Abstract
Several families of crystal proteins from Bacillus thuringiensis exhibit nematicidal activity. Cry5B protein, a pore-forming toxin, has been intensively studied yielding many insights into the mode of action of crystal protein at molecular level and pathogenesis of pore-forming toxins. However, little attention was paid to Cry6A, another representative nematicidal crystal protein. Cry6A shares very low homology with Cry5B at amino acid sequence and probably acts in a distinct pathway from Cry5B and even the other main commercial crystal proteins. In the current study, we comprehensively investigated the nematicidal properties of Cry6Aa2 against the free-living soil nematode Caenorhabditis elegans and examined the physical response of C. elegans to Cry6Aa2 attack. Our results indicate that Cry6Aa2 exhibits high lethal activity to C. elegans and could cause detrimental effects on C. elegans, including obviously suppressed growth, decreased brood size, and even abnormal motility. Meanwhile, our study additionally shows that C. elegans could defend against the Cry6Aa2 toxin harmful threat through behavioral defense responses, such as reduced oral uptake and physical avoidance. In general, this study suggests that Cry6Aa2 possesses diverse nematicidal properties, which strongly indicates that Cry6Aa2 is a promising potential candidate of nematicidal agent. Moreover, this study highlights the importance of behavioral responses in defense of C. elegans for survival and demonstrates the key role of crystal protein in the interaction of B. thuringiensis–C. elegans. These findings could shed light on understanding the interaction of C. elegans with B. thuringiensis and provide a perfect model to study the role of pathogenic factor in the interaction of pathogen–host.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Able DJ (1996) The contagion indicator hypothesis for parasite-mediated sexual selection. Proc Natl Acad Sci U S A 93:2229–2233
Aroian R, van der Goot FG (2007) Pore-forming toxins and cellular non-immune defenses (CNIDs). Curr Opin Microbiol 10:57–61
Bird DM, Williamson VM, Abad P, McCarter J, Danchin EG, Castagnone-Sereno P, Opperman CH (2009) The genome of root-knot nematodes. Annu Rev Phytopathol 47:333–351
Bischof LJ, Huffman DL, Aroian RV (2006) Assays for toxicity studies in C. elegans with Bt crystal proteins. In: Strange K (ed) C. elegans methods and application (1st edn). Humana, Totowa, pp 139–154
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Bravo A, Likitvivatanavong S, Gill SS, Soberón M (2011) Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem Mol Biol 41:423–431
Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77:71–94
de Maagd RA, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet 17:193–199
Gregory WF, Parkinson J (2003) Caenorhabditis elegans—application to nematode genomics. Comp Funct Genom 4:194–202
Griffitts JS, Whitacre JL, Stevens DE, Aroian RV (2001) Bt toxin resistance from loss of a putative carbohydrate-modifying enzyme. Science 293:860–864
Griffitts JS, Haslam SM, Yang T, Garczynski SF, Dell A, Adang MJ, Aroian RV (2005) Glycolipids as receptor for Bacillus thruingiensis crystal toxin. Science 307:922–925
Guo SX, Liu M, Peng DH, Ji SS, Wang PX, Yu ZN, Sun M (2008) New strategy for isolating novel nematicidal crystal protein genes from Bacillus thuringiensis strain YBT-518. Appl Environ Microbiol 74:6997–7001
Hasshoff M, Böhnisch C, Tonn D, Hasert B, Schulenburg H (2007) The role of Caenorhabditis elegans insulin-like signaling in the behavioral avoidance of pathogenic Bacillus thuringiensis. FASEB J 21:1801–1812
Huffman DL, Abrami L, Sasik R, Corbeil J, van der Goot FG, Aroian RV (2004) Mitogen-activated protein kinase pathways defend against bacterial pore-forming toxins. Proc Natl Acad Sci U S A 101:10995–11000
Hui F, Scheib U, Hu Y, Sommer RJ, Aroian RV, Ghosh P (2012) Structure and glycolipid binding properties of the nematicidal protein Cry5B. Biochemistry 51:9911–9921
Irazoqui JE, Troemel ER, Feinbaoum RL, Luhachack LG, Cezairliyan BO, Ausubel FM (2010) Distinct pathogenesis and host responses during infection of C. elegans by P. aeruginosa and S. aureus. Plos Pathog 6:e1000982
Kavaliers M, Choleris E, Pfaff DW (2005) Genes, odours and the recognition of parasitized individuals by rodents. Trends Parasitol 21:423–429
Kho MF, Bellier A, Balasubramani V, Hu Y, Hsu W, Nielsen-LeRoux C, McGillivray SM, Nizet V, Aroian RV (2011) The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans. Plos One 6:e29122
Li XQ, Wei JZ, Tan A, Aroian RV (2007) Resistance to root-knot nematode in tomato roots expressing a nematicidal Bacillus thuringiensis crystal protein. Plant Biotechnol J 5:455–464
Marroquin LD, Elyassnia D, Griffitts JS, Feitelson JS, Aroian RV (2000) Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans. Genetics 156:1693–1699
Moore J (2002) Parasites and the behavior of animals. Oxford University Press, Oxford
Pujol N, Link EM, Liu LX, Kurz CL, Alloing G, Tan MW, Ray P, Solari R, Johnson CD, Ewbank JJ (2001) A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans. Curr Biol 11:809–821
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor
Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806
Schulenburg H, Müller S (2004) Natural variation in the response of Caenorhabditis elegans towards Bacillus thuringiensis. Parasitology 128:433–443
Schulenburg H, Kurz CL, Ewbank JJ (2004) Evolution of the innate immune system: the worm perspective. Immunol Rev 198:36–58
Schulte RD, Makus C, Hasert B, Michiels NK, Schulenburg H (2010) Multiple reciprocal adaptations and rapid genetic change upon experimental coevolution of an animal host and its microbial parasite. Proc Natl Acad Sci U S A 107:7359–7364
Schulte RD, Hasert B, Makus C, Michiels NK, Schulenburg H (2011a) Increased responsiveness in feeding behaviour of Caenorhabditis elegans after experimental coevolution with its microparasite Bacillus thuringiensis. Biol Lett. doi:10.1098/rsbl.2011.0684
Schulte RD, Makus C, Hasert B, Michiels NK, Schulenburg H (2011b) Host–parasite local adaptation after experimental coevolutio of Caenorhabditis elegans and its micoparasite Bacillus thuringiensis. Proc R Soc 278:2832–2839
Sicard M, Hering S, Schulte R, Gaudriault S, Schulenburg H (2007) The effect of Photorhabdus luminescens (Enterobacteriaceae) on the survival, development, reproduction and behaviour of Caenorhabditis elegans (Nematoda: Rhabditidae). Environ Microbiol 9:12–25
Smith MP, Laws TR, Atkins TP, Oyston PCF, de Pomerai DI, Titball RW (2002) A liquid-based method for the assessment of bacterial pathogenicity using the nematode Caenorhabditis elegans. FEMS Microbiol Lett 210:181–185
Tavernarakis N, Shreffler W, Wang S, Driscoll M (1997) Unc-8, a DEG/ENaC family member, encodes a subunit of a candidate mechanically gated channel that modulates C. elegans locomotion. Neuron 18:107–119
Trudgill DL, Blok VC (2001) Apomictic, polyphagous root-knot nematode: exceptionally successful and damaging biotrophic root pathogens. Annu Rev Phytopathol 39:53–77
Wei JZ, Hale K, Carta L, Platzer E, Wong C, Fang SC, Aroian RV (2003) Bacillus thuringiensis crystal proteins that target nematodes. Proc Natl Acad Sci U S A 100:2760–2765
White JG, Southgate E, Thomson JN, Brenner S (1986) The structure of the nervous system of the nematode Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci 314:1–340
Yu Z, Bai P, Ye W, Zhang F, Ruan L, Sun M (2008) A novel negative regulatory factor for nematicidal Cry protein gene expression in Bacillus thuringiensis. J Microbiol Biotechnol 18:1033–1039
Zhang Y, Lu H, Bargman CI (2005) Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans. Nature 438:179–184
Acknowledgments
We would like to thank Professor Ming Sun (Stake Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China) for providing cry6Aa2 gene, and the C. elegans Genetic Center which is supported by the NIH Center for Research Resource for providing N2 worms for use in this study. This study was supported by the National Basic Research Program (973) of China (2013CB127504); the National High Technology Research and Development Program (863) of China (2011AA10A203); the Natural Science Foundation of Hunan Province, China (10JJ6055); Scientific Research Fund of Hunan Provincial Education Department, China (09K023); Specialized Research Fund for the Doctoral Program of Higher Education of China (20114306120006); and the Industrialization Research Fund of Hunan Provincial Education Department, China (11CY024).
Author information
Authors and Affiliations
Corresponding author
Additional information
Hui Luo and Jing Xiong contributed equally to this work.
Rights and permissions
About this article
Cite this article
Luo, H., Xiong, J., Zhou, Q. et al. The effects of Bacillus thuringiensis Cry6A on the survival, growth, reproduction, locomotion, and behavioral response of Caenorhabditis elegans . Appl Microbiol Biotechnol 97, 10135–10142 (2013). https://doi.org/10.1007/s00253-013-5249-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-013-5249-3