Applied Microbiology and Biotechnology

, Volume 99, Issue 17, pp 7209–7218 | Cite as

Assembling of Holotrichia parallela (dark black chafer) midgut tissue transcriptome and identification of midgut proteins that bind to Cry8Ea toxin from Bacillus thuringiensis

  • Changlong Shu
  • Shuqian Tan
  • Jiao Yin
  • Mario Soberón
  • Alejandra Bravo
  • Chunqing Liu
  • Lili Geng
  • Fuping Song
  • Kebin Li
  • Jie Zhang
Genomics, transcriptomics, proteomics


Holotrichia parallela is one of the most severe crop pests in China, affecting peanut, soybean, and sweet potato crops. Previous work showed that Cry8Ea toxin is highly effective against this insect. In order to identify Cry8Ea-binding proteins in the midgut cells of H. parallela larvae, we assembled a midgut tissue transcriptome by high-throughput sequencing and used this assembled transcriptome to identify Cry8Ea-binding proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, we obtained de novo sequences of cDNAs from midgut tissue of H. parallela larvae and used available cDNA data in the GenBank. In a parallel assay, we obtained 11 Cry8Ea-binding proteins by pull-down assays performed with midgut brush border membrane vesicles. Peptide sequences from these proteins were matched to the H. parallela newly assembled midgut transcriptome, and 10 proteins were identified. Some of the proteins were shown to be intracellular proteins forming part of the cell cytoskeleton and/or vesicle transport such as actin, myosin, clathrin, dynein, and tubulin among others. In addition, an apolipophorin, which is a protein involved in lipid metabolism, and a novel membrane-bound alanyl aminopeptidase were identified. Our results suggest that Cry8Ea-binding proteins could be different from those characterized for Cry1A toxins in lepidopteran insects.


Bacillus thuringiensis Cry8Ea Binding Holotrichia parallela Midgut proteins 



This study was supported by the National Natural Science Foundation of China (Nos. 31301731, 31428020); the National High Technology Research and Development Program of China (863 Program) (No. 2011AA10A203); and the National Science and Technology Major Project (No. 2014ZX08009-013B)

Compliance with ethical standards

The manuscript does not contain experiments using mammals and does not contain studies on humans.

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

253_2015_6755_MOESM1_ESM.pdf (5.8 mb)
ESM 1 (PDF 5951 kb)


  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new proteinration of protein database search programs. Nucleic Acids Res 25:3389–3402. doi: 10.1093/nar/25.17.3389 PubMedCentralCrossRefPubMedGoogle Scholar
  2. Bagg J, Banks S, Baute T, Bohner H, Brown C, Cowbrough M, Hall B, Hayes A, Johnson P, Martin H, McDonald I, Quesnel G, Reid K, Spieser H, Stewart G, Tenuta A, Verhallen A (2009) Insect pests of field crops. In: Brown C (ed) Agronomic guide for field crops. Queens Printers for Ontario, Toronto, pp 195–226, ISBN 978-1-4249-8198-4 Google Scholar
  3. Bedoya-Pérez LP, Cancino-Rodezno A, Flores-Escobar B, Soberón M, Bravo A (2013) Differential role of UPR pathway in defense response of Aedes aegypti against Cry11Aa toxin from Bacillus thuringiensis. Int J Mol Sci 14:8467–8478. doi: 10.3390/ijms14048467 PubMedCentralCrossRefPubMedGoogle Scholar
  4. Bi Y, Zhang Y, Shu C, Crickmore N, Wang Q, Du L, Song F, Zhang J (2015) Genomic sequencing identifies novel Bacillus thuringiensis Vip1/Vip2 binary and Cry8 toxins that have high toxicity to Scarabaeoidea larvae. Appl Microbiol Biotechnol 99:753–760. doi: 10.1007/s00253-014-5966-2 CrossRefPubMedGoogle Scholar
  5. Bischof LJ, Kao C-Y, Los FCO, Gonzalez MR, Shen Z, Briggs SP, van der Goot FG, Aroian RV (2008) Activation of the unfolded protein response is required for defenses against bacterial pore forming toxin in vivo. PLoS Pathog 4, e1000176. doi: 10.1371/journal.ppat.1000176 PubMedCentralCrossRefPubMedGoogle Scholar
  6. Bravo A, Gomez I, Conde J, Munoz-Garay C, Sanchez J, Miranda R, Zhuang M, Gill SS, Soberon M (2004) Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochim Biophys Acta 1667:38–46. doi: 10.1016/j.bbamem.2004.08.013 CrossRefPubMedGoogle Scholar
  7. Bravo A, Gomez I, Porta H, Garcia-Gomez BI, Rodriguez-Almazan C, Pardo L, Soberon M (2013) Evolution of Bacillus thuringiensis Cry toxins insecticidal activity. Microb Biotechnol 6:17–26. doi: 10.1111/j.1751-7915.2012.00342.x PubMedCentralCrossRefPubMedGoogle Scholar
  8. Cancino-Rodezno A, Lozano L, Oppert C, Castro JI, Lanz-Mendoza H, Encarnacion S, Evans AE, Gill SS, Soberon M, Jurat-Fuentes JL, Bravo A (2012) Comparative proteomic analysis of Aedes aegypti larval midgut after intoxication with Cry11Aa toxin from Bacillus thuringiensis. PLoS One 7, e37034. doi: 10.1371/journal.pone.0037034 PubMedCentralCrossRefPubMedGoogle Scholar
  9. Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676. doi: 10.1093/bioinformatics/bti610 CrossRefPubMedGoogle Scholar
  10. Cooper GM (2000) The cell: a molecular approach, 2nd edn. Sunderland Associates, Sunderland, ISBN-10: 0-87893-106-6 Google Scholar
  11. de Maagd RA, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends proteint 17:193–199. doi: 10.1016/S0168-9525(01)02237-5 Google Scholar
  12. Fernandez LE, Aimanova KG, Gill SS, Bravo A, Soberon M (2006) A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae. Biochem J 394:77–84. doi: 10.1042/BJ20051517 PubMedCentralCrossRefPubMedGoogle Scholar
  13. Gong Y, Wang C, Yang Y, Wu S, Wu Y (2010) Characterization of resistance to Bacillus thuringiensis toxin Cry1Ac in Plutella xylostella from China. J Invertebr Pathol 104:90–96. doi: 10.1016/j.jip.2010.02.003 CrossRefPubMedGoogle Scholar
  14. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652. doi: 10.1038/nbt.1883 PubMedCentralCrossRefPubMedGoogle Scholar
  15. Guo S, Ye S, Liu Y, Wei L, Xue J, Wu H, Song F, Zhang J, Wu X, Huang D, Rao Z (2009) Crystal structure of Bacillus thuringiensis Cry8Ea1: an insecticidal toxin toxic to underground pests, the larvae of Holotrichia parallela. J Struct Biol 168:259–266. doi: 10.1016/j.jsb.2009.07.004 CrossRefPubMedGoogle Scholar
  16. Gurcel L, Abrami L, Girardin S, Tschopp J, van der Goot FG (2006) Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival. Cell 126:1135–1145. doi: 10.1016/j.cell.2006.07.033 CrossRefPubMedGoogle Scholar
  17. Hua G, Park Y, Adang MJ (2014) Cadherin AdCad1 in Alphitobius diaperinus larvae is a receptor of Cry3Bb toxin from Bacillus thuringiensis. Insect Biochem Mol Biol 45:11–17. doi: 10.1016/j.ibmb.2013.10.007 CrossRefPubMedGoogle Scholar
  18. Husmann M, Beckmann E, Boller K, Kloft N, Tenzer S, Bobkiewicz W, Neukirch C, Bayley H, Bhakdi S (2009) Elimination of a bacterial pore-forming toxin by sequential endocytosis and exocytosis. FEBS Lett 583:337–344. doi: 10.1016/j.febslet.2008.12.028 CrossRefPubMedGoogle Scholar
  19. Idone V, Tam C, Goss JW, Toomre D, Pypaert M, Andrews NW (2008) Repair of injured plasma membrane by rapid Ca2+-dependent endocytosis. J Cell Biol 180:905–914. doi: 10.1083/jcb.200708010 PubMedCentralCrossRefPubMedGoogle Scholar
  20. Iseli C, Jonproteinel CV, Bucher P (1999) ESTScan: a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences. Proc Int Conf Intell Syst Mol Biol 99:138–148. doi: 10.1038/srep097805.08 Google Scholar
  21. James C (2014) Global status of commercialized biotech/GM crops: 2014. ISAAA Brief No. 49. ISAAA, Ithaca, NY, ISBN: 978-1-892456-59-1Google Scholar
  22. Jurat-Fuentes JL, Adang MJ (2004) Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae. Eur J Biochem 271:3127–3135. doi: 10.1111/j.1432-1033.2004.04238.x CrossRefPubMedGoogle Scholar
  23. Jurat-Fuentes JL, Gould FL, Adang MJ (2003) Dual resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa toxins in Heliothis virescens suggests multiple mechanisms of resistance. Appl Environ Microbiol 69:5898–5906. doi: 10.1128/AEM.69.10.5898-5906.2003 PubMedCentralCrossRefPubMedGoogle Scholar
  24. Knight PJ, Carroll J, Ellar DJ (2004) Analysis of glycan structures on the 120 kDa aminopeptidase N of Manduca sexta and their interactions with Bacillus thuringiensis Cry1Ac toxin. Insect Biochem Mol Biol 34:101–112. doi: 10.1016/j.ibmb.2003.09.007 CrossRefPubMedGoogle Scholar
  25. Krishnamoorthy M, Jurat-Fuentes JL, McNall RJ, Andacht T, Adang MJ (2007) Identification of novel Cry1Ac binding proteins in midgut membranes from Heliothis virescens using proteomic analyses. Insect Biochem Mol Biol 37:189–201. doi: 10.1016/j.ibmb.2006.10.004 CrossRefPubMedGoogle Scholar
  26. Li X, Schuler MA, Berenbaum MR (2007) Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. Annu Rev Entomol 52:231–253. doi: 10.1146/annurev.ento.51.110104.151104 CrossRefPubMedGoogle Scholar
  27. Li H, Liu R, Shu C, Zhang Q, Zhao S, Shao G, Zhang X, Gao J (2014) Characterization of one novel cry8 protein from Bacillus thuringiensis strain Q52-7. World J Microbiol Biotechnol 30:3075–3080. doi: 10.1007/s11274-014-1734-9 CrossRefPubMedGoogle Scholar
  28. Likitvivatanavong S, Chen J, Bravo A, Soberon M, Gill SS (2011) Cadherin, alkaline phosphatase, and aminopeptidase N as receptors of Cry11Ba toxin from Bacillus thuringiensis subsp. jegathesan in Aedes aegypti. Appl Environ Microbiol 77:24–31. doi: 10.1128/AEM.01852-10 PubMedCentralCrossRefPubMedGoogle Scholar
  29. Los FC, Kao CY, Smitham J, McDonald KL, Ha C, Peixoto CA, Aroian RV (2011) RAB-5- and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin. Cell Host Microbe 9:147–157. doi: 10.1016/j.chom.2011.01.005 PubMedCentralCrossRefPubMedGoogle Scholar
  30. McNall RJ, Adang MJ (2003) Identification of novel Bacillus thuringiensis Cry1Ac binding proteins in Manduca sexta midgut through proteomic analysis. Insect Biochem Mol Biol 33:999–1010. doi: 10.1016/S0965-1748(03)00114-0 CrossRefPubMedGoogle Scholar
  31. Nagamatsu Y, Koike T, Sasaki K, Yoshimoto A, Furukawa Y (1999) The cadherin-like protein is essential to specificity determination and cytotoxic action of the Bacillus thuringiensis insecticidal CryIAa toxin. FEBS Lett 460:385–390. doi: 10.1016/S0014-5793(99)01327-7 CrossRefPubMedGoogle Scholar
  32. Pacheco S, Gomez I, Arenas I, Saab-Rincon G, Rodriguez-Almazan C, Gill SS, Bravo A, Soberon M (2009) Domain II loop 3 of Bacillus thuringiensis Cry1Ab toxin is involved in a “ping pong” binding mechanism with Manduca sexta aminopeptidase-N and cadherin receptors. J Biol Chem 284:32750–32757. doi: 10.1074/jbc.M109.024968 PubMedCentralCrossRefPubMedGoogle Scholar
  33. Pardo-Lopez L, Soberon M, Bravo A (2013) Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection. FEMS Microbiol Rev 37:3–22. doi: 10.1111/j.1574-6976.2012.00341.x CrossRefPubMedGoogle Scholar
  34. Pigott CR, Ellar DJ (2007) Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol Mol Biol Rev 71:255–281. doi: 10.1128/MMBR.00034-06 PubMedCentralCrossRefPubMedGoogle Scholar
  35. Raymond B, Johnston PR, Nielsen-LeRoux C, Lereclus D, Crickmore N (2010) Bacillus thuringiensis: an impotent pathogen? Trends Microbiol 18:189–194. doi: 10.1016/j.tim.2010.02.006 CrossRefPubMedGoogle Scholar
  36. Shu C, Yan G, Wang R, Zhang J, Feng S, Huang D, Song F (2009a) Characterization of a novel cry8 protein specific to Melolonthidae pests: Holotrichia oblita and Holotrichia parallela. Appl Microbiol Biotechnol 84:701–707. doi: 10.1007/s00253-009-1971-2 CrossRefPubMedGoogle Scholar
  37. Shu C, Yu H, Wang R, Fen S, Su X, Huang D, Zhang J, Song F (2009b) Characterization of two novel cry8 proteins from Bacillus thuringiensis strain BT185. Curr Microbiol 58:389–392. doi: 10.1007/s00284-008-9338-y CrossRefPubMedGoogle Scholar
  38. Soberon M, Gill SS, Bravo A (2009) Signaling versus punching hole: how do Bacillus thuringiensis toxins kill insect midgut cells? Cell Mol Life Sci 66:1337–1349. doi: 10.1007/s00018-008-8330-9 CrossRefPubMedGoogle Scholar
  39. Vachon V, Laprade R, Schwartz JL (2012) Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: a critical review. J Invertebr Pathol 111:1–12. doi: 10.1016/j.jip.2012.05.001 CrossRefPubMedGoogle Scholar
  40. Wightman JA, Ranga Rao GV (1994) Groundnut pest. In: Smartt J (ed) The groundnut crop: a scientific basis for improvement, Chapman and Hall, London ISBN: 0 412 408201, pp. 395–479Google Scholar
  41. Wolfersberger M, Lüthy P, Maurer A, Parenti P, Sacchi F, Giordana B, Hanozet G (1987) Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Comp Biochem Phys A 86:301–308. doi: 10.1016/0300-9629(87)90334-3 CrossRefGoogle Scholar
  42. Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L, Wang J (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34(Web Server issue):W293–W297. doi: 10.1093/nar/gkl031 PubMedCentralCrossRefPubMedGoogle Scholar
  43. Zdybicka-Barabas A, Cytrynska M (2013) Apolipophorins and insect immune response. Invertebrate Surviv J 10:58–68. doi: 10.1371/journal.pone.0015410 Google Scholar
  44. Zhang X, Candas M, Griko NB, Taussig R, Bulla LA Jr (2006) A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis. Proc Natl Acad Sci U S A 103:9897–9902. doi: 10.1073/pnas.0604017103 PubMedCentralCrossRefPubMedGoogle Scholar
  45. Zhang S, Cheng H, Gao Y, Wang G, Liang G, Wu K (2009) Mutation of an aminopeptidase N protein is associated with Helicoverpa armigera resistance to Bacillus thuringiensis Cry1Ac toxin. Insect Biochem Mol Biol 39:421–429. doi: 10.1016/j.ibmb.2009.04.003 CrossRefPubMedGoogle Scholar
  46. Zhang Y, Zheng G, Tan J, Li C, Cheng L (2013) Cloning and characterization of a novel cry8Ab1 protein from Bacillus thuringiensis strain B-JJX with specific toxicity to scarabaeid (Coleoptera: Scarabaeidae) larvae. Microbiol Res 168:512–517. doi: 10.1016/j.micres.2013.03.003 CrossRefPubMedGoogle Scholar
  47. Zhao JZ, Collins HL, Tang JD, Cao J, Earle ED, Roush RT, Herrero S, Escriche B, Ferre J, Shelton AM (2000) Development and characterization of diamondback moth resistance to transgenic broccoli expressing high levels of Cry1C. Appl Environ Microbiol 66:3784–3789. doi: 10.1016/j.ibmb.2007.09.014 PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Changlong Shu
    • 1
  • Shuqian Tan
    • 1
  • Jiao Yin
    • 1
  • Mario Soberón
    • 2
  • Alejandra Bravo
    • 2
  • Chunqing Liu
    • 3
  • Lili Geng
    • 1
  • Fuping Song
    • 1
  • Kebin Li
    • 1
  • Jie Zhang
    • 1
  1. 1.State Key Laboratory of Biology for Plant Diseases and Insect Pests, Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijingPeople’s Republic of China
  2. 2.Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico
  3. 3.Cangzhou Academy of Agricultural and Forestry SciencesCangzhouPeople’s Republic of China

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