Abstract
Globally, weeds and sap-sucking insects are major constrains in rice production. We have combined two desired traits for resistance to glyphosate and sapsuckers by transgenic overexpression of mutated rice 5-enolpyruvylshikimate3-phosphate synthase (Os-mEPSPS) and garlic leaf lectin (Allium sativum leaf agglutinin, ASAL) in a high yielding IR-64 rice cultivar. Polymerase chain reaction (PCR), southern and genome walking analyses showed stable integration of transgenes in genomes of transgenic lines. The molecular analysis of transgenic lines revealed effective expression of both Os-mEPSPS and ASAL transcripts and their respective proteins and imparted resistance to glyphosate and brown planthopper. In planta brown hopper assays in the progenies of independent rice transgenic lines exhibit enhanced ASAL expression that imparts substantial level of resistance, reduced survival and fecundity of brown hoppers by ~74 and ~68 %, respectively. The glyphosate and sap-sucking insect pest-resistant transgenic rice lines showed normal phenotype and no growth penalty or fitness costs. The double transgenic traits co-segregated as a single unit in the subsequent generations. In spite of the presence of several herbicide- and insect-resistant GM rice crops in the market, there is a need to develop new transgenic rice lines by selecting the transgenes from homologous and/or edible crop sources that circumvent the biosafety issues raised by regulatory bodies regarding the acceptance of these products for human consumption. In this scenario, the transgenic rice varieties generated in this study serve as a novel genetic resource in rice breeding aimed at durable resistance to glyphosate and sap-sucking insect pests.
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Baerson SR, Rodriguez DJ, Tran M, Feng Y, Biest NA, Dill GM (2002) Glyphosate-resistant goosegrass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Plant Physiol 129:1265–1275
Bandyopadhyay S, Roy A, Das S (2001) Binding of garlic (Allium sativum) leaf lectin to the gut receptors of homopteran pests is correlated to its insecticidal activity. Plant Sci 161:1025–1033
Bharathi Y, Vijaya KS, Pasalu IC, Balachandran SM, Reddy VD (2011) Pyramided rice lines harbouring Allium sativum (asal) and Galanthus nivalis (gna) lectin genes impart enhanced resistance against major sap-sucking pests. J Biotechnol 152:63–71
Cao MX, Huang JQ, Wei ZM, Yao QH, Wan CZ, Lu JA (2004) Engineering higher yield and herbicide resistance in rice by Agrobacterium-mediated multiple gene transformation. Crop Sci 44:2206–2213
Cao G, Liu Y, Zhang S, Yang X, Chen R, Zhang Y, Lu W, Liu Y, Wang J, Lin M, Wang G (2012) A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in Escherichia coli and tobacco plants. PLoS ONE 7:e38718
Chandrasekhar K, Vijayalakshm M, Vani K, Kaul T, Reddy MK (2014) Phloem-specific expression of the lectin gene from Allium sativum confers resistance to the sap-sucker the Nilaparvata lugens. Biotechnol Tech Lett. doi:10.1007/s10529-011-0620-x
Chen QJ, Zhou HM, Chen J, Wang XC (2006) A Gateway-based platform for multigene plant transformation. Plant Mol Biol 62:927–936
Coggins JR, Abell C, Evans LB, Frederickson M, Robinson DA, Roszak AW, Lapthorn AP (2003) Experiences with the shikimate-pathway enzymes as targets for rational drug design. Biochem Soc Trans 31:548–552
Curtis MD, Grossniklaus U (2003) A Gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Report 1:19–21
Funke T, Yang Y, Han H, Healy-Fried M, Olesen S, Becker A, Schonbrunn E (2009) Structural basis of glyphosate resistance resulting from the double mutation Thr97 –> lle and Pro101 –> Ser in 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli. J Biol Chem 284:9854–9860
Gallagher KD, Kenmore PE, Sogawa K (1994) Judicial use of insecticides deter planthopper outbreaks and extend the life of resistant varieties in Southeast Asian rice. In: Denno RK, Perfect TJ (eds) Planthoppers; their ecology and management. Chapman & Hall, London, pp 599–614
Harper SM, Crenshaw RW, Mullins MA, Privalle LSJ (1995) Lectin binding to insect brush-border membranes. J Econ Entomol 88:1197–1202
Hibberd JM, Sheehy JE, Langdale JA (2008) Using C4 photosynthesis to increase the yield of rice-rationale and feasibility. Curr Opin Plant Biol 11:228–231
Meilan R, Han K, Ma C, DiFazio S, Eaton J, Hoien E, Stanton B, Crockett R, Taylor M, James R, Skinner J, Jouanin L, Pilate G, Strauss S (2002) The CP4 transgene provides high levels of tolerance to Roundup herbicide in field-grown hybrid poplars. Can J Forest Res 32:967–976
Mochida O, Wahyu A, Surjani TK (1979) Some considerations on screening resistant cultivars/lines of rice plant to the brown planthopper, Nilaparvata lugens (Stål) (Hom., Delphacidae), pp 1–9. IRRI, Los Baños
Nagadhara D, Ramesh S, Pasalu IC, Rao YK, Krishnaiah NV, Sarma NP, Bown DP, Gatehouse JA, Reddy VD, Rao KV (2003) Transgenic indica rice resistant to sap sucking insects. Plant Biotechnol J 1:231–240
Nagadhara D, Ramesh S, Pasalu IC, Rao YK, Sarma NP, Reddy VD, Rao KV (2004) Transgenic rice plants expressing the snowdrop lectin gene (gna) exhibit high-level resistance to the whitebacked planthopper (Sogatella furcifera). Theor Appl Genet 109:1399–1405
Oerke EC (2006) Crop losses to pests. J Agric Sci 144:31–43
Padgette SR, Huyn QK, Ayken S, Sammons RD, Sikorski JA, Kishore GM (1988) Identification of the reactive cysteines of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase and their nonessentiality for enzymatic catalysis. J Biol Chem 263:1798–1802
Que Q, Chilton MD, de Fontes CM, He C, Nuccio M, Zhu T, Wu Y, Chen JS, Shi L (2010) Trait stacking in transgenic crops: challenges and opportunities. GM Crops 1:220–229
Reddy MK, Nair S, Sopory SK (2002) A new approach for efficient directional genome walking using polymerase chain reaction. Anal Biochem 306:154–158
Sadeghi A, Broeders S, De Greve H, Hernalsteens JP, Peumans WJ, Van Damme EJM, Smagghe G (2007) Expression of garlic leaf lectin under the control of the phloem-specific promoter Asus1 from Arabidopsis thaliana protects tobacco plants against the tobacco aphid (Myzus nicotianae). Pest Manag Sci 63:1215–1223
Saha P, Dasgupta I, Das S (2006) A novel approach for developing resistance in rice against phloem limited viruses by antagonizing the phloem feeding hemipteran vectors. Plant Mol Biol 62:735–752
Saxena RC, Khan ZR (1989) Factors effecting resistance to rice varieties to planthopper and leafhopper pests. Agric Zool Rev 3:97–132
Senguttuvan T, Gopalan M, Chelliah S (1991) Impact of resistance mechanisms in rice against the brown planthopper, Niluparvara lugens (Homoptera: Delphacidae). Crop Prot 10:125–128
Stalker DM, Hiatt WR, Comai L (1985) A single amino acid substitution in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase confers resistance to the herbicide glyphosate. J Biol Chem 260:4724–4728
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some application. Proc Natl Acad Sci U S A 76:4350–4354
Vajhala CSK, Sadumpati VK, Nunna HR, Puligundla SK, Reddy VD, Rao KV (2013) Development of transgenic cotton lines expressing Allium sativum agglutinin (ASAL) for enhanced resistance against major sap-sucking pests. PLoS ONE 8:e72542
Whitten MJ, Oakenshott JG (1991) Opportunities for modern biotechnology in control of insect pests and weeds with special reference to developing countries. FAO Plant Prot Bull 39:155–181
Yarasi B, Sadumpati V, Immanni CP, Reddy VD, Rao KV (2008) Transgenic rice expressing Allium sativum leaf agglutinin (ASAL) exhibits high-level resistance against major sap-sucking pests. BMC Plant Biol 8:102
Zhou M, Xu H, Wei X, Ye Z, Wei L, Gong W, Wang Y, Zhu Z (2005) Identification of a glyphosate-resistant mutant of rice 5-enolpyruvylshikimate 3-phosphate synthase using a directed evolution strategy. Plant Physiol 140:184–195
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We are grateful to the Department of Biotechnology, Government of India, New Delhi, for the generous financial assistance.
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Chandrasekhar, K., Reddy, G.M., Singh, J. et al. Development of Transgenic Rice Harbouring Mutated Rice 5-Enolpyruvylshikimate 3-Phosphate Synthase (Os-mEPSPS) and Allium sativum Leaf Agglutinin (ASAL) Genes Conferring Tolerance to Herbicides and Sap-Sucking Insects. Plant Mol Biol Rep 32, 1146–1157 (2014). https://doi.org/10.1007/s11105-014-0715-3
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DOI: https://doi.org/10.1007/s11105-014-0715-3