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Plant Cell Reports

, Volume 36, Issue 1, pp 193–201 | Cite as

Transgenic sugarcane overexpressing CaneCPI-1 negatively affects the growth and development of the sugarcane weevil Sphenophorus levis

  • Vanessa Karine Schneider
  • Andrea Soares-Costa
  • Mohan Chakravarthi
  • Carolina Ribeiro
  • Sabrina Moutinho Chabregas
  • Maria Cristina Falco
  • Flavio Henrique-SilvaEmail author
Original Article

Abstract

Key message

Transgenic sugarcane expressing CaneCPI-1 exhibits resistance to Sphenophorus levis larvae.

Abstract

Transgenic plants have widely been used to improve resistance against insect attack. Sugarcane is an economically important crop; however, great losses are caused by insect attack. Sphenophorus levis is a sugarcane weevil that digs tunnels in the stem base, leading to the destruction of the crop. This insect is controlled inefficiently by chemical insecticides. Transgenic plants expressing peptidase inhibitors represent an important strategy for impairing insect growth and development. Knowledge of the major peptidase group present in the insect gut is critical when choosing the most effective inhibitor. S. levis larvae use cysteine peptidases as their major digestive enzymes, primarily cathepsin L-like activity. In this study, we developed transgenic sugarcane plants that overexpress sugarcane cysteine peptidase inhibitor 1 (CaneCPI-1) and assessed their potential through feeding bioassays with S. levis larvae. Cystatin overexpression in the transgenic plants was evaluated using semi-quantitative RT-PCR, RT-qPCR, and immunoblot assays. A 50% reduction of the average weight was observed in larvae that fed on transgenic plants in comparison to larvae that fed on non-transgenic plants. In addition, transgenic sugarcane exhibited less damage caused by larval attack than the controls. Our results suggest that the overexpression of CaneCPI-1 in sugarcane is a promising strategy for improving resistance against this insect.

Keywords

Cysteine peptidase Cystatin Feeding bioassay Sugarcane Sugarcane weevil Sphenophorus levis 

Notes

Acknowledgements

The research was supported by the São Paulo Research Foundation (FAPESP, CBME, CEPID Proc. 98/14138-2). The authors thank Centro de Tecnologia Canavieira, Piracicaba, São Paulo, Brazil, for essential help in sugarcane transformation. FHS is recipient of a productivity scholarship from National Council for Scientific and Technological Development (CNPq) (##311745/2013-0). V.K.S. and A.S.C.F. received a Grant from FAPESP (2013/05370-0 and 2005/59833-5, respectively), and C.W.R. received a Grant from National Council for Scientific and Technological Development (CNPq).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Álvarez-Alfageme F, Martínez M, Pascual-Ruiz S, Castañera P, Diaz I, Ortego F (2007) Effects of potato plants expressing a barley cystatin on the predatory bug Podisus maculiventris via herbivorous prey feeding on the plant. Transgenic Res 16:1–13CrossRefPubMedGoogle Scholar
  2. Broadway RM, Duffey SS (1986) Plant proteinase inhibitor: mechanism of action and effect on the growth and digestive physiology of larval Heliothis zea and Spodoptera exigua. J Insect Physiol 32:827–833CrossRefGoogle Scholar
  3. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622CrossRefPubMedGoogle Scholar
  4. Campos FA, Xavier-Filho J, Silva CP, Ary MB (1989) Resolution and partial characterization of proteinases and α-amylases from midgets of larvae of the bruchid beetle Callosobruchus maculates (F.). Comp Biochem Physiol 92:51–57Google Scholar
  5. Carrillo L, Martinez M, Álvarez-Alfageme F, Castañera P, Smagghe G, Diaz I, Ortego F (2011) A barley cysteine-proteinase inhibitor reduces the performance of two aphid species in artificial diets and transgenic Arabidopsis plants. Transgenic Res 20:305–319CrossRefPubMedGoogle Scholar
  6. Chen P, Senthilkumar R, Jane W, He Y, Tian Z, Yeh K (2014) Transplastomic Nicotiana benthamiana plants expressing multiple defence genes encoding protease inhibitors and chitinase display broad-spectrum resistance against insects, pathogens and abiotic stresses. Plant Biotechnol J 12(4):503–515CrossRefPubMedGoogle Scholar
  7. Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5:213–218CrossRefPubMedGoogle Scholar
  8. Companhia Nacional de Abastecimento—CONAB (2016) Acompanhamento da Safra Brasileira de Cana-de-açúcar—Safra 2015/16—Quarto Levantamento. http://www.conab.gov.br/OlalaCMS/uploads/arquivos/16_04_14_09_06_31_boletim_cana_portugues_-_4o_lev_-_15-16.pdf. Accessed on: 20/09/2016
  9. Cristofoletti PT, Ribeiro AF, Terra WR (2005) The cathepsin L-like proteinases from the midgut of Tenebrio molitor larvae: sequence, properties, immunocytochemical localization and function. Insect Biochem Mol Biol 35(8):883–901CrossRefPubMedGoogle Scholar
  10. Degaspari N, Botelho PSM, Dealmeida LC, Castilho HJ (1987) Biology of Sphenophorus levis, Vaurie, 1978 (Col, Curculionidae), with artificial diet and in the field. Pesquisa Agropecuaria Brasil 22:553–558Google Scholar
  11. Evangelista DE, Fonseca FPP, Rodrigues A, Henrique-Silva F (2015) Pectinases from Sphenophorus levis Vaurie, 1978 (Coleoptera: Curculionidae): putative accessory digestive enzymes. J Insect Sci 15:1–8CrossRefGoogle Scholar
  12. Fonseca FPP, Soares-Costa A, Ribeiro AF, Rosa JC, Terra WR, Henrique-Silva F (2012) Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase (Sl-CathL) from sugarcane weevil, Sphenophorus levis. Insect Biochem Mol Biol 42:58–69CrossRefPubMedGoogle Scholar
  13. Gallo DN, Neto OS, Carvalho S, Baptista RPL (2002) Entomologia agrícola. Fundação de Estudos Agrários Luiz de QueirozGoogle Scholar
  14. Gatehouse JA (2011) Prospects for using proteinase inhibitors to protect transgenic plants against attack by herbivorous insects. Curr Protein Pept Sci 12:409–416CrossRefPubMedGoogle Scholar
  15. Habib H, Fazili KM (2007) Plant protease inhibitors: a defense strategy in plants. Biotechnol Mol Biol Rev 2:68–85Google Scholar
  16. Haq SK, Atif SM, Khan RH (2004) Protein proteinase inhibitor genes in combat against insects, pests, and pathogens: natural and engineered phytoprotection. Arch Biochem Biophys 431:145–159CrossRefPubMedGoogle Scholar
  17. Jouanin L, Bonadé-bottino M, Girard C, Lerin J, Pham-delegue MH (2000) Expression of protease inhibitors in rapeseed. In: Michaud D (ed) Recombinant protease inhibitors in plants, pp 179–190Google Scholar
  18. Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: the emerging molecular analysis. Annu Rev Plant Biol 53:299–328CrossRefPubMedGoogle Scholar
  19. Klein TM, Harper EC, Svab Z, Sanford JC, Fromm ME, Maliga P (1988) Stable genetic transformation of intact Nicotiana cells by the particle bombardment process. Proc Natl Acad Sci USA 85:8502–8505CrossRefPubMedPubMedCentralGoogle Scholar
  20. Koiwa H, Bressan RA, Hasegawa PM (1997) Regulation of protease inhibitors and plant defense. Trends Plant Sci 2:379–384CrossRefGoogle Scholar
  21. Lawrence PK, Koundal KR (2002) Plant protease inhibitors in control of phytophagous insects. Electron J Biotechnol 5:93–109CrossRefGoogle Scholar
  22. Leplé JC, Bonadé-Bottino M, Augustin S, Pilate G, Lê Tân VD, Delplanque A, Cornu D, Jouanin L (1995) Toxicity tochrysomela tremulae (Coleoptera: Chrysomelidae) of transgenic poplars expressing a cysteine proteinase inhibitor. Mol Breed 1(4):319–328CrossRefGoogle Scholar
  23. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆Ct method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  24. Murdock LL, Brookhart G, Dunn PE, Foard DE, Kelley S (1987) Cysteine digestive proteinase in Coleoptera. Comp Biochem Physiol 87:783–787Google Scholar
  25. Ninković S, Miljuš-Ðukić J, Radović S, Maksimović V, Lazarević J, Vinterhalter B, Nešković M, Smigocki A (2007) Phytodecta fornicata Brüggemann resistance mediated by oryzacystatin II proteinase inhibitor transgene. Plant Cell Tissue Organ Cult 91:289–294CrossRefGoogle Scholar
  26. Nogueira F, Silva CP, Alexandre D, Samuels RI, Soares EL, Aragão FJ, Palmisano G, Domont GB, Roepstorff P, Campos FA (2012) Global proteome changes in larvae of Callosobruchus maculatus Coleoptera: Chrysomelidae: Bruchinae) following ingestion of a cysteine proteinase inhibitor. Proteomics 12(17):2704–2715CrossRefPubMedGoogle Scholar
  27. OECD (2016) Safety assessment of transgenic organisms in the environment, volume 6: OECD consensus documents, harmonisation of regulatory oversight in biotechnology. OECD Publishing, ParisGoogle Scholar
  28. Oliva MLV, Carmona AK, Andrade SS, Cotrin SS, Soares-Costa A, Henrique-Silva F (2004) Inhibitory selectivity of canecystatin: a recombinant cysteine peptidase inhibitor from sugarcane. Biochem Biophys Res Commun 320:1082–1086CrossRefPubMedGoogle Scholar
  29. Outchkourov NS, de Kogel WJ, Wiegers GL, Abrahamson M, Jongsma MA (2004) Engineered multidomain cysteine protease inhibitors yield resistance against western flower thrips (Franklinielia occidentalis) in greenhouse trials. Plant Biotechnol J 2:449–458CrossRefPubMedGoogle Scholar
  30. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30(9):e36CrossRefPubMedPubMedCentralGoogle Scholar
  31. Rahbé Y, Deraison C, Bonadé-Bottino M, Girard C, Nardon C, Lise Jouanin L (2003) Effects of the cysteine protease inhibitor oryzacystatin (OC-I) on different aphids and reduced performance of Myzus persicae on OC-I expressing transgenic oilseed rape. Plant Sci 164:441–450CrossRefGoogle Scholar
  32. Ribeiro AO, Pereira EJ, Galvan TL, Picanco MC, Picoli ET, Silva D, Fari MG, Otoni WC (2006) Effect of eggplant transformed with oryzacystatin gene on Myzus persicae and Macrosiphum euphorbiae. J Appl Entomol 130(2):84–90CrossRefGoogle Scholar
  33. Ribeiro CW, Soares-Costa A, Falco MC, Chabregas SM, Ulian EC, Cotrin SS, Carmona AK, Santana LA, Oliva ML, Henrique-Silva F (2008) Production of a His-tagged canecystatin in transgenic sugarcane and subsequent purification. Biotechnol Prog 24(5):1060–1066CrossRefPubMedGoogle Scholar
  34. Rozen S, Skaletsky H (1999) Primer3 on the WWW for general users and for biologist programmers. In: Stephen M, Stephen AK (eds) Bioinformatics methods and protocols, pp 365–386Google Scholar
  35. Ryan CA (1990) Proteinase inhibitors in plants: genes for improving defenses against insects and pathogens. Ann. Rev. Phytopathol 28:425–449CrossRefGoogle Scholar
  36. Santos F, Borém A, Caldas C (2015) Sugarcane: agricultural production, bioenergy and ethanol. Academic Press, LondonGoogle Scholar
  37. Soares-Costa A, Beltramini LM, Thiemann OH, Henrique-Silva FA (2002) sugarcane cystatin: recombinant expression, purification, and antifungal activity. Biochem Biophys Res Commun 296:1194–1199CrossRefPubMedGoogle Scholar
  38. Soares-Costa A, Dias AB, Dellamano M, Fonseca FPP, Carmona AK, Terra WR, Henrique-Silva F (2011) Digestive physiology and characterization of digestive cathepsin L-like proteinase from the sugarcane weevil Sphenophorus levis. J Insect Physiol 57:462–468CrossRefPubMedGoogle Scholar
  39. Terra WR, Cristofoletti PT (1996) Midgut proteinases in three divergent species of Coleoptera. Comp Biochem Physiol B Biochem Mol Biol 113(4):725–730CrossRefGoogle Scholar
  40. Terra WR, Ferreira C (1994) Insect digestive enzymes: properties, compartmentalization and function. Comp Biochem Physiol Part B Comp Biochem 109:1–62CrossRefGoogle Scholar
  41. Tribolium Genome Sequencing Consortium (2008) The genome of the model beetle and pest Tribolium castaneum. Nature 452:949–955CrossRefGoogle Scholar
  42. Vaurie P (1978) Revision of the genus Sphenophorus in South America. American Museum of Natural History, New YorkGoogle Scholar
  43. Vettore AL et al (2003) Analysis and functional annotation of an expressed sequence tag collection for tropical crop sugarcane. Genome Res 13(12):2725–2735CrossRefPubMedPubMedCentralGoogle Scholar
  44. Vorster J, Rasoolizadeh A, Goulet MC, Cloutier C, Sainsbury F, Michaud D (2015) Positive selection of digestive Cys proteases in herbivorous Coleoptera. Insect Biochem Mol Biol 65:10–19CrossRefPubMedGoogle Scholar
  45. Zhu-Salzman K, Zeng R (2015) Insect response to plant defensive protease inhibitors. Annu Rev Entomol 60:13.1–13.20CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Vanessa Karine Schneider
    • 1
  • Andrea Soares-Costa
    • 1
  • Mohan Chakravarthi
    • 1
  • Carolina Ribeiro
    • 1
  • Sabrina Moutinho Chabregas
    • 2
  • Maria Cristina Falco
    • 2
  • Flavio Henrique-Silva
    • 1
    Email author
  1. 1.Laboratory of Molecular Biology, Department of Genetics and EvolutionFederal University of São CarlosSão CarlosBrazil
  2. 2.Centro de Tecnologia CanavieiraPiracicabaBrazil

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