Biologia plantarum 2013, 57:351-358 | DOI: 10.1007/s10535-012-0291-z

Transgenic rice lines constitutively co-expressing tlp-D34 and chi11 display enhancement of sheath blight resistance

J. M. Shah1, R. Singh1, K. Veluthambi1,*
1 Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, India

Transgenic rice (Oryza sativa L. subsp. indica cv. White Ponni) constitutively expressing the rice thaumatin-like protein gene (tlp-D34, PR-5) individually or in combination with the rice chitinase gene (chi11, PR-3) was generated using an Agrobacterium vir helper strain with multiple copies of pTiBo542 virB and virG. Transformation with the tlp-D34 gene alone and tlp-D34 + chi11 genes yielded five and seven single-copy transgenic lines, respectively. Southern blot analysis with two probes, one flanking the right T-DNA border and the second flanking the left T-DNA border, confirmed that all transgenic plants harboured single and complete T-DNA copies. Homozygous transgenic lines were first identified in the T1 generation by Southern blot analysis and were subsequently confirmed by segregation analysis of T2 plants. Accumulation of transcripts encoded by the transgenes was confirmed in T0 plants and homozygous T2 plants by Northern blot analysis. The homozygous T2 plants harbouring tlp-D34 + chi11 genes showed 2.8- to 4.2-fold higher chitinase activity. Western blot analysis revealed the accumulation of thaumatin-like protein and chitinase in the respective transgenic plants. Upon infection with Rhizoctonia solani, the disease index reduced from 100 % in control plants to 65 % in a T3 homozygous transgenic line T4 expressing the tlp-D34 gene alone. In a T2 homozygous transgenic line CT22 co-expressing tlp-D34 and chi11 genes, the disease index reduced to 39 %.

Keywords: Agrobacterium tumefaciens; homozygous transgenic lines; Oryza sativa; pathogenesis-related protein; Rhizoctonia solani; fungal resistance
Subjects: transgenic plants; sheath blight; pathogen resistance; pathogenesis-related proteins; Oryza sativa; Agrobacterium tumefaciens; Souther blot; chitinase; Northern blot; Western blot
Species: Oryza sativa; Agrobacterium tumefaciens; Rhizoctonia solani

Received: January 8, 2009; Accepted: August 15, 2012; Published: June 1, 2013Show citation

ACS AIP APA ASA Harvard Chicago IEEE ISO690 MLA NLM Turabian Vancouver
Shah, J.M., Singh, R., & Veluthambi, K. (2013). Transgenic rice lines constitutively co-expressing tlp-D34 and chi11 display enhancement of sheath blight resistance. Biologia plantarum, 57(2), 351-358. doi: 10.1007/s10535-012-0291-z.
Download citation
  • BibTeX (.bib)
  • Bookends (.ris)
  • EasyBib (.ris)
  • EndNote (.enw)
  • EndNote 8 (.xml)
  • ISI WoS (.isi)
  • Medlars (.medlars)
  • Mendeley (.ris)
  • MODS (.xml)
  • Papers (.ris)
  • RefWorks (.txt)
  • RefManager (.ris)
  • RIS (.ris)
  • MS Word (.xml)
  • Zotero (.ris)

    • Open full article

    References

    1. Anand, A., Lei, Z., Sumner, L.W., Mysore, K.S., Arakane, Y., Bockus, W.W., Muthukrishnan, S.: Apoplastic extracts from a transgenic wheat line exhibiting lesion-mimic phenotype have multiple pathogenesis-related proteins that are antifungal. - Mol. Plant Microbe Intreract. 17: 1306-1317,2004. Go to original source...
    2. Anand, A., Zhou, T., Trick, H.N., Gill, B.S., Bockus, W.W., Muthukrishnan, S.: Greenhouse and field testing of transgenic wheat plants stably expressing genes for thaumatin-like protein, chitinase and glucanase against Fusarium graminearum. - J. exp. Bot. 54: 1101-1111, 2003. Go to original source...
    3. Anzlovar, S., Serra, M.D., Dermastia, M., Menestrina, G.: Membrane permeabilizing activity of pathogenesis-related protein linusitin from flax seed. - Mol. Plant Microbe Interact. 11: 610-617, 1998. Go to original source...
    4. Chen, W.P., Chen, P.D., Liu, D.J., Kynast, R., Friebe, B., Velazhahan, R., Muthukrishnan, S., Gill, B.S.: Development of wheat scab symptoms is delayed in transgenic wheat plants that constitutively express a rice thaumatin-like protein gene. - Theor. appl. Genet. 99: 755-760, 1999. Go to original source...
    5. Chen, W.P., Gu, X., Liang, G.H., Muthukrishnan, S., Chen, P.D., Liu, D.J., Gill, B.S.: Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker. - Theor. appl. Genet. 97: 1296-1306, 1998. Go to original source...
    6. Chye, M.-L., Zhao, K.-J., He, Z.-M., Ramalingam, S., Fung, K.-L.: An agglutinating chitinase with two chitin-binding domains confers fungal protection in transgenic potato. - Planta 220: 717-730, 2005. Go to original source...
    7. Dai, S., Zheng, P., Marmey, P., Zhang, S., Tian, W., Chen, S., Beachy, R.N., Fauquet, C.: Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. - Mol. Breed. 7: 25-33, 2001. Go to original source...
    8. Datta, K., Velazhahan, R., Oliva, N., Ona, I., Mew, T., Khush, G.S., Muthukrishnan, S., Datta, S.K.: Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease. - Theor. appl. Genet. 98: 1138-1145, 1999. Go to original source...
    9. Eamens, A., Wang, M.-B., Smith, N.A., Waterhouse, P.M.: RNA silencing in plants: yesterday, today and tomorrow. - Plant Physiol. 147: 456-468, 2008. Go to original source...
    10. Ferreira, R.B., Monteiro, S., Freitas, F., Santos, C.N., Chen, C., Batista, L.M., Duarte, J., Borges, A., Teixeira, A.R.: The role of plant defence proteins in fungal pathogenesis. - Mol. Plant Pathol. 8: 677-700, 2007. Go to original source...
    11. Gelvin, S.B.: The introduction and expression of transgenes in plants. - Curr. Opin. Biotechnol. 9: 227-232, 1998. Go to original source...
    12. Iyer, L.M., Kumpatla, S.P., Chandrasekharan, M.B., Hall, T.C.: Transgene silencing in monocots. - Plant mol. Biol. 43: 323-346, 2000. Go to original source...
    13. Jach, G., Gornhardt, B., Mundy, J., Logemann, J., Pinsdorf, E., Leah, R., Schell, J., Maas, C.: Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. - Plant J. 8: 97-109, 1995. Go to original source...
    14. Jacobs, A.K., Dry, I.B., Robinson, S.P.: Induction of different pathogenesis-related cDNAs in grapevine infected with powdery mildew and treated with ethephon. - Plant Pathol. 48: 325-336, 1999. Go to original source...
    15. Jayaraj, J., Punja, Z.K.: Combined expression of chitinase and lipid transfer protein genes in transgenic carrot plants enhances resistance to foliar fungal pathogens. - Plant Cell Rep. 26: 1539-1546, 2007. Go to original source...
    16. Jongedijk, E., Tigelaar, H., Van Roekel, J.S.C., Bres-Vloemans, S.A., Dekker, I., Van den Elzen, P.J.M., Cornelissen, B.J.C., Melchers, L.S.: Synergistic activity of chitinases and β-1,3-glucanases enhances fungal resistance in transgenic tomato plants. - Euphytica 85: 173-180, 1995. Go to original source...
    17. Kalpana, K., Maruthasalam, S., Rajesh, T., Poovannan, K., Kumar, K.K., Kokiladevi, E., Raja, J.A.J., Sudhakar, D., Velazhahan, R., Samiyappan, R., Balasubramanian, P.: Engineering sheath blight resistance in elite indica rice cultivars using genes encoding defense proteins. - Plant Sci. 170: 203-215, 2006. Go to original source...
    18. Kim, J.-K., Duan, X., Wu, R., Seok, S.J., Boston, R.S., Jang, I.- C., Eun, M.-Y., Nahm, B.H.: Molecular and genetic analysis of transgenic rice plants expressing the maize ribosomeinactivating protein b-32 gene and the herbicide resistance bar gene. - Mol. Breed. 5: 85-94, 1999. Go to original source...
    19. Kim, J.-K., Jang, I.-C., Wu, R., Zuo, W.-N., Boston, R.S., Lee, Y.-H., Ahn, I.-P., Nahm, B.H.: Co-expression of a modified maize ribosome-inactivating protein and a rice basic chitinase gene in transgenic rice plants confers enhanced resistance to sheath blight. - Transgenic Res. 12: 475-484, 2003. Go to original source...
    20. Maruthasalam, S., Kalpana, K., Kumar, K.K., Loganathan, M., Poovannan, K., Raja, J.A.J., Kokiladevi, E., Samiyappan, R., Sudhakar, D., Balasubramanian, P.: Pyramiding transgenic resistance in elite indica rice cultivars against the sheath blight and bacterial blight. - Plant Cell Rep. 26: 791-804, 2007. Go to original source...
    21. Mauch, F., Hadwiger, L.A., Boller, T.: Ethylene: symptom, not signal for the induction of chitinase and β-1,3-glucanase in pea pods by pathogens and elicitors. - Plant Physiol. 76: 607-611, 1984. Go to original source...
    22. Melander, M., Kamnert, I., Happstadius, I., Liljeroth, E., Bryngelsson, T.: Stability of transgene integration and expression in subsequent generations of doubled haploid oilseed rape transformed with chitinase and β-1,3-glucanase genes in a double-gene construct. - Plant Cell Rep. 25: 942-952, 2006. Go to original source...
    23. Moravčíková, J., Matuíková, I., Libantová, J., Bauer, M., Mlynárová, L.: Expression of a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase genes in transgenic potato plants. - Plant Cell Tissue Organ Cult. 79: 161-168, 2004. Go to original source...
    24. Pawlowski, K., Kunze, R., De Vries, S., Bisseling, T.: Isolation of total, poly(A) and polysomal RNA from plant tissues. - In: Gelvin, S.B., Schilperoort, R.A. (ed.): Plant Molecular Biology Manual. Pp. D5/1-D5/13. Kluwer Academic Publishers, Dordrecht - Boston - London 1994.
    25. Perri, F., Romitelli, F., Rufini, F., Secundo, F., Di Stasio, E., Giardina, B., Vitali, A.: Different structural behaviors evidenced in thaumatin-like proteins: a spectroscopic study - Protein J. 27: 13-20, 2008. Go to original source...
    26. Punja, Z.K.: Recent developments toward achieving fungal disease resistance in transgenic plants. - Can. J. Plant Pathol. 28: 298-308, 2006. Go to original source...
    27. Ramesh Sundar, A., Velazhahan, R., Nagarathinam, S., Vidhyasekaran, P.: Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from Colletotrichum falcatum. - Biol. Plant. 52: 321-328, 2008. Go to original source...
    28. Rogers, S.O., Bendich, A.J.: Extraction of total cellular DNA from plants. - In: Gelvin, S.B., Schilperoort, R.A. (ed.): Plant Molecular Biology Manual. Pp. A6/1-A6/10. Kluwer Academic Publishers, Dordrecht - Boston - London 1988.
    29. Sarowar, S., Kim, Y.J., Kim, E.N., Kim, K.D., Choi, J.Y., Hyung, N.I., Shin, J.S.: Constitutive expression of two pathogenesis-related genes in tomato plants enhanced resistance to oomycete pathogen Phytophthora capsici. - Plant Cell Tissue Organ Cult. 86: 7-14, 2006. Go to original source...
    30. Selitrennikoff, C.P.: Antifungal proteins. - Appl. Environ. Microbiol. 67: 2883-2894, 2001. Go to original source...
    31. Sridevi, G., Dhandapani, M., Veluthambi, K.: Agrobacteriummediated transformation of White Ponni, a non-basmati variety of indica rice (Oryza sativa L.). - Curr. Sci. 88: 128-132, 2005.
    32. Sridevi, G., Parameswari, C., Rajamuni, P., Veluthambi, K.: Identification of hemizygous and homozygous transgenic rice plants in T1 generation by DNA blot analysis. - Plant Biotechnol. 23: 531-534, 2006. Go to original source...
    33. Sridevi, G., Parameswari, C., Sabapathi, N., Raghupathy, V., Veluthambi, K.: Combined expression of chitinase and β- 1,3-glucanase genes in indica rice (Oryza sativa L.) enhances resistance against Rhizoctonia solani. - Plant Sci. 175: 283-290, 2008. Go to original source...
    34. Terada, R., Asao, H., Iida, S.: A large-scale Agrobacteriummediated transformation procedure with a strong positivenegative selection for gene targeting in rice (Oryza sativa L.). - Plant Cell Rep. 22: 653-659, 2004. Go to original source...
    35. Van Loon, L.C., Rep, M., Pieterse, C.M.J.: Significance of inducible defence-related proteins in infected plants. - Annu. Rev. Phytopathol. 44: 135-162, 2006. Go to original source...
    36. Velazhahan, R., Chen-Cole, K., Anuratha, C.S., Muthukrishnan, S.: Induction of thaumatin-like proteins (TLPs) in Rhizoctonia solani-infected rice and characterization of two new cDNA clones. - Physiol. Plant. 102: 21-28, 1998. Go to original source...
    37. Wang, Y., Kausch, A.P., Chandlee, J.M., Luo, H., Ruemmele, B.A., Browning, M., Jackson, N., Goldsmith, M.R.: Cotransfer and expression of chitinase, glucanase, and bar genes in creeping bentgrass for conferring fungal disease resistance. - Plant Sci. 165: 497-506, 2003. Go to original source...
    38. Zareie, R., Melanson, D.L., Murphy, P.J.: Isolation of fungal cell wall degrading proteins from barley (Hordeum vulgare L.) leaves infected with Rhynchosporium secalis. - Mol. Plant Microbe Interact. 15: 1031-1039, 2002. Go to original source...
    39. Zhu, Q., Maher, E.A., Masoud, S., Dixon, R.A., Lamb, C.J.: Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco. - Bio/Technology 12: 807-812, 1994. Go to original source...

    Return to the content