from biological replicates. g, Detached rosette leaves of the indicated 7-week-old Arabidopsis plants grown under long-day condition were laid out. Scale bar, 1 cm. h, Chlorophyll content of 6th rosette leaves of 7-week-old Arabidopsis plants grown under long-day conditions. The Tamlo-aabbdd mutants in b–e is in the KN199 background. Data are means of three biological replicates. Error bars represent means ± s.d. P values are indicated. i, Macroscopic infection phenotypes of representative detached leaves of the indicated Arabidopsis plants seven days after inoculation with G. orontii Scale bar, 1 cm. j, Micrographs of microcolony formation by G. orontii on Arabidopsis leaves of the indicated genotypes three days post-inoculation. Powdery mildew spores and colonies were stained with Coomassie blue. Scale bars, 100 μm. k, Percentages of microcolonies formed from the total number of germinated spores of G. orontii on leaves of indicated Arabidopsis plants. More than 1000 germinated spores per genotype per experiment were examined 72 h after inoculation in e and k. Data are means of three independent experiments. Error bars represent means ± s.d. Statistical significance in e, h, k was determined by two-tailed Mann-Whitney tests or two-tailed Student’s t-tests

Source data

Extended Data Fig. 7 Detection of transgene-free mutants.

Outcome of tests for transgene-free mutants using five primer sets in 31 mutant plants in Extended Data Table 2. Lanes labelled WT and plasmid show the PCR fragments amplified from a WT plant and plasmid constructs pJIT163-Ubi-Cas9 and pU6-gRNA vector respectively. KN-WT, XN-WT, XY-WT and S-WT indicate elite wheat varieties KN199, XN511, XY60 and S4185. Experiments were repeated 3 times with the same results.

Extended Data Table 1 Frequencies of mutations generated by genome editing with CRISPR-Cas9 DNA/RNP in the T0 generation of four elite wheat varieties
Extended Data Table 2 Genotypes of the mutants generated by CRISPR-Cas9 DNA in terms of mutations in TaMLO-A1, TaMLO-D1 and the large deletion in the B genome
Extended Data Table 3 Genotypes of the mutants generated by CRISPR-Cas9 RNP with respect to mutations in TaMLO-A1, TaMLO-D1 and the large deletion in the B genome

Supplementary information

Supplementary Information

This file contains the uncropped images of agarose gels (Supplementary Fig. 1) and a list of primers used in the study (Supplementary Table 1).

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Li, S., Lin, D., Zhang, Y. et al. Genome-edited powdery mildew resistance in wheat without growth penalties. Nature 602, 455–460 (2022). https://doi.org/10.1038/s41586-022-04395-9

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