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Expression of the entomotoxic Cocculus hirsutus trypsin inhibitor (ChTI) gene in transgenic chickpea enhances its underlying resistance against the infestation of Helicoverpa armigera and Spodoptera litura

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Abstract

A trypsin inhibitor from Cocculus hirsutus, commonly known as “Farid Buti” has been demonstrated to exhibit insecticidal, fungicidal, as well as nematocidal activity. The ChTI (Cocculus hirsutus Trypsin Inhibitor) gene was designed in silico and synthesized by PCR-based gene synthesis and cloned in the plant expression vector pBI121, with kanamycin, as the selectable marker. Agrobacterium strain LBA4404 was transformed with pBI121:ChTI vector for plant transformation. For develo** insect-tolerant chickpea, Agrobacterium-mediated transformation of ChTI gene was performed in cultivar P-362. Twenty-day-old cotyledonary node (CN) explants were used for sonication-assisted Agrobacterium-mediated transformation. Three cycles of increasing concentrations of kanamycin were used for the selection of transformed shoots. In vitro grown transgenic chickpea shoots were grafted on decapitated stock of chickpea seedlings. After 45–50 days of acclimatization and hardening, pod development and its maturation occurred. After screening by PCR, seven transgenic events were confirmed to be positive by Southern blot hybridization analysis, showing 1–4 copies of the transgene. The quantitative expression of the ChTI gene by qRT-PCR analysis showed up to 12–17-fold change in the T1 progeny. Immunoblot analysis revealed the expression of 31 kDa and 15 kDa ChTI protein in E.coli and transgenic plants respectively. Trypsin activity assay was performed in the T1 generation and higher anti-trypsin activity was recorded. Insect tolerance against Helicoverpa armigera and Spodoptera litura were estimated by insect bioassay, wherein an overall mortality of 60–80% and weight loss (30–60% and 40–60% for Spodoptera litura and Helicoverpa armigera respectively) have been recorded in the plants of T1 generation.

Key Message

Expression of Cocculus hirsutus trypsin inhibitor (ChTI) gene in chickpea by Agrobacterium-mediated transformation, restricted the growth as well as the survival of two Lepidopteran insect pests Helicoverpa armigera and Spodoptera litura.

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References

  • Acharjee S, Sarmah BK (2013) Biotechnologically generating ‘super chickpea’for food and nutritional security. Plant Sci 207:108–116

    Article  CAS  PubMed  Google Scholar 

  • Acharjee S, Sarmah BK, Kumar PA, Olsen K, Mahon R, Moar WJ, Moore A, Higgins T (2010) Transgenic chickpeas (Cicer arietinum L.) expressing a sequence-modified cry2Aa gene. Plant Sci 178:333–339

    Article  CAS  Google Scholar 

  • Altpeter F, Diaz I, McAuslane H, Gaddour K, Carbonero P, Vasil IK (1999) Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor CMe. Mol Breed 5(1):53–63

    Article  CAS  Google Scholar 

  • Asharani B, Ganeshaiah K, Kumar A, Udayakumar M (2011) Transformation of chickpea lines with Cry1X using in planta transformation and characterization of putative transformants T1 lines for molecular and biochemical characters. J Plant Breed Crop Sci 3:413–423

    Article  CAS  Google Scholar 

  • Bhattacharjee C, Manjunath NH, Prasad DT (2009) Purification of a trypsin inhibitor from Cocculus hirsutus and identification of its biological activity. J Crop Sci Biotechnol 12:253–260

    Article  Google Scholar 

  • Carrière Y, Crickmore N, Tabashnik BE (2015) Optimizing pyramided transgenic Bt crops for sustainable pest management. Nat Biotechnol 33:161

    Article  PubMed  Google Scholar 

  • Chakraborti D, Sarkar A, Mondal HA, Das S (2009) Tissue specific expression of potent insecticidal, Allium sativum leaf agglutinin (ASAL) in important pulse crop, chickpea (Cicer arietinum L.) to resist the phloem feeding Aphis craccivora. Transgenic Res 18:529–544

    Article  CAS  PubMed  Google Scholar 

  • Dixit G, Srivastava A, Singh N (2019) Marching towards self-sufficiency in chickpea. Curr Sci 116:239–242

    Article  Google Scholar 

  • Dunse KM, Stevens JA, Lay FT, Gaspar YM, Heath RL, Anderson MA (2010) Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection against insect damage in the field. Proc Natl Acad Sci USA 107(34):15011–15015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Erb M, Reymond P (2019) Molecular interactions between plants and insect herbivores. Annu Rev Plant Biol 70:527–557

    Article  CAS  PubMed  Google Scholar 

  • Ganguly M, Molla KA, Karmakar S, Datta K, Datta SK (2014) Development of pod borer-resistant transgenic chickpea using a pod-specific and a constitutive promoter-driven fused cry1Ab/Ac gene. Theor Appl Genet 127:2555–2565

    Article  CAS  PubMed  Google Scholar 

  • Gassmann AJ, Petzold-Maxwell JL, Clifton EH, Dunbar MW, Hoffmann AM, Ingber DA, Keweshan RS (2014) Field-evolved resistance by western corn rootworm to multiple Bacillus thuringiensis toxins in transgenic maize. Proc Natl Acad Sci USA 111:5141–5146

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gatehouse AM, Shi Y, Powell KS, Brough C, Hilder VA, Hamilton WD, Newell CA, Merryweather A, Boulter D, Gatehouse JA (1993) Approaches to insect resistance using transgenic plants. Philos Trans R Soc Lond Ser B Biol Sci 342:279–286

    Article  Google Scholar 

  • Gowri Neelima M, Ramu S, Sreevathsa R, Rani A, Kumar A, Gayatri M (2008) In planta transformation strategy to generate transgenic plants in chickpea: proof of concept with a cry gene. J Plant Biol (New Delhi, India) 35:201–206

    Google Scholar 

  • Gupta VK, Kaushik A, Chauhan DS, Ahirwar RK, Sharma S, Bisht D (2018) Anti-mycobacterial activity of some medicinal plants used traditionally by tribes from Madhya Pradesh, India for treating tuberculosis related symptoms. J Ethnopharmacol 227:113–120

    Article  PubMed  Google Scholar 

  • Hamza R, Pérez-Hedo M, Urbaneja A, Rambla JL, Granell A, Gaddour K, Beltrán JP, Cañas LA (2018) Expression of two barley proteinase inhibitors in tomato promotes endogenous defensive response and enhances resistance to Tuta absoluta. BMC Plant Biol 18(1):1–14

    Article  Google Scholar 

  • Hilder VA, Gatehouse AM, Sheerman SE, Barker RF, Boulter D (1987) A novel mechanism of insect resistance engineered into tobacco. Nature 330:160

    Article  CAS  Google Scholar 

  • Ignacimuthu S, Prakash S (2006) Agrobacterium-mediated transformation of chickpea with α-amylase inhibitor gene for insect resistance. J Biosci 31:339–345

    Article  CAS  PubMed  Google Scholar 

  • Kansal R, Kumar M, Kuhar K, Gupta RN, Subrahmanyam B, Koundal KR, Gupta VK (2008) Purification and characterization of trypsin inhibitor from Cicer arietinum L. and its efficacy against Helicoverpa armigera. Braz J Plant Physiol 20:313–322

    Article  Google Scholar 

  • Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10:845

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Khatodia S (2017) Helicoverpa resistant chickpea plants: from Bt toxins to plant-mediated RNAi. Ekin Journal of Crop Breeding and Genetics 3:52–60

    Google Scholar 

  • Khatodia S, Kharb P, Batra P, Chowdhury VK (2014) Development and characterization of transgenic chickpea (Cicer arietinum L.) plants with cry1Ac gene using tissue culture independent protocol. Int J Adv Res 2:323–331

    Google Scholar 

  • Letunic I, Bork P (2017) 20 years of the SMART protein domain annotation resource. Nucleic Acids Res 46:D493–D496

    Article  PubMed Central  Google Scholar 

  • Luo M, Ding LW, Ge ZJ, Wang ZY, Hu BL, Yang XB, Sun QY, Xu ZF (2012) The characterization of SaPIN2b, a plant trichome-localized proteinase inhibitor from Solanum americanum. Int J Mol Sci 13(11):15162–15176

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Madeira F, Ym Park, Lee J, Buso N, Gur T, Madhusoodanan N, Basutkar P, Tivey ARN, Potter SC, Finn RD, Lopez R (2019) The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res 47:W636–W641

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Manushree V, Devaraj V, Prasad D (2020) Expression of Cocculus hirsutus trypsin inhibitor promotes endogenous defensive response against Helicoverpa armigera and enhanced levels of antioxidants. Afr J Plant Sci 14:65–82

    Article  CAS  Google Scholar 

  • Mehrotra M, Singh AK, Sanyal I, Altosaar I, Amla D (2011) Pyramiding of modified cry1Ab and cry1Ac genes of Bacillus thuringiensis in transgenic chickpea (Cicer arietinum L.) for improved resistance to pod borer insect Helicoverpa armigera. Euphytica 182:87

    Article  CAS  Google Scholar 

  • Nair M, Sandhu SS, Babbar A (2013) Purification of trypsin inhibitor from seeds of Cicer arietinum (L.) and its insecticidal potential against Helicoverpa armigera (Hübner). Theor Exp Plant Physiol 25:137–148

    Article  Google Scholar 

  • Patil SB, Goyal A, Chitgupekar SS, Kumar S, El-Bouhssini M (2017) Sustainable management of chickpea pod borer: a review. Agron Sustain Dev 37:20

    Article  Google Scholar 

  • Perlak FJ, Oppenhuizen M, Gustafson K, Voth R, Sivasupramaniam S, Heering D, Carey B, Ihrig RA, Roberts JK (2001) Development and commercial use of Bollgard® cotton in the USA—early promises versus today’s reality. Plant J 27:489–501

    Article  CAS  PubMed  Google Scholar 

  • ProtParam E (2017) ExPASy-ProtParam tool

  • Rakesh T, Prashant T (2012) Effects of Cocculus hirsutus leaves extract on Freund’s complete adjuvant and formaldehyde-induced arthritis. Int Res J Pharm 3:267–270

    Google Scholar 

  • Robles L, Vaziri ND, Li S, Takasu C, Masuda Y, Vo K, Farzaneh SH, Stamos MJ, Ichii H (2015) Dimethyl fumarate ameliorates acute pancreatitis in rodent. Pancreas 44:441–447

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Roy A, Yang J, Zhang Y (2012) COFACTOR: an accurate comparative algorithm for structure-based protein function annotation. Nucleic Acids Res 40:W471–W477

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Sanahuja G, Banakar R, Twyman RM, Capell T, Christou P (2011) Bacillus thuringiensis: a century of research, development and commercial applications. Plant Biotech J 9:283–300

    Article  CAS  PubMed  Google Scholar 

  • Sane VA, Nath P, Sane A, Sane PV (1997) Development of insect-resistant transgenic plants using plant genes: expression of cowpea trypsin inhibitor in transgenic tobacco plants. Curr Sci 72(10):741–747

    CAS  Google Scholar 

  • Sanyal I, Amla D (2008) Genetic transformation of chickpea (Cicer arietinum L) using cotyledonary node explants. Handbook of new technologies for genetic improvement of legumes. The Haworth Press, Taylor & Francis Group, Boca Raton, pp 147–158

  • Sanyal I, Singh A, Amla D (2003) Agrobacterium tumefaciens-mediated transformation of chickpea (Cicer arietinum L.) using mature embryonic axes and cotyledonary nodes. Indian J Biotechnol 2:524–532

    CAS  Google Scholar 

  • Sanyal I, Singh AK, Kaushik M, Amla DV (2005) Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) with Bacillus thuringiensis cry1Ac gene for resistance against pod borer insect Helicoverpa armigera. Plant Sci 168:1135–1146

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Thavamani BS, Mathew M, Palaniswamy DS (2014) Anticancer activity of Cocculus hirsutus against Dalton’s lymphoma ascites (DLA) cells in mice. Pharm Biol 52:867–872

    Article  PubMed  Google Scholar 

  • Tripathi L, Singh AK, Singh S, Singh R, Chaudhary S, Sanyal I, Amla DV (2013) Optimization of regeneration and Agrobacterium-mediated transformation of immature cotyledons of chickpea (Cicer arietinum L.). Plant Cell Tiss Org Cult (PCTOC) 113:513–527

    Article  CAS  Google Scholar 

  • Yamasaki A, Shimizu K, Fujisaki K (2009) Effect of host plant part on larval body-color polymorphism in Helicoverpa armigera (Lepidoptera: Noctuidae). Ann Entomol Soc Am 102:76–84

    Article  Google Scholar 

  • Yang J, Zhang Y (2015) I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res 43:W174–W181

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang Y (2009) I-TASSER: fully automated protein structure prediction in CASP8. Proteins Struct Funct Bioinform 77:100–113

    Article  CAS  Google Scholar 

  • Zhu-Salzman K, Zeng R (2015) Insect response to plant defensive protease inhibitors. Ann Rev Entomol 60:233–252

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors are grateful to the Director, CSIR-NBRI, Lucknow, for infrastructural support. The authors are grateful for fellowships to UGC, New Delhi (AP, SK), CSIR, New Delhi (RY, AK, PS), and DST (AY), New Delhi. The research work was carried out under the CSIR-NBRI In-house project OLP104. NBRI manuscript no. CSIR-NBRI_MS/2021/02/06.

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Authors and Affiliations

  1. CSIR-National Botanical Research Institute, Lucknow, 226001, India

    Ankesh Pandey, Reena Yadav, Sanoj Kumar, Anil Kumar, Priya Shukla, Ankita Yadav & Indraneel Sanyal

  2. Department of Biotechnology, Kumaun University, Bhimtal Campus, Nainital, 263001, India

    Reena Yadav & Anil Kumar

  3. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India

    Ankesh Pandey & Indraneel Sanyal

  4. Institute of Environment and Sustainable Development, Banaras Hindu University, Varanasi, 221005, India

    Sanoj Kumar

  5. Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, 221005, India

    Ankita Yadav

  6. Molecular Biology and Biotechnology Division, CSIR-National Botanical Research Institute, Lucknow, India

    Indraneel Sanyal

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  1. Ankesh Pandey
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  2. Reena Yadav
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Contributions

Conceptualization: AP, RY & IS, Formal analysis: AP, AK, RY, Funding acquisition: IS, Investigation: AP, RY, AK, SK, PS, AY, Methodology: AP & RY, Project administration: IS, Resources: IS, Supervision: IS, Validation: AP, Writing - original draft: AP, Writing - review & editing: IS and AP.

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Correspondence to Indraneel Sanyal.

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Communicated by Sergio J. Ochatt.

The original version of this article has been revised: The following missing sections have been included: Acknowledgements, Author contributions, and Declaration.

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Pandey, A., Yadav, R., Kumar, S. et al. Expression of the entomotoxic Cocculus hirsutus trypsin inhibitor (ChTI) gene in transgenic chickpea enhances its underlying resistance against the infestation of Helicoverpa armigera and Spodoptera litura. Plant Cell Tiss Organ Cult 146, 41–56 (2021). https://doi.org/10.1007/s11240-021-02041-2

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