Skip to main content

Thank you for visiting nature.com. 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.

  • Letter
  • Published:

Genomics accelerated isolation of a new stem rust avirulence gene–wheat resistance gene pair

Nature Plants volume 7, pages 1220–1228 (2021)Cite this article

Subjects

Abstract

Stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt) is a devastating disease of the global staple crop wheat. Although this disease was largely controlled in the latter half of the twentieth century, new virulent strains of Pgt, such as Ug99, have recently evolved1,2. These strains have caused notable losses worldwide and their continued spread threatens global wheat production. Breeding for disease resistance provides the most cost-effective control of wheat rust diseases3. A number of rust resistance genes have been characterized in wheat and most encode immune receptors of the nucleotide-binding leucine-rich repeat (NLR) class4, which recognize pathogen effector proteins known as avirulence (Avr) proteins5. However, only two Avr genes have been identified in Pgt so far, AvrSr35 and AvrSr50 (refs. 6,7), and none in other cereal rusts8,9. The Sr27 resistance gene was first identified in a wheat line carrying an introgression of the 3R chromosome from Imperial rye10. Although not deployed widely in wheat, Sr27 is widespread in the artificial crop species Triticosecale (triticale), which is a wheat–rye hybrid and is a host for Pgt11,12. Sr27 is effective against Ug99 (ref. 13) and other recent Pgt strains14,15. Here, we identify both the Sr27 gene in wheat and the corresponding AvrSr27 gene in Pgt and show that virulence to Sr27 can arise experimentally and in the field through deletion mutations, copy number variation and expression level polymorphisms at the AvrSr27 locus.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Pgt mutants with virulence to Sr27.
Fig. 2: The AvrSr27 locus encodes two related secreted proteins.
Fig. 3: Differential expression of AvrSr27 alleles.
Fig. 4: Identification of the Sr27 resistance gene.

Similar content being viewed by others

Data availability

All sequence data from this study are available in NCBI under BioProject PRJNA695305 (Sr27 MutRenSeq data) and PRJNA698655 (Pgt21-0 mutants). This includes raw data for Fig. 2b and Supplementary Figs. 2 and 8. Figure 3 is derived from raw sequence data available in NCBI BioProject PRJNA253722 and PRJNA415866. All other relevant data are available on request from the corresponding author.

Code availability

Scripts and files for MutRenSeq analysis are available at https://github.com/TC-Hewitt/MuTrigo.

References

  1. Singh, R. P. et al. Emergence and spread of new races of wheat stem rust fungus: continued threat to food security and prospects of genetic control. Phytopathology 105, 872–884 (2015).

    Article  PubMed  Google Scholar 

  2. Li, F. et al. Emergence of the Ug99 lineage of the wheat stem rust pathogen through somatic hybridisation. Nat. Commun. 10, 5068 (2019).

  3. Ellis, J. G., Lagudah, E. S., Spielmeyer, W. & Dodds, P. N. The past, present and future of breeding rust resistant wheat. Front. Plant Sci. https://doi.org/10.3389/fpls.2014.00641 (2014).

  4. Periyannan, S., Milne, R. J., Figueroa, M., Lagudah, E. S. & Dodds, P. N. An overview of genetic rust resistance: from broad to specific mechanisms. PloS Pathog. https://doi.org/10.1371/journal.ppat.1006380 (2017).

  5. Garnica, D. P., Nemri, A., Upadhyaya, N. M., Rathjen, J. P. & Dodds, P. N. The ins and outs of rust haustoria. PloS Pathog. https://doi.org/10.1371/journal.ppat.1004329 (2014).

  6. Chen, J. et al. Loss of AvrSr50 by somatic exchange in stem rust leads to virulence for Sr50 resistance in wheat. Science 358, 1607–1610 (2017).

    Article  CAS  PubMed  Google Scholar 

  7. Salcedo, A. et al. Variation in the AvrSr35 gene determines Sr35 resistance against wheat stem rust race Ug99. Science 358, 1604–1606 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bakkeren, G. & Szabo, L. J. Progress on molecular genetics and manipulation of rust fungi. Phytopathology 110, 532–543 (2020).

  9. Figueroa, M., Dodds, P. N. & Henningsen, E. C. Evolution of virulence in rust fungi—multiple solutions to one problem. Curr. Opin. Plant Biol. 56, 20–27 (2020).

    Article  CAS  PubMed  Google Scholar 

  10. McIntosh, R. A., Wellings, C. R. & Park, R. F. Wheat Rusts: An Atlas of Resistance Genes (CSIRO Publications, 1995).

    Book  Google Scholar 

  11. McIntosh, R. A., Luig, N. H., Milne, D. L. & Cusick, J. Vulnerability of triticales to wheat stem rust. Can. J. Plant. Pathol. 5, 61–69 (1983).

    Article  Google Scholar 

  12. Zhang, J., Wellings, C. R., McIntosh, R. A. & Park, R. F. Seedling resistances to rust diseases in international triticale germplasm. Crop Pasture Sci. 61, 1036–1048 (2010).

    Article  Google Scholar 

  13. Jin, Y., Pretorius, Z. A. & Singh, R. P. New virulence within race TTKS (Ug99) of the stem rust pathogen and effective resistance genes. Phytopathology 97, S137–S137 (2007).

    Google Scholar 

  14. Olivera, P. et al. Phenotypic and genotypic characterization of race TKTTF of Puccinia graminis f. sp. tritici that caused a wheat stem rust epidemic in southern Ethiopia in 2013–14. Phytopathology 105, 917–928 (2015).

    Article  PubMed  Google Scholar 

  15. Olivera, P. D. et al. Presence of a sexual population of Puccinia graminis f. sp. tritici in Georgia provides a hotspot for genotypic and phenotypic diversity. Phytopathology 109, 2152–2160 (2019).

    Article  PubMed  Google Scholar 

  16. Upadhyaya, N. M. et al. Comparative genomics of Australian isolates of the wheat stem rust pathogen Puccinia graminis f. sp. tritici reveals extensive polymorphism in candidate effector genes. Front. Plant Sci. https://doi.org/10.3389/fpls.2014.00759 (2015).

  17. Zhang, J., Zhang, P., Karaoglu, H. & Park, R. F. Molecular characterization of australian isolates of Puccinia graminis f. sp. tritici supports long-term clonality but also reveals cryptic genetic variation. Phytopathology 107, 1032–1038 (2017).

    Article  CAS  PubMed  Google Scholar 

  18. Lewis, C. M. et al. Potential for re-emergence of wheat stem rust in the United Kingdom. Commun. Biol. 1, 13 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Visser, B. et al. Microsatellite analysis and urediniospore dispersal simulations support the movement of Puccinia graminis f. sp. tritici from Southern Africa to Australia. Phytopathology 109, 133–144 (2019).

    Article  CAS  PubMed  Google Scholar 

  20. Visser, B., Herselman, L. & Pretorius, Z. A. Genetic comparison of Ug99 with selected South African races of Puccinia graminis f. sp. tritici. Mol. Plant Pathol. 10, 213–222 (2009).

    Article  CAS  PubMed  Google Scholar 

  21. Lee, W. S., Hammond-Kosack, K. E. & Kanyuka, K. Barley stripe mosaic virus-mediated tools for investigating gene function in cereal plants and their pathogens: virus-induced gene silencing, host-mediated gene silencing, and virus-mediated overexpression of heterologous protein. Plant Physiol. 160, 582–590 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Qutob, D. et al. Copy number variation and transcriptional polymorphisms of Phytophthora sojae RXLR effector genes Avr1a and Avr3a. PLoS ONE 4, e5066 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Pais, M. et al. Gene expression polymorphism underpins evasion of host immunity in an asexual lineage of the Irish potato famine pathogen. BMC Evol. Biol. 18, 93 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Wu, W. et al. Flax rust infection transcriptomics reveals a transcriptional profile that may be indicative for rust Avr genes. PLoS ONE 4, e0226106 (2019).

    Article  CAS  Google Scholar 

  25. Steuernagel, B. et al. Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture. Nat. Biotechnol. 34, 652–655 (2016).

  26. Appels, R. et al. Shifting the limits in wheat research and breeding using a fully annotated reference genome. Science 361, eaar7191 (2018).

    Article  CAS  Google Scholar 

  27. Saur, I. M. L., Bauer, S., Lu, X. L. & Schulze-Lefert, P. A cell death assay in barley and wheat protoplasts for identification and validation of matching pathogen AVR effector and plant NLR immune receptors. Plant Methods https://doi.org/10.1186/s13007-019-0502-0 (2019).

  28. Luo, M. et al. A five-transgene cassette confers broad-spectrum resistance to a fungal rust pathogen in wheat. Nat. Biotechnol. 39, 561–566 (2020).

    Article  CAS  Google Scholar 

  29. Mago, R. et al. The wheat Sr50 gene reveals rich diversity at a cereal disease resistance locus. Nat. Plants 1, 15186 (2015).

    Article  CAS  PubMed  Google Scholar 

  30. Marais, G. F. An evaluation of three Sr27-carrying wheat × rye translocations. South Afr. J. Plant Soil 18, 135–136 (2001).

    Article  Google Scholar 

  31. Mago, R. et al. Transfer of stem rust resistance gene SrB from Thinopyrum ponticum into wheat and development of a closely linked PCR-based marker. Theor. Appl Genet. 132, 371–382 (2019).

    Article  CAS  PubMed  Google Scholar 

  32. Zhang, J. et al. A recombined Sr26 and Sr61 disease resistance gene stack in wheat encodes unrelated NLR genes. Nat. Commun. 12, 3378 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wulff, B. B., Horvath, D. M. & Ward, E. R. Improving immunity in crops: new tactics in an old game. Curr. Opin. Plant Biol. 14, 468–476 (2011).

    Article  CAS  PubMed  Google Scholar 

  34. Hubbard, A. et al. Field pathogenomics reveals the emergence of a diverse wheat yellow rust population. Genome Biol. 16, 23 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Sperschneider, J. et al. The stem rust fungus Puccinia graminis f. sp. tritici induces centromeric small RNAs during late infection that direct genome-wide DNA methylation. Preprint at bioRxiv https://doi.org/10.1101/469338 (2020).

  36. Park, R. F. Stem rust of wheat in Australia. Aust. J. Agric. Res. 58, 558–566 (2007).

    Article  Google Scholar 

  37. Singh, S. J. & Mcintosh, R. A. Allelism of 2 genes for stem rust resistance in triticale. Euphytica 38, 185–189 (1988).

    Article  Google Scholar 

  38. Acosta, A. C. The transfer of stem rust resistance from rye to wheat. Dissertation Abstr. 23, 34–35 (1962).

    Google Scholar 

  39. Garrison, E. & Marth, G. Haplotype-based variant detection from short-read sequencing. Preprint at https://arxiv.org/abs/1207.3907 (2012).

  40. Stamatakis, A., Ludwig, T. & Meier, H. RAxML-III: a fast program for maximum likelihood-based inference of large phylogenetic trees. Bioinformatics 21, 456–463 (2005).

    Article  CAS  PubMed  Google Scholar 

  41. Paradis, E., Claude, J. & Strimmer, K. APE: analyses of phylogenetics and evolution in R language. Bioinformatics 20, 289–290 (2004).

    Article  CAS  PubMed  Google Scholar 

  42. Heibl, C. PHYLOCH: R language tree plotting tools and interfaces to diverse phylogenetic software packages (R Foundation for Statistical Computing, 2008); http://www.christophheibl.de/Rpackages.html

  43. Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer, 2016).

  44. Yin, T., Cook, D. & Lawrence, M. ggbio: an R package for extending the grammar of graphics for genomic data. Genome Biol. 13, R77 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evolution 35, 1547–1549 (2018).

    Article  CAS  Google Scholar 

  46. Lee, W. S., Rudd, J. J. & Kanyuka, K. Virus induced gene silencing (VIGS) for functional analysis of wheat genes involved in Zymoseptoria tritici susceptibility and resistance. Fungal Genet. Biol. 79, 84–88 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Franco-Orozco, B. et al. A new proteinaceous pathogen-associated molecular pattern (PAMP) identified in Ascomycete fungi induces cell death in Solanaceae. New Phytol. 214, 1657–1672 (2017).

    Article  CAS  PubMed  Google Scholar 

  48. Hen-Avivi, S. et al. A metabolic gene cluster in the wheat W1 and the barley cer-cqu loci determines beta-diketone biosynthesis and glaucousness. Plant Cell 28, 1440–1460 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Lee, W. S., Rudd, J. J., Hammÿond-Kosack, K. E. & Kanyuka, K. Mycosphaerella graminicola LysM effector-mediated stealth pathogenesis subverts recognition through both CERK1 and CEBiP homologues in wheat. Mol. Plant Microbe Interact. 27, 236–243 (2014).

    Article  CAS  PubMed  Google Scholar 

  50. Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. https://doi.org/10.1186/s13059-014-0550-8 (2014).

  52. Mago, R. et al. Generation of loss-of-function mutants for wheat rust disease resistance gene cloning. Wheat Rust. Dis.: Methods Protoc. 1659, 199–205 (2017).

    Article  CAS  Google Scholar 

  53. Mago, R. et al. Development of PCR markers for the selection of wheat stem rust resistance genes Sr24 and Sr26 in diverse wheat germplasm. Theor. Appl. Genet. 111, 496–504 (2005).

    Article  CAS  PubMed  Google Scholar 

  54. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Rabanus-Wallace, M. T. et al. Chromosome-scale genome assembly provides insights into rye biology, evolution and agronomic potential. Nat. Genet. 53, 564–573 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Akamatsu, A. et al. An OsCEBiP/OsCERK1-OsRacGEF1-OsRac1 module is an essential early component of chitin-induced rice immunity. Cell Host Microbe 13, 465–476 (2013).

    Article  CAS  PubMed  Google Scholar 

  59. Arndell, T. et al. gRNA validation for wheat genome editing with the CRISPR-Cas9 system. BMC Biotech. 19, 71 (2019).

    Article  CAS  Google Scholar 

  60. Bemoux, M. et al. Structural and functional analysis of a plant resistance protein TIR domain reveals interfaces for self-association, signaling, and autoregulation. Cell Host Microbe 9, 200–211 (2011).

    Article  CAS  Google Scholar 

  61. Cesari, S. et al. Cytosolic activation of cell death and stem rust resistance by cereal MLA-family CC-NLR proteins. Proc. Natl Acad. Sci. USA 113, 10204–10209 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Cesari, S. et al. The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding. Plant Cell 25, 1463–1481 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Richardson, T., Thistleton, J., Higgins, T. J., Howitt, C. & Ayliffe, M. Efficient Agrobacterium transformation of elite wheat germplasm without selection. Plant Cell, Tissue Organ Cult. 119, 647–659 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Work described here was supported by funding from the 2Blades foundation to P.N.D. J.C. was supported by a Chinese Scholarship Council postgraduate fellowship. J.S. was supported by an Australian Research Council Discovery Early Career Researcher Award (no. DE190100066). K.K. and V.P. acknowledge financial support by the Institute Strategic Program Grant ‘Designing Future Wheat’ (grant no. BB/P016855/1) from the Biotechnology and Biological Sciences Research Council of the UK. H.N.-P. was supported by USDA-NIFA grant no. 2018-67013-27819.

Author information

Author notes

  1. Diana Ortiz

    Present address: Génétique et Amélioration des Fruits et Légumes, INRA, Montfavet Cedex, France

  2. These authors contributed equally: Narayana M. Upadhyaya, Rohit Mago.

Authors and Affiliations

  1. Commonwealth Scientific and Industrial Research Organisation, Agriculture and Food, Canberra, Australian Capital Territory, Australia

    Narayana M. Upadhyaya, Rohit Mago, Tim Hewitt, Ming Luo, Jian Chen, Aihua Wang, Diana Ortiz, Luch Hac, Dhara Bhatt, Jianping Zhang, Michael Ayliffe, Melania Figueroa, Jeffrey G. Ellis & Peter N. Dodds

  2. Biointeractions and Crop Protection, Rothamsted Research, Harpenden, UK

    Vinay Panwar & Kostya Kanyuka

  3. Research School of Biology, Australian National University, Canberra, Australian Capital Territory, Australia

    Jian Chen

  4. Biological Data Science Institute, Australian National University, Canberra, Australian Capital Territory, Australia

    Jana Sperschneider

  5. Department of Ecology and Evolutionary Biology, Vietnam National University, Ho Chi Minh, Vietnam

    Hoa Nguyen-Phuc

  6. Department of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN, USA

    Feng Li

Authors
  1. Narayana M. Upadhyaya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rohit Mago
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vinay Panwar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tim Hewitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ming Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jian Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jana Sperschneider
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hoa Nguyen-Phuc
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Aihua Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Diana Ortiz
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Luch Hac
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Dhara Bhatt
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Feng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Jianping Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Michael Ayliffe
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Melania Figueroa
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Kostya Kanyuka
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Jeffrey G. Ellis
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Peter N. Dodds
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.N.D. conceptualized the project, acquired funding and supervised the work. N.M.U., R.M., V.P., M.L., A.W., D.O., J.C., J.Z., D.B., M.A. and L.H. acquired experimental data. N.M.U., R.M., J.S., H.N.-P., T.H., M.F., K.K., J.G.E. and P.N.D conducted data analysis. P.N.D. drafted the manuscript and all authors contributed to review and editing.

Corresponding author

Correspondence to Peter N. Dodds.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Beat Keller, Simon Krattinger and Brande Wulff 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. 1–11, Tables 1 and 2 and source data as uncropped gel/blot images for Supplementary Figs. 3, 5a, 6 and 11.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Upadhyaya, N.M., Mago, R., Panwar, V. et al. Genomics accelerated isolation of a new stem rust avirulence gene–wheat resistance gene pair. Nat. Plants 7, 1220–1228 (2021). https://doi.org/10.1038/s41477-021-00971-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41477-021-00971-5

This article is cited by

Search

Advanced search

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