Theoretical and Applied Genetics

, Volume 128, Issue 11, pp 2213–2225 | Cite as

Functional divergence of duplicated genes results in a novel blast resistance gene Pi50 at the Pi2/9 locus

  • Jing Su
  • Wenjuan Wang
  • Jingluan Han
  • Shen Chen
  • Congying Wang
  • Liexian Zeng
  • Aiqing Feng
  • Jianyuan Yang
  • Bo ZhouEmail author
  • Xiaoyuan ZhuEmail author


Key message

We characterized a novel blast resistance gene Pi50 at the Pi2/9 locus; Pi50 is derived from functional divergence of duplicated genes. The unique features of Pi50 should facilitate its use in rice breeding and improve our understanding of the evolution of resistance specificities.


Rice blast disease, caused by the fungal pathogen Magnaporthe oryzae, poses constant, major threats to stable rice production worldwide. The deployment of broad-spectrum resistance (R) genes provides the most effective and economical means for disease control. In this study, we characterize the broad-spectrum R gene Pi50 at the Pi2/9 locus, which is embedded within a tandem cluster of 12 genes encoding proteins with nucleotide-binding site and leucine-rich repeat (NBS–LRR) domains. In contrast with other Pi2/9 locus, the Pi50 cluster contains four duplicated genes (Pi50_NBS4_1 to 4) with extremely high nucleotide sequence similarity. Moreover, these duplicated genes encode two kinds of proteins (Pi50_NBS4_1/2 and Pi50_NBS4_3/4) that differ by four amino acids. Complementation tests and resistance spectrum analyses revealed that Pi50_NBS4_1/2, not Pi50_NBS4_3/4, control the novel resistance specificity as observed in the Pi50 near isogenic line, NIL-e1. Pi50 shares greater than 96 % amino acid sequence identity with each of three other R proteins, i.e., Pi9, Piz-t, and Pi2, and has amino acid changes predominantly within the LRR region. The identification of Pi50 with its novel resistance specificity will facilitate the dissection of mechanisms behind the divergence and evolution of different resistance specificities at the Pi2/9 locus.


Blast Resistance Sequence Exchange Resistance Specificity Blast Resistance Gene Resistance Spectrum 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank Y. L. Peng and S. Q. Wu for providing rice blast isolates. This research is supported by grants from the National Transgenic Research Projects (2014ZX0800904B), the National Natural Science Foundation (31301304, 31461143019), the Guangzhou sciences and technology project (2012J2200066, 2012J4300059), Earmarked Fund for Modern Agro-Industry Technology Research System (CARS-01-24).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

122_2015_2579_MOESM1_ESM.pdf (25 kb)
Supplementary Fig. S1 Pairwise comparison of Pi50_NBS4_1 respectively with Pi50_NBS4_3 (top panel) and Pi50_NBS2 (lower panel). The sequence alignment was conducted using BLAST2 ( and the graphic summary was captured in scale. The sequence of Pi50_NBS4_1 was used as the subject for each comparison. The promoter/exon/intron are indicated corresponding to their positions
122_2015_2579_MOESM2_ESM.pdf (55 kb)
Supplementary Fig. S2 Overall good synteny with respect to gene order and composition between the Pi2 and Pi50 loci. The X-axis displays the genomic context of NBS–LRR genes at the Pi50 locus and the Y-axis displays the one at the Pi2 locus. The pseudomolecule of the Pi50 locus is composed of three fragments, i.e., the NIP side (GenBank accession no. KP985759), the central genomic block (GenBank accession no. KP985761), and the PK side (GenBank accession no. KP985760) was compared to the sequence of the Pi2 locus (GenBank accession no. DQ352453) using BLAST2. Nine orthologous groups (Pi50_NBS1-4, Pi50_NBS8-12) each indicated in different colors are named only for the Pi50 locus as an example. (PDF 54 kb)
122_2015_2579_MOESM3_ESM.pdf (14 kb)
Supplementary Fig. S3 Phylogenetic analysis of different NBS–LRR genes at the Pi2/9 locus. Pi2_NBS4, Pi9_NBS3, and Piz-t_NBS4 correspond to functional genes Pi2, Pi9, and Piz-t respectively. The Pi2/9 homologues in Nipponbare were named with the clone name AP005659. The tree was constructed using a neighbor-joining algorithm based on the predicted full-length sequence of proteins. Numbers on the branches indicate the percentage of 1000 bootstrap replicates. The unit branch length is equivalent to 0.1 amino acid substitutions per site, as indicated by the bar at the upper left corner. (PDF 24 kb) (PDF 13 kb)
122_2015_2579_MOESM4_ESM.doc (73 kb)
Supplementary material 4 (DOC 73 kb)


  1. Ameline-Torregrosa C, Wang B, O’Bleness M, Deshpande S, Zhu H, Roe B, Young N, Cannon S (2008) Identification and characterization of nucleotide-binding site-leucine-rich repeat genes in the model plant Medicago truncatula. Plant Physiol 146:5–21PubMedCentralCrossRefPubMedGoogle Scholar
  2. Ashikawa I, Hayashi N, Yamane H, Kanamori H, Wu J, Matsumoto T, Ono K, Yano M (2008) Two adjacent nucleotide-binding site-leucine-rich repeat class genes are required to confer Pikm-specific rice blast resistance. Genetics 180:2267–2276PubMedCentralCrossRefPubMedGoogle Scholar
  3. Bai J, Pennill L, Ning J, Lee S, Ramalingam J, Webb C, Zhao B, Sun Q, Nelson J, Leach J, Hulbert S (2002) Diversity in nucleotide binding site-leucine-rich repeat genes in cereals. Genome Res 12:1871–1884PubMedCentralCrossRefPubMedGoogle Scholar
  4. Bakker E, Toomajian C, Kreitman M, Bergelson J (2006) A genome-wide survey of R gene polymorphisms in Arabidopsis. Plant Cell 18:1803–1818PubMedCentralCrossRefPubMedGoogle Scholar
  5. Chen X, Ronald P (2011) Innate immunity in rice. Trends Plant Sci 16:451–459PubMedCentralCrossRefPubMedGoogle Scholar
  6. Chen X, Shang J, Chen D, Lei C, Zou Y, Zhai W, Liu G, Xu J, Ling Z, Cao G, Ma B, Wang Y, Zhao X, Li S, Zhu L (2006) A B-lectin receptor kinase gene conferring rice blast resistance. Plant J 46:794–804CrossRefPubMedGoogle Scholar
  7. Chen Q, Han Z, Jiang H, Tian D, Yang S (2010) Strong positive selection drives rapid diversification of R-genes in Arabidopsis relatives. J Mol Evol 70:137–148CrossRefPubMedGoogle Scholar
  8. Dai L, Wu J, Li X, Wang X, Liu X, Jantasuriyarat C, Kudrna D, Yu Y, Wing R, Han B, Zhou B, Wang G (2010) Genomic structure and evolution of the Pi2/9 locus in wild rice species. Theor Appl Genet 121:295–309CrossRefPubMedGoogle Scholar
  9. Davies PA, Gray G (2002) Long-range PCR. Methods Mol Biol 187:51–55PubMedGoogle Scholar
  10. Deng Y, Zhu X, Shen Y, He Z (2006) Genetic characterization and fine mapping of the blast resistance locus Pigm(t) tightly linked to Pi2 and Pi9 in a broad-spectrum resistant Chinese variety. Theor Appl Genet 113:705–713CrossRefPubMedGoogle Scholar
  11. Dodds P, Lawrence G, Catanzariti A, Teh T, Wang C, Ayliffe M, Kobe B, Ellis J (2006) Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes. Proc Natl Acad Sci USA 103:8888–8893PubMedCentralCrossRefPubMedGoogle Scholar
  12. Ellis J, Lawrence G, Luck J, Dodds P (1999) Identification of regions in alleles of the flax rust resistance gene L that determine differences in gene-for-gene specificity. Plant Cell 11:495–506PubMedCentralCrossRefPubMedGoogle Scholar
  13. Ellis J, Lagudah E, Spielmeyer W, Dodds P (2014) The past, present and future of breeding rust resistant wheat. Front Plant Sci 5:641PubMedCentralCrossRefPubMedGoogle Scholar
  14. Fukuoka S, Saka N, Koga H, Ono K, Shimizu T, Ebana K, Hayashi N, Takahashi A, Hirochika H, Okuno K, Yano M (2009) Loss of function of a proline-containing protein confers durable disease resistance in rice. Science 325:998–1001CrossRefPubMedGoogle Scholar
  15. Fukuoka S, Mizobuchi R, Saka N, Suprun I, Matsumoto T, Okuno K, Yano M (2012) A multiple gene complex on rice chromosome 4 is involved in durable resistance to rice blast. Theor Appl Genet 125:551–559PubMedCentralCrossRefPubMedGoogle Scholar
  16. Hiei Y, Komari T, Kubo T (1997) Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol Biol 35:205–218CrossRefPubMedGoogle Scholar
  17. Hua L, Wu J, Chen C, Wu W, He X, Lin F, Wang L, Ashikawa I, Matsumoto T, Wang L, Pan Q (2012) The isolation of Pi1, an allele at the Pik locus which confers broad spectrum resistance to rice blast. Theor Appl Genet 125:1047–1055CrossRefPubMedGoogle Scholar
  18. Hulbert S, Webb C, Smith S, Sun Q (2001) Resistance gene complexes: evolution and utilization. Annu Rev Phytopathol 39:285–312CrossRefPubMedGoogle Scholar
  19. Jeung J, Kim B, Cho Y, Han S, Moon H, Lee Y, Jena K (2007) A novel gene, Pi40(t), linked to the DNA markers derived from NBS–LRR motifs confers broad spectrum of blast resistance in rice. Theor Appl Genet 115:1163–1177CrossRefPubMedGoogle Scholar
  20. Jiang N, Li Z, Wu J, Wang Y, Wu L, Wang S, Wang D, Wen T, Liang Y, Sun P, Liu J, Dai L, Wang Z, Wang C, Luo M, Liu X, Wang G (2012) Molecular mapping of the Pi2/9 allelic gene Pi2-2 conferring broad-spectrum resistance to Magnaporthe oryzae in the rice cultivar Jefferson. Rice 5:29CrossRefGoogle Scholar
  21. Jones J, Dangl J (2006) The plant immune system. Nature 444:323–329CrossRefPubMedGoogle Scholar
  22. Jung Y, Agrawal G, Rakwal R, Kim J, Lee M, Choi P, Kim Y, Kim M, Shibato J, Kim S, Iwahashi H, Jwa N (2006) Functional characterization of OsRacB GTPase–a potentially negative regulator of basal disease resistance in rice. Plant Physiol Biochem 44:68–77CrossRefPubMedGoogle Scholar
  23. Jupe F, Pritchard L, Etherington G, Mackenzie K, Cock P, Wright F, Sharma S, Bolser D, Bryan G, Jones J, Hein I (2012) Identification and localization of the NB-LRR gene family within the potato genome. BMC Genom 13:75CrossRefGoogle Scholar
  24. Kanzaki H, Yoshida K, Saitoh H, Fujisaki K, Hirabuchi A, Alaux L, Fournier E, Tharreau D, Terauchi R (2012) Arms race co-evolution of Magnaporthe oryzae AVR-Pik and rice Pik genes driven by their physical interactions. Plant J 72:894–907PubMedGoogle Scholar
  25. Karasov T, Kniskern J, Gao L, DeYoung B, Ding J, Dubiella U, Lastra R, Nallu S, Roux F, Innes R, Barrett L, Hudson R, Bergelson J (2014) The long-term maintenance of a resistance polymorphism through diffuse interactions. Nature 512:436–440CrossRefPubMedGoogle Scholar
  26. Kohler A, Rinaldi C, Duplessis S, Baucher M, Geelen D, Duchaussoy F, Meyers B, Boerjan W, Martin F (2008) Genome-wide identification of NBS resistance genes in Populus trichocarpa. Plant Mol Biol 66:619–636CrossRefPubMedGoogle Scholar
  27. Kuang H, Woo S, Meyers B, Nevo E, Michelmore R (2004) Multiple genetic processes result in heterogeneous rates of evolution within the major cluster disease resistance genes in lettuce. Plant Cell 16:2870–2894PubMedCentralCrossRefPubMedGoogle Scholar
  28. Kuang H, Caldwell K, Meyers B, Michelmore R (2008) Frequent sequence exchanges between homologs of RPP8 in Arabidopsis are not necessarily associated with genomic proximity. Plant J 54:69–80CrossRefPubMedGoogle Scholar
  29. Leister D (2004) Tandem and segmental gene duplication and recombination in the evolution of plant disease resistance gene. Trends Genet 20:116–122CrossRefPubMedGoogle Scholar
  30. Li W, Wang B, Wu J, Lu G, Hu Y, Zhang X, Zhang Z, Zhao Q, Feng Q, Zhang H, Wang Z, Wang G, Han B, Wang Z, Zhou B (2009) The Magnaporthe oryzae avirulence gene AvrPiz-t encodes a predicted secreted protein that triggers the immunity in rice mediated by the blast resistance gene Piz-t. Mol Plant Microbe Interact 22:411–420CrossRefPubMedGoogle Scholar
  31. Lin F, Chen S, Que Z, Wang L, Liu X, Pan Q (2007) The blast resistance gene Pi37 encodes a nucleotide binding site leucine-rich repeat protein and is a member of a resistance gene cluster on rice chromosome 1. Genetics 177:1871–1880PubMedCentralCrossRefPubMedGoogle Scholar
  32. Liu G, Lu G, Zeng L, Wang G (2002) Two broad-spectrum blast resistance genes, Pi9(t) and Pi2(t), are physically linked on rice chromosome 6. Mol Genet Genomics 267:472–480CrossRefPubMedGoogle Scholar
  33. Liu W, Liu J, Triplett L, Leach J, Wang G (2014) Novel insights into rice innate immunity against bacterial and fungal pathogens. Annu Rev Phytopathol 52:213–241CrossRefPubMedGoogle Scholar
  34. Luck J, Lawrence G, Dodds P, Shepherd K, Ellis J (2000) Regions outside of the leucine-rich repeats of flax rust resistance proteins play a role in specificity determination. Plant Cell 12:1367–1377PubMedCentralCrossRefPubMedGoogle Scholar
  35. Luo S, Zhang Y, Hu Q, Chen J, Li K, Lu C, Liu H, Wang W, Kuang H (2012) Dynamic nucleotide-binding site and leucine-rich repeat-encoding genes in the grass family. Plant Physiol 159:197–210PubMedCentralCrossRefPubMedGoogle Scholar
  36. Ma J, Lei C, Xu X, Hao K, Wang J, Cheng Z, Ma X, Ma J, Zhou K, Zhang X, Guo X, Wu F, Lin Q, Wang C, Zhai H, Wang H, Wan J (2015) Pi64, encoding a novel CC–NBS–LRR protein, confers resistance to leaf and neck blast in rice. Mol Plant Microbe Interact (Epub ahead of print) Google Scholar
  37. Marone D, Russo M, Laido G, De Leonardis A, Mastrangelo A (2013) Plant nucleotide binding site-leucine-rich repeat (NBS–LRR) genes: active guardians in host defense responses. Int J Mol Sci 14:7302–7326PubMedCentralCrossRefPubMedGoogle Scholar
  38. Martin G, Bogdanove A, Sessa G (2003) Understanding the functions of plant disease resistance proteins. Annu Rev Plant Biol 54:23–61CrossRefPubMedGoogle Scholar
  39. Meyers B, Kozik A, Griego A, Kuang H, Michelmore R (2003) Genome-wide analysis of NBS–LRR-encoding genes in Arabidopsis. Plant Cell 15:809–834PubMedCentralCrossRefPubMedGoogle Scholar
  40. Michelmore R, Meyers B (1998) Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process. Genome Res 8:1113–1130PubMedGoogle Scholar
  41. Mondragon-Palomino M, Gaut B (2005) Gene conversion and the evolution of three leucine-rich repeat gene families in Arabidopsis thaliana. Mol Biol Evol 22:2444–2456CrossRefPubMedGoogle Scholar
  42. Moytri R, Jia Y, Richard DC (2012) Structure, function, and co-evolution of rice blast resistance genes. Acta Agron Sin 38:381–393CrossRefGoogle Scholar
  43. Nguyen T, Koizumi S, La T, Zenbayashi K, Ashizawa T, Yasuda N, Imazaki I, Miyasaka A (2006) Pi35(t), a new gene conferring partial resistance to leaf blast in the rice cultivar Hokkai 188. Theor Appl Genet 113:697–704CrossRefPubMedGoogle Scholar
  44. Ou S (1985) Rice disease. Second Edition, Commonwealth Mycological Institute, Kew Surrey, The Cambrian News Ltd, UK, pp 109–201Google Scholar
  45. Qu S, Liu G, Zhou B, Bellizzi M, Zeng L, Dai L, Han B, Wang G (2006) The broad-spectrum blast resistance gene Pi9 encodes a nucleotide-binding site-leucine-rich repeat protein and is a member of a multigene family in rice. Genetics 172:1901–1914PubMedCentralCrossRefPubMedGoogle Scholar
  46. Ravensdale M, Bernoux M, Ve T, Kobe B, Thrall P, Ellis J, Dodds P (2012) Intramolecular interaction influences binding of the Flax L5 and L6 resistance proteins to their AvrL567 ligands. PLoS Pathog 8:e1003004PubMedCentralCrossRefPubMedGoogle Scholar
  47. Richter T, Ronald P (2000) The evolution of disease resistance genes. Plant Mol Biol 42:195–204CrossRefPubMedGoogle Scholar
  48. Sun Q, Collins N, Ayliffe M, Smith S, Drake J, Pryor T, Hulbert S (2001) Recombination between paralogues at the Rp1 rust resistance locus in maize. Genetics 158:423–438PubMedCentralPubMedGoogle Scholar
  49. Takahashi A, Hayashi N, Miyao A, Hirochika H (2010) Unique features of the rice blast resistance Pish locus revealed by large scale retrotransposon-tagging. BMC Plant Biol 10:175PubMedCentralCrossRefPubMedGoogle Scholar
  50. Tan S, Wu S (2012) Genome Wide analysis of nucleotide-binding site disease resistance genes in Brachypodium distachyon. Comp Funct Genomics 2012:418208PubMedCentralCrossRefPubMedGoogle Scholar
  51. Thompson J, Gibson T, Plewniak F, Jeanmougin F, Higgins D (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 25:4876–4882PubMedCentralCrossRefPubMedGoogle Scholar
  52. Wang W, Su J, Zhang J, Li Y, Chen S, Zeng L, Yang J, Zhu X (2012a) Pathogenicity analysis of the rice blast fungus isolated from the blast panicles of Yuejingsimiao 2. Guangdong Agric Sin 23:59–61 (in Chinese) Google Scholar
  53. Wang Y, Wang D, Deng X, Liu J, Sun P, Liu Y, Huang H, Jiang N, Kang H, Ning Y, Wang Z, Xiao Y, Liu X, Liu E, Dai L, Wang G (2012b) Molecular mapping of the blast resistance genes Pi2-1 and Pi51(t) in the durably resistant rice ‘Tianjingyeshengdao’. Phytopathology 102:779–786CrossRefPubMedGoogle Scholar
  54. Wu K, Xu T, Guo C, Zhang X, Yang S (2012) Heterogeneous evolutionary rates of Pi2/9 homologs in rice. BMC Genet 13:73PubMedCentralCrossRefPubMedGoogle Scholar
  55. Wu W, Wang L, Zhang S, Li Z, Zhang Y, Lin F, Pan Q (2014) Stepwise arms race between AvrPik and Pik alleles in the rice blast pathosystem. Mol Plant Microbe Interact 27:759–769CrossRefPubMedGoogle Scholar
  56. Wu J, Kou Y, Bao J, Li Y, Tang M, Zhu X, Ponaya A, Xiao G, Li J, Li C, Song M, Cumagun C, Deng Q, Lu G, Jeon J, Naqvi N, Zhou B (2015) Comparative genomics identifies the Magnaporthe oryzae avirulence effector AvrPi9 that triggers Pi9-mediated blast resistance in rice. N Phytol. doi: 10.1111/nph.13310 (Epub ahead of print) Google Scholar
  57. Xu X, Lv Q, Shang J, Pang Z, Zhou Z, Wang J, Jiang G, Tao Y, Xu Q, Li X, Zhao X, Li S, Xu J, Zhu L (2014) Excavation of Pid3 orthologs with differential resistance spectra to Magnaporthe oryzae in rice resource. PLoS One 9:e93275PubMedCentralCrossRefPubMedGoogle Scholar
  58. Yang Z (1997) PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci 13:555–556PubMedGoogle Scholar
  59. Yang Z, Nielsen R, Goldman N, Pedersen AM (2000) Codon-substitution models for heterogeneous selection pressure at amino acid sites. Genetics 155:431–449PubMedCentralPubMedGoogle Scholar
  60. Yang J, Chen S, Zeng L, Li Y, Chen Z, Li C, Zhu X (2008) Race specificity of major rice blast resistance genes to Magnaporthe grisea isolates collected from indica Rice in Guangdong, China. Rice Sci 15:311–318CrossRefGoogle Scholar
  61. Yuan B, Zhai C, Wang W, Zeng X, Xu X, Hu H, Lin F, Wang L, Pan Q (2011) The Pik-p resistance to Magnaporthe oryzae in rice is mediated by a pair of closely linked CC–NBS–LRR genes. Theor Appl Genet 122:1017–1028CrossRefPubMedGoogle Scholar
  62. Yue J, Meyers B, Chen J, Tian D, Yang S (2012) Tracing the origin and evolutionary history of plant nucleotide-binding site-leucine-rich repeat (NBS–LRR) genes. N Phytol 193:1049–1063CrossRefGoogle Scholar
  63. Zhai C, Lin F, Dong Z, He X, Yuan B, Zeng X, Wang L, Pan Q (2011) The isolation and characterization of Pik, a rice blast resistance gene which emerged after rice domestication. N Phytol 189:321–334CrossRefGoogle Scholar
  64. Zhou T, Wang Y, Chen J, Araki H, Jing Z, Jiang K, Shen J, Tian D (2004) Genome-wide identification of NBS genes in japonica rice reveals significant expansion of divergent non-TIR NBS–LRR genes. Mol Genet Genomics 271:402–415CrossRefPubMedGoogle Scholar
  65. Zhou B, Qu S, Liu G, Dolan M, Sakai H, Lu G, Bellizzi M, Wang G (2006) The eight amino-acid differences within three leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the resistance specificity to Magnaporthe grisea. Mol Plant Microbe Interact 19:1216–1228CrossRefPubMedGoogle Scholar
  66. Zhou B, Dolan M, Sakai H, Wang G (2007) The genomic dynamics and evolutionary mechanism of the Pi2/9 locus in rice. Mol Plant Microbe Interact 20:63–71CrossRefPubMedGoogle Scholar
  67. Zhu X, Yang J, Chen Y, Yang W, Chen X, Zeng L, Chen S (2008) Race identification and pathogenicity test of the blast fungus causing the resistance breakdown of hybrid rice Tianyou 998. Guangdong Agric Sin 12:84–86 (in Chinese) Google Scholar
  68. Zhu X, Chen S, Yang J, Zhou S, Zeng L, Han J, Su J, Wang L, Pan Q (2012) The identification of Pi50(t), a new member of the rice blast resistance Pi2/Pi9 multigene family. Theor Appl Genet 124:1295–1304CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Plant Protection Research InstituteGuangdong Academy of Agricultural SciencesGuangzhouChina
  2. 2.Plant Breeding, Genetics, and Biotechnology DivisionInternational Rice Research InstituteMetro ManilaPhilippines
  3. 3.College of Life SciencesSouth China Agricultural UniversityGuangzhouChina

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