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Transgenic potato lines expressing hairpin RNAi construct of molting-associated EcR gene exhibit enhanced resistance against Colorado potato beetle (Leptinotarsa decemlineata, Say)

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Abstract

Most of the commercialized insect resistant transgenic crops express cry gene(s) isolated from Bacillus thuringiensis; however, intensive cultivation of Bt crops over almost two decades has been questioned regarding its sustainability and durability in pest management. The present study focused on silencing of highly specific molting-associated Ecdysone receptor (EcR) gene of Colorado potato beetle (CPB) using RNA interference (RNAi) approach. The partial cDNA of EcR gene of CPB was amplified using specific primers in sense and anti-sense orientations, and cloned in pRNAi-GG vector flanked by an intronic sequence (pdk). Leaf and internodal explants of Agria and Lady Olympia potato cultivars were infected with Agrobacterium strain LBA4404 harboring constructs under the control of CaMV 35S promoter. Standard molecular analysis of primary transformants showed proper integration of T-DNA in plant genome. The transgenic plants of both cultivars were evaluated for their efficacy against first, second and third instar CPB larvae. The leaf biotoxicity assays revealed 15–80% of CPB mortality. A significantly lower fold-change (0.87–4.14×) in larval weight was observed in insects fed on transgenic plants compared to the ones fed on control plants (1.87–6.53×). Furthermore, CPB larvae fed on transgenic plants exhibited reduced EcR transcripts, indicating the functionality of dsRNA EcR in silencing EcR gene expression. This study is an excellent example of the integration of an alternative, effective and reliable method to cope with potato insect pests that incur significant losses to potato production in the world.

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References

  • Asokan R, Chandra GS, Manamohan M, Kumar NK, Sita T (2014) Response of various target genes to diet-delivered dsRNA mediated RNA interference in the cotton bollworm, Helicoverpa armigera. J Pest Sci 87:163–172

    Article  Google Scholar 

  • Bagri DS, Upadhyay DC, Jain SK, Upadhyay CP (2018) Biotechnological improvement of nutritional and therapeutic value of cultivated potato. Front Biosci 10:217–228

    Article  Google Scholar 

  • Bakhsh A, Siddique S, Husnain T (2012) A molecular approach to combat spatiotemporal variation in insecticidal gene (Cry1Ac) expression in cotton”. Euphytica 183:65–74

    Article  CAS  Google Scholar 

  • Bakhsh A, Anayol E, Ozcan SF (2014) Comparison of transformation efficiency of five Agrobacterium tumefaciens strains in Nicotiana tabacum L. Emir J Food Agric 26:259–264

    Article  Google Scholar 

  • Bakhsh A, Baloch FS, Hatipoğlu R, Özkan H (2015) Use of genetic engineering: benefits and health concerns. In: Hui YH, Evranuz EÖ (eds) Handbook of vegetable preservation and processing, 2nd edn. CRC Press, pp 100–131

  • Bakhsh A, Dinc T, Hussain T, Demirel U, Aasim M, Çalışkan ME (2018) Development of transgenic tobacco lines with pyramided insect resistant genes. Turkish J Biol 42:174–186

    Article  CAS  Google Scholar 

  • Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O, Johnson S, Plaetinck G, Munyikwa T, Pleau M, Vaughn T, Roberts J (2007) Control of coleopteran insect pests through RNA interference. Nat Biotechnol 25:1322–1326

    Article  CAS  PubMed  Google Scholar 

  • Beaujean A, Sangwan RS, Lecardonnel A, Sangwan-Norreel BS (1998) Agrobacterium-mediated transformation of three economically important potato cultivars using sliced internodal explants: an efficient protocol of transformation. J Exp Bot 49:1589–1595

    Article  CAS  Google Scholar 

  • Belles X, Piulachs MD (2015) Ecdysone signalling and ovarian development in insects: from stem cells to ovarian follicle formation. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms 1849(2):181–186

    Article  CAS  Google Scholar 

  • Çalışkan ME, Onaran H, Arıoğlu H (2010) Overview of the Turkish potato sector: challenges, achievements and expectations. Potato Res 53:255–266

    Article  Google Scholar 

  • Carrière Y, Fabrick JA, Tabashnik BE (2016) Can pyramids and seed mixtures delay resistance to Bt crops? Trends Biotechnol 34:291–302

    Article  CAS  PubMed  Google Scholar 

  • Coleman AD, Wouters RH, Mugfor ST, Hogenhout SA (2015) Persistence and transgenerational effect of plant-mediated RNAi in aphids. J Exp Bot 66:541–548

    Article  CAS  PubMed  Google Scholar 

  • Coleman AD, Mugford ST, Hogenhout SA (2016) Silencing of aphid genes by dsRNA feeding from plants. In: Czosnek H, Ghanim M (eds) Management of insect pests to agriculture. Springer, Cham, pp 245–251

    Chapter  Google Scholar 

  • Douches DS, Maas D, Jastrzebski K, Chase RW (1996) Assessment of potato breeding progress in the USA over the last century. Crop Sci 36:1544–1552

    Article  Google Scholar 

  • Douglas CJ, Staneloni RJ, Rubin RA, Nester EW (1985) Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region. J Bacteriol 161:850–860

    CAS  PubMed  PubMed Central  Google Scholar 

  • FAOSTAT data (2017) http://www.fao.org/home/en/. Retrieved 30 July 2018

  • Ferro DN, Lyon SM (1991) Colorado potato beetle (Coleoptera: Chrysomelidae) larval mortality: operative effects of Bacillus thuringiensis subsp. San diego. J Econ Entomol 84:806–809

    Article  Google Scholar 

  • Flanders KL, Hawkes JG, Radcliffe EB, Lauer FI (1992) Insect resistance in potatoes: sources, evolutionary relationships, morphological and chemical defenses, and eco-geographical associations. Euphytica 61:83–111

    Article  CAS  Google Scholar 

  • Frary A, Earle ED (1996) An examination of factors affecting the efficiency of Agrobacterium-mediated transformation of tomato. Plant Cell Rep 16:235–240

    CAS  PubMed  Google Scholar 

  • Gassmann AJ, Petzold-Maxwell JL, Keweshan RS, Dunbar MW (2011) Field evolved resistance to Bt maize by western corn rootworm. PLoS ONE 6:e22629

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gould F, Brown ZS, Kuzma J (2018) Wicked evolution: Can we address the sociobiological dilemma of pesticide resistance? Science 360(6390):728–732

    Article  CAS  PubMed  Google Scholar 

  • Guo P, Lee TJ (2007) Viral nanomotors for packaging of dsDNA and dsRNA. Mol Microbiol 64:886–903

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hameed A, Tahir MN, Asad S, Bilal R, Van Eck J, Jander G, Mansoor S (2017) RNAi-mediated simultaneous resistance against three RNA viruses in potato. Molecular Biotechnol 59:73–83

    Article  CAS  Google Scholar 

  • Hopkins P (2009) Crustacean ecdysteroids and their receptors. In: Smagghe G (ed) Ecdysone: structures and functions. Springer, pp 73–97

    Chapter  Google Scholar 

  • Jin S, Singh ND, Li L, Zhang X, Daniell H (2015) Engineered chloroplast dsRNA silences cytochrome p450 monooxygenase, V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa armigera larval development and pupation. Plant Biotechnol J 13:435–446

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kumar P, Pandit SS, Baldwin IT (2012) Tobacco rattle virus vector: a rapid and transient means of silencing Manduca sexta genes by plant mediated RNA interference. PLoS ONE 7:e31347

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • LeBlanc GA (2007) Crustacean endocrine toxicology: a review. Ecotoxicol 16:61–81

    Article  CAS  Google Scholar 

  • Li Y, Wang K, Lu Q, Du J, Wang Z, Wang D, Sun B, Li H (2017) Transgenic Nicotiana benthamiana plants expressing a hairpin RNAi construct of a nematode Rs-cps gene exhibit enhanced resistance to Radopholus similis. Sci Rep 7:13126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lindbo JA, Dougherty WG (2005) Plant pathology and RNAi: a brief history. Annu Rev Phytopathol 43:191–204

    Article  CAS  PubMed  Google Scholar 

  • Liu F, Wang XD, Zhao YY, Li YJ, Liu YC, Sun J (2015) Silencing the HaAK gene by transgenic plant-mediated RNAi impairs larval growth of Helicoverpa armigera. Int J Biol Sci 11:67–74

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real time quantitative PCR and the 2ΔΔC(T) Method. Methods 25:402–408

    Article  CAS  Google Scholar 

  • Mamta B, Rajam MV (2017) RNAi technology: a new platform for crop pest control. Physiology and Molecular Biology of Plants 23(3):487–501

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mamta B, Reddy KRK, Rajam MV (2015) Targeting chitinase gene of Helicoverpa armigera by host-induced RNA interference confers insect resistance in tobacco and tomato. Plant Mol Biol 90:281–292

    Article  CAS  PubMed  Google Scholar 

  • Mansoor S, Amin I, Hussain M, Zafar Y, Briddon RW (2006) Engineering novel traits in plants through RNA interference. Trends Plant Sci 11:559–565

    Article  CAS  PubMed  Google Scholar 

  • Mao J, Zeng F (2014) Plant-mediated RNAi of a gap gene-enhanced tobacco tolerance against the Myzus persicae. Transgenic Res 23:389–396

    Article  CAS  Google Scholar 

  • Mao YB, Cai WJ, Wang JW, Hong GJ, Tao XY (2007) Silencing a cotton bollworm P450 monooxygenase gene by plantmediated RNAi impairs larval tolerance of gossypol. Nat Biotechnol 25:1307–1313

    Article  CAS  PubMed  Google Scholar 

  • Mao YB, Tao XY, Xue XY, Wang LJ, Chen XY (2011) Cotton plants expressing CYP6AE14 double-stranded RNA show enhanced resistance to bollworms. Transgenic Res 20:665–673

    Article  CAS  PubMed  Google Scholar 

  • Michaud D, Bernier-Vadnais N, Overney S, Yelle S (1995) Constitutive expression of digestive cysteine proteinase forms during development of the Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Insect Bioch Mole Biol 25:1041–1048

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Nain V, Jaiswal R, Dalal M, Ramesh B, Kumar PA (2005) Polymerase chain reaction analysis of transgenic plants contaminated by Agrobacterium. Plant Mol Biol Report 23:59–65

    Article  CAS  Google Scholar 

  • Nakagawa Y, Henrich VC (2009) Arthropod nuclear receptors and their role in molting. FEBS J 276:6128–6157

    Article  CAS  PubMed  Google Scholar 

  • Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Botany 56:2907–2914

    Article  CAS  Google Scholar 

  • Oerke EC (2006) Crop losses to pests. J Agric Sci 144:31–43

    Article  Google Scholar 

  • Pitino M, Coleman AD, Maffei ME, Ridout CJ, Hogenhout SA (2011) Silencing of aphid genes by dsRNA feeding from plants. PLoS ONE 6(10):e25709

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rao AQ, Bakhsh A, Kiani S, Shahzad K, Shahid AA, Husnain T, Riazuddin S (2009) The myth of plant transformation. Biotechnol Adv 27:753–763

    Article  PubMed  Google Scholar 

  • Sambrook J, Russell DW, Maniatis T (2001) Molecular cloning, vol 1-3. Cold Spring Habour Laboratory Press, New York

    Google Scholar 

  • Schwenke RA, Lazzaro BP, Wolfner MF (2016) Reproduction–immunity trade-offs in insects. Annu Rev Entomol 61:239–256

    Article  CAS  Google Scholar 

  • Sehnal F (1989) Hormonal role of ecdysteroids in insect larvae and during metamorphosis. In: Koolman J (ed) Ecdysone. From chemistry to mode of action. Georg thieme Verlag, Stuttgart, pp 271–278

    Google Scholar 

  • Soto N, Enríquez GA, Ferreira A, Corrada M, Fuentes A, Tiel K, Pujol M (2007) Efficient transformation of potato stems segments from cultivar Désirée using phosphinothricin as selection marker. Biotecnol Apl 24:139–144

    Google Scholar 

  • Southern EM (1975) Detection of Specific sequence among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517

    Article  CAS  Google Scholar 

  • Swift ML (1997) GraphPad prism, data analysis, and scientific graphing. J Chem Inf Comput Sci 37:411–412

    Article  CAS  Google Scholar 

  • Tabashnik BE, Carrière Y (2017) Surge in insect resistance to transgenic crops and prospects for sustainability. Nat Biotechnol 35:926–935

    Article  CAS  PubMed  Google Scholar 

  • Tabashnik BE, Brévault T, Carrière Y (2013) Insect resistance to Bt crops: lessons from the first billion acres. Nat Biotechnol 31:510–521

    Article  CAS  PubMed  Google Scholar 

  • Upadhyay SK, Chandrashekar K, Thakur N, Verma PC, Borgio JF, Singh PK, Tuli R (2011) RNA interference for the control of whiteflies (Bemisia tabaci) by oral route. J Bio 36:153–161

    CAS  Google Scholar 

  • Van den Berg J, Hilbeck A, Bøhn T (2013) Pest resistance to Cry 1Ab Bt maize: field resistance, contributing factors and lessons from South Africa. Crop Prot 54:154–160

    Article  Google Scholar 

  • Van Eck J (2018) Genome editing and plant transformation of solanaceous food crops. Curr Opin Biotechnol 49:35–41

    Article  CAS  PubMed  Google Scholar 

  • Veale MA, Slabbert MM, Van Emmenes L (2012) Agrobacterium-mediated transformation of potato cv. Mnandi for resistance to the potato tuber moth (Phthorimaea operculella). S Afr J Bot 80:7–74

    Article  CAS  Google Scholar 

  • Wang B, Ma Y, Zhang Z, Wu Z, Wu Y, Wang Q, Li M (2011) Potato viruses in China. Crop Prot 30:1117–1123

    Article  Google Scholar 

  • Wuriyanghan H, Rosa C, Falk BW (2011) Oral delivery of double-stranded RNAs and siRNAs induces RNAi effects in the potato/tomato psyllid. Bactericerca cockerelli. PloS one 6:27736

    Article  CAS  Google Scholar 

  • Xiong YH, Zeng HM, Zhang YL, Xu DW, Qiu DW (2013) Silencing the HaHR3 gene by transgenic plant-mediated RNAi to disrupt Helicoverpa armigera development. Int J Biol Sci 9:370–381

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu L, Duan X, Lv Y, Zhang X, Nie Z, Xie C, Ni Z, Liang R (2014) Silencing of an aphid carboxylesterase gene by use of plant-mediated RNAi impairs Sitobion avenae tolerance of Phoxim insecticides. Transgenic Res 23:389–396

    Article  CAS  PubMed  Google Scholar 

  • Yan P, Shen W, Gao X, Li X, Zhou P, Duan J (2012) High-throughput construction of intron-containing hairpin RNA vectors for RNAi in plants. PLoS ONE 7:e38186

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yang C, Pan H, Noland JE, Zhang D, Zhang Z, Liu Y, Zhou X (2015) Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae). Scientific reports 5:18201

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yao J, Rotenberg D, Afsharifar A, Barandoc-Alviar K, Whitfield AE (2013) Development of RNAi Methods for Peregrinus maidis, the Corn Planthopper. PLoS ONE 8:e70243

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yu R, Xu X, Liang Y, Tian H, Pan Z, Jin S, Wang N, Zhang W (2014) The insect ecdysone receptor is a good potential target for RNAi-based pest control. Int J Biol Sci 10:1171

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zha W, Peng X, Chen R, Du B, Zhu L (2011) Knockdown of midgut genes by dsRNA-transgenic plant-mediated RNA interference in the hemipteran insect Nilaparvata lugens. PLoS ONE 6:e20504

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang H, Yin W, Zhao J, Jin L, Yang Y (2011) Early warning of cotton bollworm resistance associated with intensive planting of Bt cotton in China. PLoS ONE 6:e22874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang J, Khan SA, Heckel DG, Bock R (2017) Next-generation insect-resistant plants: RNAi-mediated crop protection. Trends Biotechnol 35(9):871–882

    Article  CAS  PubMed  Google Scholar 

  • Zhu F, Xu JJ, Palli R, Ferguson J, Palli SR (2011) Ingested RNA: interference for managing the populations of the Colorado potato beetle (Leptinotarsa decemlineata). Pest Manag Sci 67:175–182

    Article  CAS  PubMed  Google Scholar 

  • Zhu JQ, Liu S, Ma J, Zhang JQ, Qi HS, Wei ZJ, Yao Q, Zhang WQ, Li S (2012) Improvement of pest resistance in transgenic tobacco plants expressing dsRNA of an insect-associated gene EcR. PLoS ONE 7:e38572

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The present research project was supported by “The Scientific and Technological Council of Turkey” (Tübitak, Project No. 215O520). The authors are highly thankful to Tübitak for providing PhD fellowship to Ms. Tahira Hussain from the project budget. Authors acknowledge the contributions of Prof. Dr. Ayhan GÖKÇE, Muhammad Saleem and Mr. Nadir Naqqash for allowing us to use facilities of entomology laboratory in Department of Plant Production and Technologies.

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AB designed the study and secured funding for the project. TH constructed recombinant vectors, optimized transformation protocol for potato cultivars, conducted leaf bioassays. AB, EA and TH interpreted the results, and prepared the manuscript. AB and MEÇ supervised overall activities of the work.

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Correspondence to Allah Bakhsh.

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Hussain, T., Aksoy, E., Çalışkan, M.E. et al. Transgenic potato lines expressing hairpin RNAi construct of molting-associated EcR gene exhibit enhanced resistance against Colorado potato beetle (Leptinotarsa decemlineata, Say). Transgenic Res 28, 151–164 (2019). https://doi.org/10.1007/s11248-018-0109-7

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