Functional & Integrative Genomics

, Volume 12, Issue 2, pp 215–228 | Cite as

A novel blast resistance gene, Pi54rh cloned from wild species of rice, Oryza rhizomatis confers broad spectrum resistance to Magnaporthe oryzae

  • Alok Das
  • D. Soubam
  • P. K. Singh
  • S. Thakur
  • N. K. Singh
  • T. R. Sharma
Original Paper


The dominant rice blast resistance gene, Pi54 confers resistance to Magnaporthe oryzae in different parts of India. In our effort to identify more effective forms of this gene, we isolated an orthologue of Pi54 named as Pi54rh from the blast-resistant wild species of rice, Oryza rhizomatis, using allele mining approach and validated by complementation. The Pi54rh belongs to CC-NBS-LRR family of disease resistance genes with a unique Zinc finger (C3H type) domain. The 1,447 bp Pi54rh transcript comprises of 101 bp 5′-UTR, 1,083 bp coding region and 263 bp 3′-UTR, driven by pathogen inducible promoter. We showed the extracellular localization of Pi54rh protein and the presence of glycosylation, myristoylation and phosphorylation sites which implicates its role in signal transduction process. This is in contrast to other blast resistance genes that are predicted to be intracellular NBS-LRR-type resistance proteins. The Pi54rh was found to express constitutively at basal level in the leaves, but upregulates 3.8-fold at 96 h post-inoculation with the pathogen. Functional validation of cloned Pi54rh gene using complementation test showed high degree of resistance to seven isolates of M. oryzae collected from different geographical locations of India. In this study, for the first time, we demonstrated that a rice blast resistance gene Pi54rh cloned from wild species of rice provides broad spectrum resistance to M. oryzae hence can be used in rice improvement breeding programme.


Rice blast NBS-LRR Pi54 Pikh Allele mining Magnaporthe oryzae Oryza rhizomatis 



TRS is thankful to ICAR and National Agricultural Innovation Project (code C4/C1071) for financial support. We thank Dr. S.R. Bhat for his help in conducting experiments on subcellular localization studies. Thanks are due to the in-charge, National Phytotron Facility, IARI, New Delhi, for providing facilities for maintaining wild species of rice and transgenic lines. We sincerely thank Dr. R. Rathour, Dr. M. Variar and Dr. U.D. Singh for providing fungal strains for phenotyping. A.D. and D.S. were supported by PG School, IARI, New Delhi in the form of fellowships for their Ph.D. and M.Sc. degrees, respectively.


  1. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programmes. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  2. Ashikawa I, Hayashi N, Yamane H, Kanamori H, Wu J et al (2008) Two adjacent nucleotide-binding site-leucine rich repeat class genes are required to confer Pikm-specific rice blast resistance. Genetics 180:2267–2276PubMedCrossRefGoogle Scholar
  3. Barclay A (2004) Feral Play. Rice Today 3:14–19Google Scholar
  4. Bonmann JM, Dedios TV, Khin MM (1986) Physiologic specialization of Pyricularia oryzae in Phillippines. Plant Dis 70:767–769CrossRefGoogle Scholar
  5. Bryan GT, Wu KS, Farrall L, Jia Y, Hershey HP et al (2000) A single amino acid difference distinguishes resistance and susceptible alleles of the rice blast resistance gene Pi-ta. Plant Cell 12:2033–2046PubMedCrossRefGoogle Scholar
  6. Chen XW, Shang JJ, Chen DX, Lei CL, Zou Y (2006) A β-lectin receptor kinase gene conferring rice blast resistance. Plant J 46:794–804PubMedCrossRefGoogle Scholar
  7. Chujo T, Takai R, Akimoto-Tomiyama C, Ando S, Minami E et al (2007) Involvement of the elicitor-induced gene OsWRKY53 in the expression of defense-related genes in rice. Biochim Biophys Acta 1769:497–505PubMedGoogle Scholar
  8. Dangl JJ, Jones JD (2001) Plant pathogen and integrated defence responses to infection. Nature 411:826–833PubMedCrossRefGoogle Scholar
  9. Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK et al (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980–986PubMedCrossRefGoogle Scholar
  10. Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206PubMedCrossRefGoogle Scholar
  11. Ewing B, Green P (1998) Base calling sequencer traces using Phred II. Error probabilities. Genome Res 8:186–194PubMedGoogle Scholar
  12. Farnham G, Baulcombe DC (2006) Artificial evolution extends the spectrum of viruses that are targeted by a disease resistance gene from potato. Proc Natl Acad Sci USA 103:18828–18833PubMedCrossRefGoogle Scholar
  13. Fukuoka S, Saka N, Koga H, Ono K, Shimizu T et al (2009) Loss of function of a proline-containing protein confers durable disease resistance in rice. Science 325(5943):998–1001PubMedCrossRefGoogle Scholar
  14. Gupta PK, Rustogi S (2004) Molecular markers from the transcribed/expressed region of the genome in higher plants. Funct Integr Genomics 4:139–162PubMedCrossRefGoogle Scholar
  15. Gupta SK, Rai AK, Kanwar SS, Chand D, Singh NK, Sharma TR (2012) The single functional blast resistance gene Pi54 activates a complex defense mechanism in rice. J Exp Bot 63(2):757–772PubMedCrossRefGoogle Scholar
  16. Hayashi K, Yasuda N, Fujita Y, Koizumi S, Yoshida H (2010a) Identification of the blast resistance gene Pit in rice cultivars using functional markers. Theor Appl Genet 121(7):1357–1367PubMedCrossRefGoogle Scholar
  17. Hayashi N, Inoue H, Kato T, Funao T, Shirota M et al (2010b) Durable panicle blast-resistance gene Pb1 encodes an atypical CC-NBS-LRR protein and was generated by acquiring a promoter through local genome duplication. Plant J 64(3):498–510PubMedCrossRefGoogle Scholar
  18. Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of T-DNA. Plant J 6:271–282PubMedCrossRefGoogle Scholar
  19. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Res 27:297–300PubMedCrossRefGoogle Scholar
  20. Horton P, Park KJ, Obayashi T, Fujita N, Harada H, Adams-Collier CJ, Nakai K (2007) WoLF PSORT: protein localization predictor. Nucleic Acids Res 35(Web Server Issue):W585–W587PubMedCrossRefGoogle Scholar
  21. Hulbert SH, Webb CA, Smith SM, Sun Q (2001) Resistance gene complexes: evolution and utilization. Annu Rev Phytopathol 39:285–312PubMedCrossRefGoogle Scholar
  22. Hwang C, Williamson VM (2003) Leucine-rich-repeat mediated intramolecular interactions in nematode recognition and cell death signalling by the tomato resistance protein Mi. Plant J 34:585–593PubMedCrossRefGoogle Scholar
  23. IRGSP (International Rice Genome Sequencing Project) (2005) The map-based sequence of the rice genome. Nature 436:793–800CrossRefGoogle Scholar
  24. Jones DA, Jones JDG (1997) The role of leucine rich repeat protein in plant defense. Adv Bot Res 24:89–167CrossRefGoogle Scholar
  25. Kankanala P, Czymmek K, Valent B (2007) Roles of rice membrane dynamics and plasmodesmata during biotrophic invasion by the blast fungus. Plant Cell 19:706–724PubMedCrossRefGoogle Scholar
  26. Lee SK, Song MY, Seo YS, Kim HK, Ko S et al (2009) Rice Pi5-mediated resistance to Magnaporthe oryzae requires the presence of two CC-NB-LRR genes. Genetics 181:1627–1638PubMedCrossRefGoogle Scholar
  27. Lehman P (2002) Structure and evolution of plant disease resistance genes. J Appl Genet 43:403–413Google Scholar
  28. Lescot M, Dhais P, Thijs G, Marchal K, Moreau Y, de Peer YV et al (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal of tools for in silico analysis of promoter sequences. Nucleic Acids Res 30:325–327PubMedCrossRefGoogle Scholar
  29. 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 177:1871–1880PubMedCrossRefGoogle Scholar
  30. Liu X, Lin F, Wang L, Pan Q (2007) The in silico map-based cloning of Pi36, a rice coiled-coil-nucleotide-binding site-leucine-rich repeat gene that confers race-specific resistance to the blast fungus. Genetics 176:2541–2549PubMedCrossRefGoogle Scholar
  31. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T) method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  32. Luo H, Song F, Goodman RM, Zheng Z (2005) Up-regulation of OsBIHD1, a rice gene encoding BELL homeodomain transcriptional factor, in disease resistance responses. Plant Biol (Stuttg) 7(5):459–468CrossRefGoogle Scholar
  33. Lupas A, Van Dyke M, Stock J (1991) Predicting coiled coils from protein sequences. Science (New York, NY) 252(5010):1162–1164CrossRefGoogle Scholar
  34. Martin GB, Bogdanove AJ, Sessa G (2003) Understanding the functions of plant disease resistance proteins. Annu Rev Plant Biol 54:23–61PubMedCrossRefGoogle Scholar
  35. Mazumdar B, Seshadri V, Fox PL (2003) Translation control by the 3′UTR: the ends specify the means. Trends Biochem Sci 28:91–98CrossRefGoogle Scholar
  36. Menkens AE, Schindler U, Cashmore AR (1995) The G-box: a ubiquitous regulatory DNA element in plants bound by the GBF family of bZIP proteins. Trends Biochem Sci 20:506–510PubMedCrossRefGoogle Scholar
  37. Meyers BC, Dickerman AW, Michelmore RW, Sivaramakrishnan S, Sobral BW, Young ND (1999) Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide binding superfamily. Plant J 20(3):317–332PubMedCrossRefGoogle Scholar
  38. Okuyama Y, Kanzaki H, Abe A, Yoshida K, Tamiru M et al (2011) A multifaceted genomics approach allows the isolation of the rice Pia-blast resistance gene consisting of two adjacent NBS-LRR protein genes. Plant J 66:467–479PubMedCrossRefGoogle Scholar
  39. Parker D, Beckmann M, Enot DP, Overy DP, Rios ZC et al (2008) Rice blast infection of Brachypodium distachyon as a model system to study dynamic host/pathogen interactions. Nat Protoc 3:435–445PubMedCrossRefGoogle Scholar
  40. Pickering BM, Willis AE (2005) The implication of structured 5′ untranslated region on translation and disease. Semin Cell Dev Biol 16:39–47PubMedCrossRefGoogle Scholar
  41. Qu S, Liu G, Zhou B, Bellizzi M, Zeng L (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 of rice. Genetics 172:1901–1914PubMedCrossRefGoogle Scholar
  42. Rafalski JA (2002) Applications of single nucleotide polymorphism in crop genetics. Curr Opin Plant Biol 5:94–100PubMedCrossRefGoogle Scholar
  43. Rai AK, Kumar SP, Gupta SK, Gautam N, Singh NK, Sharma TR (2011) Functional complementation of rice blast resistance gene Pi-k h (Pi54) conferring resistance to diverse strains of Magnaporthe oryzae. J Plant Biochem Biotechnol 20(1):55–65CrossRefGoogle Scholar
  44. Rakshit S, Rakshit A, Matsumura H, Takahashi Y, Hasegawa Y et al (2007) Large scale DNA polymorphism study of Oryza sativa and Oryza rufipogon reveals the origin and divergence of Asian rice. Theor Appl Genet 114:731–743PubMedCrossRefGoogle Scholar
  45. Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, NJ, pp 365–386Google Scholar
  46. Rushton PJ, Torres JT, Parniske M, Wernert P, Hahlbrock K et al (1996) Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J 15:5690–5700PubMedGoogle Scholar
  47. Shang J, Tao Y, Chen X, Zou Y, Lei C et al (2009) Identification of a new rice blast resistance gene, Pid3, by genome-wide comparison of paired NBS-LRR genes and their pseudogene alleles between the two sequenced rice genomes. Genetics 182:1303–1311PubMedCrossRefGoogle Scholar
  48. Sharma TR, Chauhan RS, Singh BM, Paul R, Sagar V, Rathour R (2002) RAPD and pathotype analyses of Magnaporthe grisea population from North-Western Himalayan Region of India. J Phytopathol 150:649–656CrossRefGoogle Scholar
  49. Sharma TR, Madhav MS, Singh BK, Shanker P, Jana TK et al (2005a) High resolution mapping, cloning and molecular characterization of the Pi-k h gene of rice, which confers resistance to Magnaporthe grisea. Mol Genet Genomics 274:569–578PubMedCrossRefGoogle Scholar
  50. Sharma TR, Shanker P, Singh BK, Jana TK, Madhav MS et al (2005b) Molecular mapping of rice blast resistance gene Pi-k h in rice variety Tetep. J Plant Biochem Biotechnol 14:127–133Google Scholar
  51. Sharma TR, Rai AK, Gupta SK, Singh NK (2010) Broad-spectrum blast resistance gene Pi-k h cloned from rice line Tetep designated Pi54. J Plant Biochem Biotechnol 19(1):87–89Google Scholar
  52. Sharma TR, Rai AK, Gupta SK, Vijayan J, Devanna BN, Ray S (2012) Rice blast management through host resistance: retrospect and prospects. Agric Sci 1:37–52Google Scholar
  53. Shen Y, Ji G, Haas BJ, Wu X, Zheng J et al (2008) Genome level analysis of rice mRNA 3′end processing signals and alternative polyadenylation. Nucleic Acids Res 36:3150–3161PubMedCrossRefGoogle Scholar
  54. Silue D, Notteghem JL, Tharreau D (1992) Evidence for a gene-for-gene relationship in the Oryza sativa-Magnaporthe grisea pathosystem. Phytopathology 82:577–582CrossRefGoogle Scholar
  55. Song WY, Wang GL, Chen LL, Kim HS, Pi LY et al (1995) A receptor kinase like protein encoded by rice disease resistance gene Xa21. Science 270:1804–1806PubMedCrossRefGoogle Scholar
  56. Takahasi 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 10:175–189CrossRefGoogle Scholar
  57. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA 4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  58. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedCrossRefGoogle Scholar
  59. Vij S, Tyagi AK (2008) A20/AN1 zinc-finger domain-containing proteins in plants and animals represent common elements in stress response. Funct Integr Genomics 8:301–307PubMedCrossRefGoogle Scholar
  60. Vos P, Simons G, Jesse T, Wijbrandi J, Heinen L (1998) The tomato Mi-1 gene confers resistance to both root-knot nematodes and potato aphids. Nat Biotechnol 16:1365–1369PubMedCrossRefGoogle Scholar
  61. Wang ZX, Yano M, Yamanouchi U, Iwamoto M, Monna L et al (1999) The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine rich repeat class of plant disease resistance genes. Plant J 19:55–64PubMedCrossRefGoogle Scholar
  62. Yang P, Chen C, Wang Z, Fan B, Chen Z (1999) A pathogen- and salicylic acid-induced WRKY DNA-binding activity recognizes the elicitor response element of the tobacco class I chitinase gene promoter. Plant J 18(2):141–149CrossRefGoogle Scholar
  63. Yoshimura S, Yamanouchi U, Katayose Y, Toki S, Wang ZX et al (1998) Expression of Xa1, a bacterial blight resistance gene in rice, is induced by bacterial inoculation. Proc Natl Acad Sci USA 95:1663–1668PubMedCrossRefGoogle Scholar
  64. Yu D, Chen C, Chen Z (2001) Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression. Plant Cell 13:1527–1540PubMedCrossRefGoogle Scholar
  65. Yuan B, Zhai C, Wang W, Zeng X, Xu X et al (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(5):1017–1028PubMedCrossRefGoogle Scholar
  66. Zhai C, Lin F, Dong Z, He X, Yuan B et al (2011) The isolation and characterization of Pik, a rice blast resistance gene which emerged after rice domestication. New Phytol 189(1):321–334PubMedCrossRefGoogle Scholar
  67. Zhou B, Qu S, Liu G, Dolan M, Sakai H, Lu G, Bellizzi M, Wang GL (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(11):1216–1228PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Alok Das
    • 1
    • 2
  • D. Soubam
    • 1
  • P. K. Singh
    • 1
  • S. Thakur
    • 1
  • N. K. Singh
    • 1
  • T. R. Sharma
    • 1
  1. 1.National Research Centre on Plant BiotechnologyIndian Agricultural Research InstituteNew DelhiIndia
  2. 2.Division of Crop ImprovementIndian Institute of Pulses ResearchKanpurIndia

Personalised recommendations