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A pigeonpea gene confers resistance to Asian soybean rust in soybean

Nature Biotechnology volume 34, pages 661–665 (2016)Cite this article

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Abstract

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most economically important crop diseases, but is only treatable with fungicides, which are becoming less effective owing to the emergence of fungicide resistance. There are no commercial soybean cultivars with durable resistance to P. pachyrhizi, and although soybean resistance loci have been mapped, no resistance genes have been cloned. We report the cloning of a P. pachyrhizi resistance gene CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) from pigeonpea (Cajanus cajan) and show that CcRpp1 confers full resistance to P. pachyrhizi in soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement.

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Figure 1: C.cajan accessions display different reactions to P. pachyrhizi field isolates from Brazil.
Figure 2: Characterization of the CcRpp1 locus in four accessions of C. cajan.
Figure 3: Characterization of soybean plants heterologously expressing CcRpp1.

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Acknowledgements

This research was supported by grants from the 2Blades Foundation to The Sainsbury Laboratory and the Universidade Federal de Viçosa. 2Blades support was funded in part by DuPont-Pioneer. We wish to thank R. Godoy for providing the seeds of the C. cajan accessions used in this work. Work performed by D.R.C. and J.B. was supported by NSF award DBI 0605251. Next-generation and BAC sequencing was financed by the 2Blades Foundation. We thank G. Etherington and C. Schudoma for bioinformatics support, J. Pike for assisting in the preparation of the Solexa sequencing libraries, S. Perkins and the JIC horticultural team for excellent plant care, and the TSL support team for media preparation. We thank the DuPont Pioneer Soybean Transformation group for generating CcRpp1 transgenic events and the Controlled Environment group for growth, maintenance and seed advancement of transgenic material. We thank N. Penido, E. Feitosa Araújo and T. Castro Silva for excellent technical support during the high-resolution genotyping screenings and E.S. Mizubuti for helping with drawing Figure 1b. We thank D. Da Silva Toledo and L. Carlos Costa for plant care at UFV. We thank J. Pacheco Badel for implanting and curating a searchable seed database to support our genetic studies. We thank Y. Yamaoka for kindly providing the Japanese isolate T1-4. We acknowledge D. Horvath, M. Moscou and B. Staskawicz for many helpful discussions.

Author information

Author notes

  1. Burkhard Steuernagel, Costas Bouyioukos, Brande B H Wulff & Eric Ward

    Present address: Present addresses: The John Innes Centre, Norwich, U.K. (B.S. and B.B.H.W.); Institute of Systems & Synthetic Biology, Évry, France (C.B.); and AgBiome, Inc., Research Triangle Park, North Carolina, USA (E.W.).,

  2. H Peter van Esse, Jonathan D G Jones and Sérgio H Brommonschenkel: These authors contributed equally to this work.

Authors and Affiliations

  1. The Sainsbury Laboratory, Norwich, U.K.

    Cintia G Kawashima, Dan MacLean, Burkhard Steuernagel, Costas Bouyioukos, Brande B H Wulff, H Peter van Esse & Jonathan D G Jones

  2. Dep. de Fitopatologia, Universidade Federal de Viçosa, Viçosa, Brazil

    Gustavo Augusto Guimarães, Sônia Regina Nogueira, Bernardo do V A Melo, Gustavo Tristão, Jamile Camargos de Oliveira & Sérgio H Brommonschenkel

  3. Department of Plant Pathology, University of California Davis, Davis, California, USA

    Doug R Cook & Jongmin Baek

  4. Agricultural Biotechnology, DuPont-Pioneer, Wilmington, Delaware, USA

    Gilda Rauscher, Shipra Mittal, Lisa Panichelli, Karen Bacot, Ebony Johnson, Geeta Iyer, Girma Tabor, Gregory J Rairdan, Karen E Broglie & Gusui Wu

  5. 2Blades Foundation, Evanston, Illinois, USA

    Eric Ward

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Contributions

C.G.K., G.A.G., S.R.N., B.d.V.A.M., G.T., J.C.d.O., G.R., S.M., L.P., K.B., E.J., G.I., G.T. and S.H.B. performed research. D.R.C., J.B., C.B., B.S. and D.M. contributed bioinformatic tools. C.G.K., S.H.B., B.S., G.R., G.J.R., K.E.B., D.R.C., B.B.H.W., J.D.G.J., H.P.v.E. and E.W. analyzed the data. E.W., J.D.G.J., S.H.B., K.E.B., B.B.H.W., B.S., D.R.C., G.J.R., G.R., C.B. and G.W. edited the manuscript. C.G.K. and H.P.v.E. wrote the paper. K.E.B., G.W., E.W., B.B.H.W., H.P.v.E., J.D.G.J. and S.H.B. directed aspects of the project.

Corresponding author

Correspondence to H Peter van Esse.

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Competing interests

A patent application covering this work has been filed. 2Blades Foundation support was funded in part by DuPont-Pioneer.

Integrated supplementary information

Supplementary Figure 1 Fine-mapping of the CcRpp1 locus in the Cajanus cajan accession G119-99

(a) Physical and genetic map interval for the CcRpp1 locus based on the C. cajan reference genome scaffold (variety “Asha”). (b) Gain and loss of functions key recombinants closing the CcRpp1 genetic interval.

Supplementary Figure 2 Southern blot analyses to verify BAC clone integrity and NB-LRR copy number

(a) Southern blot of BglII- and HindIII-digested DNA of BAC 6G on the identified NB-LRR genes (NB 1-4) and, as a control, digested genomic DNA from the resistant parent G119-99. (b) Southern blot of HindIII- digested DNA of BACs 3F, 6G and the BAC clone obtained from DNA isolated from the resistant parent G59-95. The biotin labelled probe was designed on the region coding for the so-called “P-loop”, a highly variable structural feature present in NB-LRR proteins. The gene coding for CcRpp1 is indicated in bold.

Supplementary Figure 3 Visualization of RNAseq reads aligned to the four NB-LRR candidate genes NB-1, -2, -3 and -4.

(a) RNAseq reads from the resistant parent G119-99 aligned against the CcRpp1 locus sequence of 205,344 bp. (b) Close-up of the NB-LRR genes -1 to -4 paired-end read coverage.

Supplementary Figure 4 CcRpp1 expressing plants are not more resistant against another fungal plant pathogen

Disease score on CcRpp1 expressing plants homozygous for the transgene compared with segregating null plants when challenged with Fusarium virguliforme. Each dot represents a technical replicate consisting of 4-5 plants; different colours indicate different biological replicates; different symbols indicate technical replicates. Blue error bars indicate non-parametric (bootstrapped) limited 95% confidence interval of the mean. (see Supplementary Files 8-10 for details on the statistic analyses and figure generation).

Supplementary Figure 5 Expression of CcRpp1 does not impact basic agronomic traits

(a) Null segregants compared to plants expressing CcRpp1 at 8 weeks after germination. (b) Canopy area*of null segregants and plants homozygous for the CcRpp1 transgene (event CcRpp1 7.1) (c) Germination rate plotted against the expected germination rate. Numbers below zero indicate a lower than expected germination rate. Blue error bars indicate non-parametric (bootstrapped) limited 95% confidence interval of the mean. (see Supplementary Files 11-16 for details on the statistic analyses and figure generation). *Surface area determined with ImageJ 1.48a.

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Kawashima, C., Guimarães, G., Nogueira, S. et al. A pigeonpea gene confers resistance to Asian soybean rust in soybean. Nat Biotechnol 34, 661–665 (2016). https://doi.org/10.1038/nbt.3554

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