Abstract
Blast disease of rice, caused by Magnaporthe oryzae is an explosive disease that can spread rapidly in conducive conditions. R-gene mediated resistance offers an environmentally sustainable solution for management of this important disease of rice. We have earlier identified a unique R-gene of rice, on chromosome 11 of Oryza sativa ssp. indica cultivar Tetep. In this study we report functional validation of the Pi-k h (Pi54) gene using complementation assay. The blast resistance candidate gene Pi-k h (Pi54) was cloned into a plant transformation vector and the construct was used to transform a japonica cultivar of rice Taipei 309, which is susceptible to M. oryzae. Transgenic lines containing Pi-k h (Pi54) gene were found to confer high degree of resistance to diverse isolates of M. oryzae. The callose deposition was analyzed and compared between the transgenic and non-transgenic rice plants and widespread deposition was observed at the infection sites in plants showing incompatible interaction. Successful complementation of Pi-k h (Pi54) gene confirmed that the gene is responsible for resistance to M. oryzae in transgenic lines developed during this study. Expression analysis of the gene in resistant plants revealed that the gene is pathogen inducible in nature and is not expressed constitutively. Detection of callose deposition in resistant plants containing Pi-k h (Pi54) gene implicates its involvement in the initiation of defense response cascade.
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Abbreviations
- Avr:
-
Avirulence
- CTAB:
-
Cetyl trimethyl ammonium bromide
- ETI:
-
Effector triggered immunity
- hpi:
-
Hours post inoculation
- RT-PCR:
-
Real time PCR
- NBS-LRR:
-
Nucleotide binding site-leucine rich repeat
References
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 180:2267–2276
Ballini E, Morel JB, Droc G, Price A, Courtois B, Notteghem JL, Tharreau D (2008) A genome-wide meta-analysis of rice blast resistance genes and quantitative trait loci provides new insights into partial and complete resistance. Mol Plant-Microbe Interact 21:859–868
Bonman JM, Vergel TI, De D, Khin MM (1986) Physiological specialization of Pyricularia oryzae in the Philippines. Plant Dis 70:767–769
Bryan GT, Wu KS, Farrall L, Jia Y, Hershey HP, McAdams SA, Faulk KN, Donaldson GK, Tarchini R, Valent B (2000) A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta. Plant Cell 12:2033–2045
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 46:794–804
Dixit R, Bhargava A, Dalal V, Plaha P, Singh NK, Sharma TR (2009) Accumulation of defence response-related and unique expressed sequence tags during the incompatible interaction in the Oryza sativa–Magnaporthe oryzae pathosystem. J Phytopathol 157:483–489
Flor HH (1971) Current status of the gene-for-gene concept. Annu Rev Phytopathol 9:275–296
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 325:998–1001
Hardham AR, Jones DA, Tokemoto D (2007) Cytoskeleton and cell wall function in penetration resistance. Curr Opin Plant Biol 10:342–348
Hayashi K, Yoshida H (2009) Refunctionalization of the ancient rice blast disease resistance gene Pit by the recruitment of a retrotransposon as a promoter. Plant J 57:413–425
International Rice genome sequencing project-IRGSP (2005) The map based sequence of the rice genome. Nature 436:793–800
Jain M, Nijhawan A, Tyagi AK, Khurana JP (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun 345:646–651
Jones JDG, Dangl J (2006) The plant immune system. Nature 444:323–329
Kim MG, Mackey D (2008) Measuring cell-wall-based defenses and their effect on bacterial growth in Arabidopsis. Methods Mol Biol 415:443–452
Kiyosawa S, Murty VVS (1969) The inheritance of blast-resistance in Indian rice variety, HR-22. Jpn J Breed 4:269–276
Kumar SP, Dalal V, Singh NK, Sharma TR (2007) Comparative analysis of the 100 kb region containing the Pi-k(h) locus between indica and japonica rice lines. Genomics Proteomics Bioinform 5:35–44
Lee SK, Song MY, Seo YS, Kim HK, Ko S, Cao PJ, Suh JP, Yi G, Roh JH, Lee S, An G, Hahn TR, Wang GL, Ronald P, Jeon JS (2009) Rice Pi5-mediated resistance to Magnaporthe oryzae requires the presence of two coiled-coil-nucleotide-binding-leucine-rich repeat genes. Genetics 181:1627–1638
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 177:1871–1880
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 176:2541–2549
Madhav MS, Plaha P, Singh NK, Sharma TR (2008) Molecular characterization of a genomic fragment containing Pi-k h gene from the genomic library of indica rice line Tetep. J Phytopathol 157:322–324
McCouch SR, Nelson RJ, Thome J, Zeigler RS (1994) In: Zeigler RS, Leong SA, Teng PS (eds) Rice blast disease. CAB International, Wallingford, pp 167–187
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325
Qu S, Liu G, Zhou B, Bellizzi M, Zeng L, Dai L, Han B, Wang GL (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 172:1901–1914
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Sanford JC, Klein TM, Wolf E, Allen N (1987) Delivery of substance into cells and tissues using particle bombardment process. Part Sci Technol 5:27–37
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 150:649–656
Sharma TR, Madhav MS, Singh BK, Shanker P, Jana TK, Dalal V, Pandit A, Singh A, Gaikwad K, Upreti HC, Singh NK (2005a) High resolution mapping, cloning and molecular characterization of the Pi-k h gene of rice, which confers resistance to M. grisea. Mol Genet Genomics 274:569–578
Sharma TR, Shanker P, Singh BK, Jana TK, Madhav MS, Gaikwad K, Singh NK, Plaha P, Rathour R (2005b) Molecular mapping of rice blast resistance gene Pi-k h in the rice variety Tetep. J Plant Biochem Biotech 14:127–133
Sharma TR, Rai AK, Gupta SK, Singh NK (2010) Broad spectrum blast resistance gene Pi-k h designated as Pi-54. J Plant Biochem Biotech 987–989
Skalamera D, Jibodh S, Heath M (1997) Callose deposition during the interaction between cowpea (Vigna unguiculata) and the monokaryotic stage of the cowpea rust fungus (Uromyces vignae). New Phytol 136:511–524
Skamnioti P, Gurr SJ (2009) Against the grain: safeguarding rice from rice blast disease. Trends Biotechnol 693:1–10
Stuckenbrock EH, McDonald BA (2009) Population genetics of fungal and oomycete effectors involved in gene-for-gene interactions. Mol Plant-Microbe Interact 22:371–380
Wang ZX, Yano M, Yamanouchi U, Iwamoto M, Monna L, Hayasaka H, Katayose Y, Sasaki T (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 19:55–64
Yun MH, Torres PS, Oirdi ME, Rigano LA, Lamothe RG, Marano MR, Castagnaro AP, Dankert MA, Bouarab K, Vojnov AA (2006) Xanthan induces plant susceptibility by suppressing callose deposition. Plant Physiol 141:178–187
Zhang S, Song W, Chen Y, Ruan L, Taylor D, Ronald P, Beachy R, Fauquet C (1998) Transgenic elite Indica rice varieties resistant to Xanthomonas oryzae pv. Oryzae. Mol Breed 4:551–558
Zhou B, Qu S, Li 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 19:1216–1228
Acknowledgements
Financial assistance received from the Department of Biotechnology, Govt. of India by TRS (Project code: BT/AB/FG-2(PH-II)/2009) is gratefully acknowledged. We also thank Dr. K.V. Prabhu, National Phytotron Facility, Indian Agricultural Research Institute for providing basic facilities for transgenic research. Authors are also thankful to Dr. Gautam Chawla, Department of Nematology, IARI for valuable suggestions on histo-chemical experiments.
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Rai, A.K., Kumar, S.P., Gupta, S.K. et al. Functional complementation of rice blast resistance gene Pi-k h (Pi54) conferring resistance to diverse strains of Magnaporthe oryzae . J. Plant Biochem. Biotechnol. 20, 55–65 (2011). https://doi.org/10.1007/s13562-010-0026-1
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DOI: https://doi.org/10.1007/s13562-010-0026-1