Enhanced resistance to Rhizoctonia solani by combined expression of chitinase and Ribosome Inactivating Protein in transgenic potatoes (Solanum tuberosum L.)

  • M. M'hamdi Higher Institute of Agronomy- Chott Mariem, 4042 Sousse
  • H. Chikh-Rouhou Higher Institute of Agronomy- Chott Mariem, 4042 Sousse
  • N. Boughalleb Higher Institute of Agronomy- Chott Mariem, 4042 Sousse
  • J. I. Ruiz de Galarreta NEIKER- Basque Institute for Agricultural Research. PO. Box. 46.01080 Vitoria
Keywords: fungal resistance, genes, proteins, southern blot, western blot

Abstract

Potato (Solanum tuberosum L.) is susceptible to many fungal pathogens including Rhizoctonia solani. In the present study, the potato cultivar Desirée was transformed via Agrobacterium tumefaciens strain GV3101 containing the binary plasmid pGJ132 harboring both the chitinase (chiA) and rip30 genes. The potato leaf disc was used as an explant for transformation. PCR, Southern blot and Western blot were used for characterization of the transgenic plants. In this study it was shown that not all the plants developed in selective medium were positive for the corresponding gene using the PCR technique. Southern blot analysis confirmed that transgenic plants integrated 2-3 copies of chiA and rip30 genes respectively into their genome. The expression of the CHIA and RIP30 proteins was confirmed in the leaf extracts of the transgenic clones by Western blot analysis. Transgenic potato plants expressing rip30 and chiA genes showed enhanced resistance to R. solani in a greenhouse assay.

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Author Biography

H. Chikh-Rouhou, Higher Institute of Agronomy- Chott Mariem, 4042 Sousse
Plant protection department

References

Agrios GN, 1997. Plant Pathology, 4th edn. Academic Press, NY.

Almasia NI, Bazzini AA, Hopp HE, Vazquez-Rovero C, 2008. Overexpression of snakin-1 gene enhances resistance to Rhizoctonia solani and Erwinia carotovora in transgenic potato plants. Mol Plant Pathology 9: 329-338.
http://dx.doi.org/10.1111/j.1364-3703.2008.00469.x
PMid:18705874

Anand A, Zhou T, Trick HN, Gill BS, Bockus WW, Muthukrishnan S, 2003. 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.
http://dx.doi.org/10.1093/jxb/erg110
PMid:12598580

Anonymous, 1976. Manual of plant growth stages and disease assessment keys. Ministry of Agriculture Fisheries and Food (Publications), Pinner, Key N 2.4.1. Alnwick, UK.

Banerjee AK, Prat S, Hannapel DJ, 2006. Efficient production of transgenic potato (S. tuberosum L. ssp. andigena) plants via Agrobacterium tumefaciens-mediated transformation. Plant Sci 170: 732-738.
http://dx.doi.org/10.1016/j.plantsci.2005.11.007

Beaujean A, Sangwan RS, Lecardonnel A, Sangwan-Norreel BS, 1998. Agrobacterium-mediated transformation of three economically important potato cultivars using sliced intermodal explants: an efficient protocol of transformation. J Exp Bot 49: 589-1595.

Bieri S, Potrykus I, Futterer J, 2000. Expression of active barley seed ribosome-inactivating protein in transgenic wheat. Theor Appl Genet 100: 755-763.
http://dx.doi.org/10.1007/s001220051349

Carling DE, Leiner RH, 1986. Isolation and characterization of Rhizoctonia solani and binucleate R. solani-like fungi from aerial stems and subterranean organs of potato plants. Phytopathology 76: 725-729.
http://dx.doi.org/10.1094/Phyto-76-725

Chand T, Logan C, 1983. Cultural and pathogenic variation in potato isolates of Rhizoctonia solani in Northern Ireland. T Brit Mycol Soc 81: 585590.
http://dx.doi.org/10.1016/S0007-1536(83)80129-6

Chye ML, Zhao KJ, He ZM, Ramalingam S, Fung KL, 2005. An agglutinating chitinase BjCHI1 with two chitin-binding domains confers fungal protection in transgenic potato. Planta 220: 717-730.
http://dx.doi.org/10.1007/s00425-004-1391-6
PMid:15490228

Datta K, Tu J, Oliva N, Ona I, Velazhahan R, Wew TW, Muthukrishnan S, Datta SK, 2001. Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite indica rice cultivars. Plant Sci 160: 405-414.
http://dx.doi.org/10.1016/S0168-9452(00)00413-1

Ducreux LJM, Morris WL, Taylor MA, Millam S, 2005. Agrobacterium-mediated transformation of Solanum phureja. Plant Cell Rep 24, 10-14.
http://dx.doi.org/10.1007/s00299-004-0902-z
PMid:15666166

Edwards K, Johnstone C, Thompson C, 1991. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19: 1349.
http://dx.doi.org/10.1093/nar/19.6.1349
PMid:2030957 PMCid:333874

Esfahani K, Motallebi M, Zamani MR, Sohi HH, Jourabchi E, 2010. Transformation of potato (Solanum tuberosum cv. Savalan) by chitinase and ?-1,3 glucanase genes of mycoparasitic fungi towards improving resistance to Rhizoctonia solani AG-3. Iranian J Biotechnol 8(2): 3-81.

Hartley MR, Chaddock JA, Bonness MS, 1996. The structure and function of ribosome inactivating proteins. Trends Plant Sci 1: 254260.
http://dx.doi.org/10.1016/1360-1385(96)10030-3

Hide GA, Welham SJ, Read PJ, Ainsley AE, 1996. The yield of potato plants as affected by stem canker (Rhizoctonia solani), blackleg (Erwinia carotovora subsp. atroseptica) and by neighbouring plants. J Agric Sci 126: 429-440.
http://dx.doi.org/10.1017/S0021859600075511

Hooykaas PJJ, Roobol C, Schilperoort RA, 1979. Regulation of the transfer of Ti-plasmids of Agrobacterium tumefaciens. J Gen Microbiol 110: 99-109.
http://dx.doi.org/10.1099/00221287-110-1-99

Howie W, Joe L, Newbigin E, Suslow T, Dunsmuir P, 1994. Transgenic tobacco plants which express the chiA gene from Serratia marcescens have enhanced tolerance to Rhizoctonia solani. Transgenic Res 3: 90-98.
http://dx.doi.org/10.1007/BF01974086

Jach G, Gornhardt B, Mundy J, Logemann J, Pinsdorf E, Leah R, Schell J, Maas C, 1995. Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J 8: 97-109.
http://dx.doi.org/10.1046/j.1365-313X.1995.08010097.x
PMid:7655510

Jayaraj J, Punja ZK, 2007. 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.
http://dx.doi.org/10.1007/s00299-007-0368-x
PMid:17508215

Kato A, Nakamura S, Ibrahim H, Matsumi T, Tsumiyama C, Kato M, 1998. Production of genetically modified lysozymes having extreme heat stability and antimicrobial activity against gram negative bacteria in yeast and in plants. Mol Nutr Food Res 42: 128-130.

Khan RS, Sjahril R, Nakamura I, Mii M, 2008. Production of transgenic potato exhibiting enhanced resistance to fungal infections and herbicide applications. Plant Biotechnol Rep 2: 13-20.
http://dx.doi.org/10.1007/s11816-008-0043-x

Kumar A, Miller M, Whitty P, Lyon J, Davie P, 1995. Agrobacterium-mediated transformation of five wild Solanum species using in vitro microtubersin. Plant Cell Rep 14: 324-328.
http://dx.doi.org/10.1007/BF00232037

Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680685.
http://dx.doi.org/10.1038/227680a0
PMid:5432063

Lehtonen MJ, Ahvenniemi P, Wilson PS, German-Kinnari M, Valkonen JPT, 2008. Biological diversity of Rhizoctonia solani (AG-3) in a northern potato-cultivation environment in Finland. Plant Pathol 57: 141151.

Liu D, Raghothama KG, Hasegawa PM, Bressan RA, 1994. Osmotin overexpression in potato delays development of disease symptoms. Proc Natl Acad Sci USA 91: 1888-1892.
http://dx.doi.org/10.1073/pnas.91.5.1888

Logeman J, Jach G, Tommerup H, Mundy J, Schell J, 1992. Expression of a barley ribosome inactivating protein leads to increased fungal protection in transgenic tobacco plants. Biotechnology 10: 305-308.
http://dx.doi.org/10.1038/nbt0392-305

Lund P, Dunsmuir P, 1992. A plant signal sequence enhances the secretion of bacterial Chi A in transgenic tobacco. Plant Mol Biol 18: 47-53.
http://dx.doi.org/10.1007/BF00018455
PMid:1731977

McDowell JM, Woffenden BJ, 2003. Plant disease resistance genes: recent insights and potential applications. Trends Biotechnol 21: 178-183.
http://dx.doi.org/10.1016/S0167-7799(03)00053-2

Melchers LS, Stuiver MH, 2000. Novel genes for disease-resistance breeding. Curr Opin Plant Biol 3: 147-152.
http://dx.doi.org/10.1016/S1369-5266(99)00055-2

MHamdi M, Rouviere C, Rojas-Beltran J, Du Jardin P, 2003. Optimisation de la transformation gntique de la pomme de terre par Agrobacterium tumefaciens. Utilisation de la rsistance lhygromycine comme marqueur slectif. Biotechnol Agron Soc Environ 7: 183-188.

Murashige TH, Skoog F, 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 15: 473-497.
http://dx.doi.org/10.1111/j.1399-3054.1962.tb08052.x

Osusky M, Osuska L, Kay W, Misra S, 2005. Genetic modification of potato against microbial diseases: in vitro and in planta activity of a dermaseptin B1 derivative, MsrA2. Theor Appl Genet 111: 711-722.
http://dx.doi.org/10.1007/s00122-005-2056-y
PMid:15947906

Ouyang B, Chen YH, Li HX, Qian CJ, Huang SL, Ye ZB, 2005. Transformation of tomatoes with osmotin and chitinase genes and their resistance to Fusarium wilt. J Hortic Sci Biotechnol 80: 517-522.

Read PJ, Hide GA, Firmager JP, Hall SM, 1989. Growth and yield of potatoes as affected by severity of stem canker (Rhizoctonia solani). Potato Res 32: 9-15.
http://dx.doi.org/10.1007/BF02365813

Rozen S, Skalestsky H, 1999. Primer3 on the www for general users and for biologist programmers. In Methods in molecular biology (Misener S & Krawetz SA, eds.). Humana Press Inc., Totowa, NJ, USA, pp: 365-386.

Stirpe F, Barbieri L, Battelli LG, Soria M, Lappi DA, 1992. Ribosome-inactivating proteins from plants: present status and future prospects. Biotechnology 10: 405412.
http://dx.doi.org/10.1038/nbt0492-405
PMid:1368484

Tabei Y, Kitade S, Nishizawa N, Kikuchi N, Kayano T, Hibi T, Akutsu K, 1997. Transgenic cucumber plants harboring a rice chitinase gene exhibit enhanced resistance to gray mold (Botrytis cinerea). Plant Cell Rep 17: 159-164.
http://dx.doi.org/10.1007/s002990050371

Walter RS, Rosemary L, Gary FD, Weingartner DP, 2001. Compendium of potato diseases. APS Compendium of plant disease series, American Phytopathological Society, St. Paul, MN, USA.

Wang K, Herrera-Estrella L, Van Montagu M, Zambryskie P, 1984. Right 25 bp terminus sequence of the nopaline T-DNA is essential for and determines direction of DNA transfer Agrobacterium to the plant genome. Cell 38: 455-462.
http://dx.doi.org/10.1016/0092-8674(84)90500-2

Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhold DL, Alexander DC, Ahl-Goy P, Metraux JP, Ryals JA, 1991. Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell 3: 1085-1094.
PMid:12324583 PMCid:160074

Xiao YH, Li XB, Yang XY, Luo M, Hou L, Guo SH, Luo XY, Pei Y, 2007. Cloning and characterization of a balsam pear class I chitinase gene (Mcchit1) and its ectopic expression enhances fungal resistance in transgenic plants. Biosci Biotechnol Biochem 71: 1211-1219.
http://dx.doi.org/10.1271/bbb.60658
PMid:17485855

Zhu Q, Maher EA, Masoud S, Dixon RA, Lamb CJ, 1994. Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco. Biotechnology 12: 807-812.
http://dx.doi.org/10.1038/nbt0894-807

Zhu H, Xu X, Xiao G, Yuan L, Li B, 2007. Enhancing disease resistances of super hybrid rice with four antifungal genes. Sci China Life Sci 50: 31-39.
http://dx.doi.org/10.1007/s11427-007-0001-9
PMid:17393080

Published
2012-07-11
How to Cite
M’hamdi, M., Chikh-Rouhou, H., Boughalleb, N., & Ruiz de Galarreta, J. I. (2012). Enhanced resistance to Rhizoctonia solani by combined expression of chitinase and Ribosome Inactivating Protein in transgenic potatoes (Solanum tuberosum L.). Spanish Journal of Agricultural Research, 10(3), 778-785. https://doi.org/10.5424/sjar/2012103-362-11
Section
Plant protection