Plant Cell Reports

, Volume 36, Issue 11, pp 1747–1755 | Cite as

Co-transformation mediated stacking of blast resistance genes Pi54 and Pi54rh in rice provides broad spectrum resistance against Magnaporthe oryzae

  • Mandeep Kumari
  • Amit Kumar Rai
  • B. N. Devanna
  • Pankaj Kumar Singh
  • Ritu Kapoor
  • H. Rajashekara
  • G. Prakash
  • Vinay Sharma
  • Tilak Raj Sharma
Original Article


Key message

This is the first report of stacking two major blast resistance genes in blast susceptible rice variety using co-transformation method to widen the resistance spectrum against different isolates of Magnaporthe oryzae.


Single resistance (R-) gene mediated approach for the management of rice blast disease has met with frequent breakdown in resistance response. Besides providing the durable resistance, gene pyramiding or stacking also imparts broad spectrum resistance against plant pathogens, including rice blast. In the present study, we stacked two R-genes; Pi54 and Pi54rh having broad spectrum resistance against multiple isolates of Magnaporthe oryzae (M. oryzae). Both Pi54 and Pi54rh expressed under independent promoters were transferred into the blast susceptible japonica rice Taipei 309 (TP309) using particle gun bombardment method. Functional complementation analysis of stacked transgenic rice lines showed higher level of resistance to a set of highly virulent M. oryzae isolates collected from different rice growing regions. qRT-PCR analysis has shown M. oryzae induced expression of both the R-genes in stacked transgenic lines. The present study also demonstrated the effectiveness of the strategy for rapid single step gene stacking using co-transformation approach to engineer durable resistance against rice blast disease and also this is the first report in which two blast R-genes are stacked together using co-transformation approach. The two-gene-stacked transgenic line developed in this study can be used further to understand the molecular aspects of defense-related pathways vis-a-vis single R-gene containing transgenic lines.


Magnaporthe oryzae Rice blast Resistance gene Co-transformation Gene stacking 



TRS is thankful to the Department of Biotechnology, Govt. of India and Indian Council of Agricultural Research for funding and Department of Science and Technology, Govt. of India for JC Bose National Fellowship. MK is thankful to CSIR for providing fellowship.

Compliance with ethical standards

Conflict of interest

All the authors declare no conflict of interest.

Supplementary material

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  1. Abhilash Kumar V, Balachiranjeevi CH, Bhaskar Naik S, Rambabu R, Rekha G, Harika G, Hajira SK, Pranathi K, Anila M, Kousik M et al (2016) Development of gene-pyramid lines of the elite restorer line, RPHR-1005 possessing durable bacterial blight and blast resistance. Front Plant Sci 7:1195. doi: 10.3389/fpls.2016.01195 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Azizi P, Rafii MY, Abdullah SN, Hanafi MM, Maziah M, Sahebi M, Ashkani S, Taheri S, Jahromi MF (2016) Over-expression of the Pikh gene with a CaMV 35S promoter leads to improved blast disease (Magnaporthe oryzae) tolerance in rice. Front Plant Sci 7:773. doi: 10.3389/fpls.2016.00773 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bonman J, Vergel de Dios T, Khin M (1986) Physiologic specialization of Pyricularia oryzae in the Philippines. Plant Dis 70:767–769CrossRefGoogle Scholar
  4. Brunner S, Hurni S, Streckeisen P, Mayr G, Albrecht M, Yahiaoui N, Keller B (2010) Intragenic allele pyramiding combines different specificities of wheat Pm3 resistance alleles. Plant J 64(3):433–445. doi: 10.1111/j.1365-313X.2010.04342.x CrossRefPubMedGoogle Scholar
  5. Dafny-Yelin M, Tzfira T (2007) Delivery of multiple transgenes to plant cells. Plant Physiol 145:1118–1128CrossRefPubMedPubMedCentralGoogle Scholar
  6. Dai Y, Jia Y, Correll J, Wang X, Wang Y (2010) Diversification and evolution of the avirulence gene AVR-Pita1 in field isolates of Magnaporthe oryzae fungal. Genet Biol 47:973–980CrossRefGoogle Scholar
  7. Das A, Soubam D, Singh P, Thakur S, Singh N, Sharma T (2012) A novel blast resistance gene, Pi54rh cloned from wild species of rice, Oryza rhizomatis confers broad spectrum resistance to Magnaporthe oryzae. Funct Integr Genom 12:215–228CrossRefGoogle Scholar
  8. Devanna NB, Vijayan J, Sharma TR (2014) The blast resistance gene Pi54of cloned from Oryza officinalis interacts with Avr-Pi54 through its novel non-LRR domains. PLoS One 9:e104840CrossRefPubMedPubMedCentralGoogle Scholar
  9. Douglas E, Halpin C (2010) Gene stacking. In: Mohan Jain S, Brar DS (eds) Molecular techniques in crop improvement, 2nd edn. Springer, Heidelberg, pp 613–629CrossRefGoogle Scholar
  10. Ellur RK, Khanna A, Yadav A, Pathania S, Rajashekara H, Singh VK, Gopala Krishnan S, Bhowmick PK, Nagarajan M, Vinod KK, Prakash G, Mondal KK, Singh NK, Vinod Prabhu K, Singh AK (2016) Improvement of Basmati rice varieties for resistance to blast and bacterial blight diseases using marker assisted backcross breeding. Plant Sci 242:330–341. doi: 10.1016/j.plantsci.2015.08.020 CrossRefPubMedGoogle Scholar
  11. Flor H (1955) Host-parasite interaction in flax rust-its genetics and other implications. Phytopathology 45:680–685Google Scholar
  12. Fukuoka S, Saka N, Mizukami Y, Koga H, Yamanouchi U, Yoshioka Y, Hayashi N, Ebana K, Mizobuchi R, Yano M (2015) Gene pyramiding enhances durable blast disease resistance in rice. Sci Rep 5:7773CrossRefPubMedPubMedCentralGoogle Scholar
  13. Gao L, Cao Y, Xia Z, Jiang G, Liu G, Zhang W, Zhai W (2013) Do transgenesis and marker-assisted backcross breeding produce substantially equivalent plants?-A comparative study of transgenic and backcross rice carrying bacterial blight resistant gene Xa21. BMC Genom 14(1):738CrossRefGoogle Scholar
  14. Gnanamanickam SS (2009) Rice and its importance to human life. Biological control of rice diseases. Springer, Netherlands, pp 1–11CrossRefGoogle 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 defence mechanism in rice. J Exp Bot 63:757–772CrossRefPubMedGoogle Scholar
  16. Hittalmani S, Parco A, Mew T, Zeigler R, Huang N (2000) Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in rice. Theor Appl Genet 100:1121–1128CrossRefGoogle Scholar
  17. Jha S, Chattoo BB (2009) Transgene stacking and coordinated expression of plant defensins confer fungal resistance in rice. Rice 2:143–154CrossRefGoogle Scholar
  18. Khanna A, Sharma V, Ellur RK, Shikari AB, Gopala Krishnan S, Singh UD, Prakash G, Sharma TR, Rathour R, Variar M et al (2015) Development and evaluation of near-isogenic lines for major blast resistance gene(s) in Basmati rice. Theor Appl Genet 128(7):1243–1259CrossRefPubMedGoogle Scholar
  19. Kumari A, Das A, Devanna BN, Thakur S, Singh PK, Singh NK, Sharma TR (2013) Mining of rice blast resistance gene Pi54 shows effect of single nucleotide polymorphisms on phenotypic expression of the alleles. Eur J Plant Pathol 137:55. doi: 10.1007/s10658-013-0216-5 CrossRefGoogle Scholar
  20. Lin L, Liu Y-G, Xu X, Li B (2003) Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system. Proc Natl Acad Sci 100:5962–5967CrossRefPubMedPubMedCentralGoogle Scholar
  21. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  22. Mao B, Liu X, Hu D, Li D (2014) Co-expression of RCH10 and AGLU1 confers rice resistance to fungal sheath blight Rhizoctonia solani and blast Magnorpathe oryzae and reveals impact on seed germination. World J Microbiol Biotechnol 30:1229–1238CrossRefPubMedGoogle Scholar
  23. Matzke AJ, Matzke MA (1998) Position effects and epigenetic silencing of plant transgenes. Curr Opin Plant Biol 1:14–148CrossRefGoogle Scholar
  24. Nascimento-Gavioli MC, Agapito-Tenfen SZ, Nodari RO, Welter LJ, Mora FD, Saifert L, da Silva AL, Guerra MP (2017) Proteome of Plasmopara viticola-infected Vitis vinifera provides insights into grapevine Rpv1/Rpv3 pyramided resistance to downy mildew. J Proteom 151:264–274CrossRefGoogle Scholar
  25. Osborn RW, De Samblanx GW, Thevissen K, Goderis I, Torrekens S, Van Leuven F et al (1995) Isolation and characterisation of plant defensins from seeds of asteraceae, fabaceae, hippocastanaceae and saxifragaceae. FEBS Lett 368:257–262CrossRefPubMedGoogle Scholar
  26. Quilis J, López -García B, Meynard D, Guiderdoni E, San Segundo B (2014) Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice. Plant Biotechnol J 12:367–377CrossRefPubMedGoogle Scholar
  27. Rai AK, Kumar SP, Gupta SK, Gautam N, Singh NK, Sharma TR (2011) Functional complementation of rice blast resistance gene Pi-kh (Pi54) conferring resistance to diverse strains of Magnaporthe oryzae. J Plant Biochem Biotechnol 20:55–65CrossRefGoogle Scholar
  28. Ray S, Singh PK, Gupta DK, Mahato AK, Sarkar C, Rathour R, Singh NK, Sharma TR (2016) Analysis of Magnaporthe oryzae genome reveals a fungal effector, which is able to induce resistance response in transgenic rice line containing resistance gene, Pi54. Front Plant Sci 7:1140. doi: 10.3389/fpls.2016.01140 PubMedPubMedCentralGoogle Scholar
  29. Sagar KrishnaMurthy P, Deshmukh DB, Yashvanth Kumar KJ, Patil S, Jakkeral S, Nemappa GH, Singh UD, Variar M, Rathour R, Subbaiyan G et al (2017) Introgression of Pi2 and Pi5 genes for blast (Magnaporthe oryzae) resistance in rice and field evaluation of introgression lines for resistance and yield traits. J Phytopathol 165:397–405. doi: 10.1111/jph.12573 CrossRefGoogle Scholar
  30. Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual, 2d edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  31. Sanford JC, Klein TM, Wolf ED, Allen N (1987) Delivery of substances into cells and tissues using a particle bombardment process particulate. Sci Technol 5:27–37Google Scholar
  32. Scheuermann KK, de Andrade A, Wickert E, Raimondi JV, Marschalek R (2012) Magnaporthe oryzae genetic diversity and its outcomes on the search for durable resistance. In: Caliskan M (ed) The molecular basis of plant genetic diversity. InTech Europe, Rijeka, Croatia, pp 331–356Google Scholar
  33. Senthilkumar R, Cheng CP, Yeh KW (2010) Genetically pyramiding protease inhibitor genes for dual broad spectrum resistance against insect and phytopathogens in transgenic tobacco. Plant Biotechnol J 8:65–75CrossRefPubMedGoogle Scholar
  34. Shahzad R, Harlina PW, Conghua X, Ewas M, Nishawy E, Zhenyuan P, Foly MM (2016) Overexpression of potato transcription factor (StWRKY1) conferred resistance to Phytophthora infestans and improved tolerance to water stress. Plant Omics 9:149CrossRefGoogle Scholar
  35. Sharma TR, Chauhan RS, Singh BM, Paul R, Sagar B, Rathour R (2002) RAPD and pathotype analysis of Magnaporthe grisea population from North-western Himalayan region of India. J Phytopathol 150:649–656CrossRefGoogle Scholar
  36. Sharma TR, Shanker P, Singh BK, Jana TK, Madhav MS, Gaikwad K, Singh NK, Plaha P, Rathour R (2005a) Molecular mapping of rice blast resistance gene Pi-kh in the rice variety Tetep. J Plant Biochem Biotechnol 14:127–133CrossRefGoogle Scholar
  37. Sharma TR, Madhav MS, Singh BK, Shanker P, Jana TK, Dalal V, Pandit A, Singh A, Gaikwad K, Upreti HC, Singh NK (2005b) High-resolution mapping, cloning and molecular characterization of the Pi-kh gene of rice, which confers resistance to Magnaporthe grisea. Mol Genet Genom 274:569CrossRefGoogle Scholar
  38. Sharma TR, Rai A, Gupta S, Vijayan J, Devanna B, Ray S (2012) Rice blast management through host-plant resistance: retrospect and prospects. Agric Res 1:37–52CrossRefGoogle Scholar
  39. Singh S, Sidhu JS, Huang N, Vikal Y, Li Z, Brar DS, Dhaliwal HS, Khush GS (2001) Pyramiding three bacterial blight resistance genes (xa5, xa13 and Xa21) using marker-assisted selection into indica rice cultivar PR106. Theor Appl Genet 102:1011–1015CrossRefGoogle Scholar
  40. Singh A, Singh VK, Singh SP, Pandian RT, Ellur RK, Singh D, Bhowmick PK, Krishnan SG, Nagarajan M, Vinod KK, Singh UD, Prabhu KV, Sharma TR, Mohapatra T, Singh AK (2012) Molecular breeding for the development of multiple disease resistance in Basmati rice. AoB Plants 2012:pls029. doi: 10.1093/aobpla/pls029 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Singh PK, Thakur S, Rathour R, Variar M, Prashanthi SK, Singh AK, Singh UD, Sharma V, Singh NK, Sharma TR (2014) Transposon-based high sequence diversity in Avr-Pita alleles increases the potential for pathogenicity of Magnaporthe oryzae populations. Funct Integr Genom 14:419–429CrossRefGoogle Scholar
  42. Sprague SJ, Balesdent MH, Brun H, Hayden HL, Marcroft SJ, Pinochet X, Rouxel T, Howlett BJ (2006) Major gene resistance in Brassica napus (oilseed rape) is overcome by changes in virulence of populations of Leptosphaeria maculans in France and Australia. Eur J Plant Pathol 114:33–40CrossRefGoogle Scholar
  43. Tanweer FA, Rafii MY, Sijam K, Rahim HA, Ahmed F, Ashkani S, Latif MA (2015) Introgression of Blast Resistance Genes (Putative Pi-b and Pi-kh) into Elite Rice Cultivar MR219 through Marker-Assisted Selection. Front Plant Sci 6:1002. doi: 10.3389/fpls.2015.01002 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Rees SB, Vanderleyden J et al (1992) Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 267:15301–15309PubMedGoogle Scholar
  45. Thakur S, Singh PK, Das A, Rathour R, Variar M, Prashanthi SK, Singh AK, Singh UD, Chand D, Singh NK, Sharma TR (2015) Extensive sequence variation in rice blast resistance gene Pi54 makes it broad spectrum in nature. Front Plant Sci 6:345. doi: 10.3389/fpls.2015.00345 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Vasudevan K, Gruissem W, Bhullar NK (2015) Identification of novel alleles of the rice blast resistance gene Pi54. Sci Rep 5:15678CrossRefPubMedPubMedCentralGoogle Scholar
  47. Widawsky DA, O’Toole JC (1996) Prioritizing the rice research agenda for eastern India. Rice Research in Asia: Progress and Priorities. CAB International Wallingford, Oxon, pp 109–130Google Scholar
  48. Wilson RA, Talbot NJ (2009) Under pressure: investigating the biology of plant infection by Magnaporthe oryzae. Nat Rev Microbiol 7(3):185–195CrossRefPubMedGoogle Scholar
  49. Wu Y, Xiao N, Yu L, Pan C, Li Y, Zhang X, Liu G, Dai Z, Pan X, Li A (2015) Combination patterns of major R genes determine the level of resistance to the M. oryzae in Rice (Oryza sativa L.). PLoS One 10:e0126130CrossRefPubMedPubMedCentralGoogle Scholar
  50. Xiao N, Wu Y, Pan C, Yu L, Chen Y, Li Y, Dai Z, Liang C, Li A (2016) Improving of rice blast resistances in japonica by pyramiding major R genes. Front Plant Sci 7:1918PubMedGoogle Scholar
  51. Zhang C, Chen H, Cai T, Deng Y, Zhuang R, Zhang N, Zeng Y, Zheng Y, Tang R, Pan R, Zhuang W (2016) Overexpression of a novel peanut NBS-LRR gene AhRRS5 enhances disease resistance to Ralstonia solanacearum in tobacco. Plant Biotechnol J 15(1):39–55. doi: 10.1111/pbi.12589 CrossRefPubMedPubMedCentralGoogle Scholar
  52. Zhu S, Li Y, Vossen JH, Visser RG, Jacobsen E (2012) Functional stacking of three resistance genes against Phytophthora infestans in potato. Transgenic Res 21:89–99CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Mandeep Kumari
    • 1
    • 4
  • Amit Kumar Rai
    • 1
  • B. N. Devanna
    • 1
  • Pankaj Kumar Singh
    • 1
  • Ritu Kapoor
    • 1
  • H. Rajashekara
    • 2
  • G. Prakash
    • 3
  • Vinay Sharma
    • 4
  • Tilak Raj Sharma
    • 5
  1. 1.ICAR-National Research Centre on Plant BiotechnologyNew DelhiIndia
  2. 2.Crop Protection SectionVivekananda Institute of Hill AgricultureAlmoraIndia
  3. 3.Division of Plant PathologyIndian Agricultural Research InstituteNew DelhiIndia
  4. 4.Department of Bioscience and BiotechnologyBanasthali VidyapithRajasthanIndia
  5. 5.National Agri-Food Biotechnology Institute (NABI)MohaliIndia

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