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A five-transgene cassette confers broad-spectrum resistance to a fungal rust pathogen in wheat


Breeding wheat with durable resistance to the fungal pathogen Puccinia graminis f. sp. tritici (Pgt), a major threat to cereal production, is challenging due to the rapid evolution of pathogen virulence. Increased durability and broad-spectrum resistance can be achieved by introducing more than one resistance gene, but combining numerous unlinked genes by breeding is laborious. Here we generate polygenic Pgt resistance by introducing a transgene cassette of five resistance genes into bread wheat as a single locus and show that at least four of the five genes are functional. These wheat lines are resistant to aggressive and highly virulent Pgt isolates from around the world and show very high levels of resistance in the field. The simple monogenic inheritance of this multigene locus greatly simplifies its use in breeding. However, a new Pgt isolate with virulence to several genes at this locus suggests gene stacks will need strategic deployment to maintain their effectiveness.

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Fig. 1: Generating transgenic Fielder wheat lines containing a multi-transgene resistance gene cassette.
Fig. 2: Multi-transgene lines are highly resistant to Pgt without evidence of pleiotropic effects.
Fig. 3: Expression and function of resistance genes in multi-transgene lines.

Data availability

See the Nature Research Reporting Summary. All raw data presented in Fig. 3a, Supplementary Figs. 1215 (RenSeq sequence data) and Fig. 3b (Sr55 quantitative expression sequencing data) were deposited at NCBI under Bioproject accession no. PRJNA624003. The following DNA sequences were also deposited at NCBI, that is, the Sr45Sr55Sr50Sr35Sr22 T-DNA sequence (GenBank accession no. MT165900), the p380HH vector (region from attR1 to attR2; GenBank accession no. MT180324), the pDONOR1 (D1) vector (GenBank accession no. MT180325) and the pDONOR2 (D2) vector (GenBank accession no. MT180326).


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We thank the 2Blades Foundation for financial support, Y.G. Liu for the 380H vector and C. Chen for assistance with figures.

Author information




Experimental design: M.A., M.L., B.J.S., T.L.R., R.F.; manuscript preparation: M.A., M.L., B.J.S., T.L.R., R.F., B.B.H.W.; field trials and pathology assays: B.J.S., M.J., O.M., J.A.K., M.P., C.S.; protoplast assays: M.L., A.W., D.O., P.D.; molecular analysis: S.C., A.W., M.L., M.H.; construct production: M.L., L.X., A.W., S.C., B.S., B.B.H.W., R.M., S.P., E.L.; bioinformatics: N.M.U., B.S., M.L.; wheat transformation: T.R., D.B., M.A.

Corresponding author

Correspondence to Michael Ayliffe.

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Competing interests

T.L.R. was employed by the 2Blades Foundation, while R.F. is chairman of the 2Blades board. Both were involved in the conceptualization, design, analysis and preparation of this research manuscript. The 2Blades Foundation cofunded the research presented.

Additional information

Peer review information Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–16.

Reporting Summary

Supplementary Table 1

Presence of full-length copies of each transgene present in selected transgenic plants using DNA blot analysis.

Supplementary Table 2

Inheritance of the multi-transgene locus in T1 progeny.

Supplementary Table 3

Seedling rust resistance assays.

Supplementary Table 4

Rust disease scores from field trials undertaken in 2018 and 2019 comparison.

Supplementary Table 5

Comparison of select growth, development, and yield parameters of five homozygous multi-transgene lines versus controls grown in the greenhouse.

Supplementary Table 6

Resistance of transgenic Fielder lines and control lines to Italian Puccinia graminis f.sp. tritici isolate IT76a of rate TTRTF.

Supplementary Table 7

Primer sequences.

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Luo, M., Xie, L., Chakraborty, S. et al. A five-transgene cassette confers broad-spectrum resistance to a fungal rust pathogen in wheat. Nat Biotechnol 39, 561–566 (2021).

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