Research Paper

Genetic Transformation of Rice with Pi-d2 Gene Enhances Resistance to Rice Blast Fungus Magnaporthe oryzae

Expand
  • 1)Rice Research Institute, Sichuan Agricultural University, Wenjiang 611130, China; 2)Key Laboratory of Crop Genetic and Improvement, Ministry of Education, Sichuan Agricultural University, Ya’an 625014, China; 3)Institute of Plant Protection, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China; 4Institute of Genetic and Development Biology, Chinese Academy of Sciences, Beijing 100101, China

Received date: 2009-04-30

  Online published: 2010-03-28

Supported by

the Excellent Doctor Paper Foundation of the Ministry of Education of China (Grant No. 200054), the Program for Innovative Research Team in University of China (Grant No. NCET-04-0907) and the Program for New Century Excellent Talent in University of China (Grant No. IRT0453).

Abstract

The gene Pi-d2, conferring gene-for-gene resistance to the Chinese blast strain ZB15, was isolated from a rice variety (Digu) by the map-based cloning strategy. Here, we constructed a control plasmid pZH01-pi-d2tp309 (pZH01-tp309) and three different expression constructs, pCB-Pi-d25.3kb (pCB5.3kb), pCB-Pi-d26.3kb (pCB6.3kb) and pZH01-Pi-d22.72kb (pZH01-2.72kb) of Pi-d2, driven by Pi-d2 gene’s own promoter or CaMV35S promoter. These constructs were separately introduced into japonica rice varieties Lijiangxintuanhegu, Taipei 309, Nipponbare and Zhonghua 9 through Agrobacterium- mediated transformation. A total of 150 transgenic rice plants were obtained from the regenerated calli selected on hygromycin. PCR, RT-PCR and Southern-blotting assay showed that the gene of interest had been integrated into rice genome and stably inherited. Thirty-five transgenic lines independently derived from T1 progeny were inoculated with the rice blast strain ZB15. Transformants exhibited resistance to rice blast at various levels. The lesions on the transgenic plant leaves were less severe than those on the controls and the resistance level of transgenic plants harboring the gene of interest from three vectors had no difference. The own promoter of Pi-d2, about 2.2 kb or 3.2 kb, had the similar promoter function as CaMV35S. Field evaluation for three successive years supported the results of artificial trial, and some lines with high resistance to rice leaf blast and neck blast were obtained.

Cite this article

CHEN De-xi, CHEN Xue-wei, MA Bing-tian, WANG Yu-ping, ZHU Li-huang, LI Shi-gui, . Genetic Transformation of Rice with Pi-d2 Gene Enhances Resistance to Rice Blast Fungus Magnaporthe oryzae[J]. Rice Science, 2010 , 17(1) : 19 -27 . DOI: 10.1016/S1672-6308(08)60100-6

References

1 Dong J X, Dong H T, Li D B. Recent advances on rice blast disease resistance. J Agric Biotech, 2000, 8: 99–101. (in Chinese with English abstract)
2 Zhu Y Y, Chen H R, Fan J H, Wang Y Y, Li Y, Chen J B, Fan J X, Yang S S, Hu L P, Leung H, Mew T W, Teng P S, Wang Z H, Mundt C C. Genetic diversity and disease control in rice. Nature, 2000, 406: 718–722.
3 Cornelissen B J C, Melchers L S. Strategies for control of fungi diseases with transgenic plants. Plant Physiol, 1993, 101: 709–712.
4 Nishizawa Y, Nishio Z, Nakazono K, Soma M, Nakajima E, Ugaki M, Hibi T. Enhanced resistance to blast (Magnaporthe grisea) in transgenic japonica rice by constitutive expression of rice chitinase. Theor Appl Genet, 1999, 99: 383–390.
5 Feng D R, Wei J W, Xu X P, Xu Y, Li B J. Introduction of multiple antifungal protein genes into rice and preliminary study on resistance to Pyricularia oryzae of transgenic rices. Acta Sci Nat Univ Sunyatsen, 1999, 38: 62–66. (in Chinese with English abstract)
6 Xu M H, Tang Z S, Tan Y L, Tian Y C, Li C Y, Zhang S H, Chen Z H, Tian W Z. A study on introduction of chitinase gene and β-1,3-glucanase gene into restorer line of Dian-type hybrid rice (Oryza sativa L.) and enhanced resistance to blast (Magnaporthe grisea). Acta Genet Sin, 2003, 30: 330–334. (in Chinese with English abstract)
7 Ming X T, Wang L J, An C C, Yuan H Y, Zheng H H, Chen Z L. Introducing trichosanthin gene into rice mediated by Agrobacterium tumefacien and testing the activity of resistance to blast. Chinese Sci Bull, 2000, 45: 1080–1084.
8 Yuan H, Ming X, Wang L, Hu P, An C, Chen Z. Expression of a gene encoding trichosanthin in transgenic rice plants enhances resistance to fungus blast disease. Plant Cell Rep, 2002, 20: 992–998.
9 Peng H, Wang Z X, Dou D L, Zhu S W, Lu T G, Sun J S. Introducing glucose oxidase gene into rice mediated by Agrobacterium tumefaciens. J Agric Biotech, 2003, 11: 16–19. (in Chinese with English abstract)
10 Xu M H, Li C Y, Li J B, Tan X L, Tian W Z, Tang Z S. Analysis of resistant spectrum to rice blast in transgenic rice lines introduced lysozyme gene from T4 phage. Sci Agric Sin, 2003, 36: 387–392. (in Chinese with English abstract)
11 Ulrich S, Felix M, Ernst F, Patrick S, Robert D. Constitutive expression of the defense-related Rir1b gene in transgenic rice plants confers enhanced resistance to the rice blast fungus Magnaporthe grisea. Plant Mol Biol, 2000, 43: 59–66.
12 Gandikota M, de Kochko A, Chen L, Ithal N, Fauquet C, Reddy A R. Development of transgenic rice plants expressing maize anthocyanin genes and increased blast resistance. Mol Breeding, 2001, 7: 73–83.
13 Kanzaki H, Nirasasawa S, Saitoh H, Ito M. Over expression of the wasabi defensin gene confers enhanced resistance to blast fungus (Magnaporthe grisea) in transgenic rice. Theor Appl Genet, 2002, 105: 809–814.
14 Matthew A C, Heather A F, Pamela C R. Engineering pathogen resistance in crop plants. Transg Res, 2002, 11: 599–613.
15 Tan Y N, Yi Z L, Jiang J X, Qin J P, Xiao L. Strategies and advances in improving resistance to rice blast by transgenic approaches. Mol Plant Breeding, 2004, 2: 847–852. (in Chinese with English abstract)
16 Strittmatter G, Janssens J, Opsomer C, Botterman J. Inhibition of fungal disease development in plants by engineering controlled cell death. Biotechnology, 1995, 13: 1085–1089.
17 Mao S J, Gu H Y, Qu L J, Chen Z L. Producing the rice resistant to blast fungus used the engineering by controlling cell death strategy. Chinese Sci Bull, 2003, 48: 1169–1175. (in Chinese)
18 Liu M, Sun Z X, Zhu J, Xu T, Harman G E, Lorito M. Enhancing rice resistance to fungal pathogens by transformation with cell wall degrading enzyme genes from Trichoderma atroviride. J Zhejiang Univ Sci, 2004, 5: 133–136.
19 Coca M, Bortolotti C, Rufat M, Peñas G, Eritja R, Tharreau D, del Pozo A M, Messeguer J, San Segundo B. Transgenic rice plants expressing the antifungal AFP protein from Aspergillus giganteus show enhanced resistance to the rice blast fungus Magnaporthe grisea. Plant Mol Biol, 2004, 54: 245–259.
20 Wang Z X, Yano M, Yamanouchi U, Iwamoto M, Monna L, Hayasaka H, Katayose Y, Sasaki T. The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes. Plant J, 1999, 19: 55–64.
21 Bryan G T, Wu K S, Farrall L, Hershey H P, McAdams S A, Faulk K N, Donaldson G K, Tarchini R, Valent B. A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta. Plant Cell, 2000, 12: 2033–2045.
22 Qu S H, Liu G F, Zhou B, Bellizzi M, Zeng L R, Dai L Y, Han B, Wang G L. The broad-spectrum blast resistance gene Pi9 encodes an NBS-LRR protein and is a member of a multigene family in rice. Genetics, 2006, 172: 1901–1914.
23 Zhou B, Qu S H, Liu G F, Dolan M, Sakai H, Lu G D, Bellizzi M, Wang G L. 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, 2006, 19: 1216–1228.
24 Chen X W, Shang J J, Chen D X, Lei C L, Zou Y, Zhai W X , Liu G Z, Xu J H, Ling Z Z, Cao G, Ma B T, Wang Y P, Zhao X F, Li S G, Zhu L H. A B-lectin receptor kinase gene conferring rice blast resistance. Plant J, 2006, 46: 794–804.
25 Xiao H, Wang Y, Liu D F, Wang W M, Li X B, Zhao X F, Xu J C, Zhai W X, Zhu L H. Functional analysis of the rice AP3 homologue OsMADS16 by RNA interference. Plant Mol Biol, 2003, 52: 957–966.
26 Chen D X, Xu Z J, Ma B T, Li S G. Effect of phytosulfokine-α on Agrobacterium-mediated transformation in rice. Rice Sci, 2005, 12(4): 255–260.
27 Murray M G, Thompson W F. Rapid isolation of high molecular weight DNA. Nucl Acids Res, 1980, 8: 4321–4325.
28 Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J, 1994, 6: 271–282.
29 Pan Q H, Wang L, Ikehashi H, Tanisaka T. Identification of a new blast resistance gene in the indica rice cultivar Kasalath using Japanese differential cultivars and isozyme markers. Phytopathology, 1996, 86: 1071–1075.
30 Zeigler R S, Leong S A, Teng P S. Rice Blast Disease. Wallingford: CAB International, 1994: 137–154.
31 Hart C M, Fischer B, Neuhaus J M, Meins F. Regulated inactivation of homologous gene expression in transgenic Nicotiana sylvestris plants containing a defense-related tobacco chitinase gene. Mol Gen Genet, 1992, 235: 179–188.Chareonporwattana S, Thara K V, Wang L, Datta S K, Panbangred W, Muthukrishnan S. Inheritance, expression, and silencing of a chitinase transgene in rice. Theor Appl Genet, 1999, 98: 371–378.
Options
Outlines

/

浙ICP备05004719号-15   公安备案号:33010302003355
Copyright © Editorial office of Rice Science
Tel: 0571-63371017 E-mail: crrn@fy.hz.zn.cn; cjrs278@gmail.com
Supported by Beijing Magtech Co., Ltd.
Total visitors:  Visitors of today:  Now online: