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Pyrosequencing of the midgut transcriptome of the poplar leaf beetle Chrysomela tremulae reveals new gene families in Coleoptera Yannick Pauchet, Paul Wilkinson, Manuella van Munster, Sylvie Augustin, David Pauron, Richard Ffrench-Constant To cite this version: Yannick Pauchet, Paul Wilkinson, Manuella van Munster, Sylvie Augustin, David Pauron, et al.. Pyrosequencing of the midgut transcriptome of the poplar leaf beetle Chrysomela tremulae reveals new gene families in Coleoptera. Insect Biochemistry and Molecular Biology, Elsevier, 2009, 39 (5-6), pp.403-413. 10.1016/j.ibmb.2009.04.001. hal-02668241 HAL Id: hal-02668241 https://hal.inrae.fr/hal-02668241 Submitted on 31 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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ARTICLE IN PRESS IB2039_proof 16 April 2009 1/11 Insect Biochemistry and Molecular Biology xxx (2009) 1–11 Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb 55 1 56 2 Pyrosequencing of the midgut transcriptome of the poplar leaf beetle 57 3 58 4 Chrysomela tremulae reveals new gene families in Coleoptera 59 5 60 6 Yannick Pauchet a,*, Paul Wilkinson a, Manuella van Munster b, Sylvie Augustin c, 61 62 7 David Pauron b, Richard H. ffrench-Constant a 8 63 9 a School of Biosciences, University of Exeter in Cornwall, Tremough Campus, Penryn TR10 9EZ, UK 64 b 10 INRA, UMR 1301 Interactions Biotiques et Sante´ Ve´ge´tale, 06903 Sophia Antipolis cedex, France 65 c INRA, UR 633 Zoologie Forestie`re, F-45000 Orle´ans, France 11 66 12 67 13 68 article info abstract 14 69 15 70 Article history: The insect midgut is the primary target site for Bt-derived insecticides and Bt alternatives. However, 16 71 Received 10 February 2009 despite extensive recent study, the precise role and nature of different Bt receptors remains a subject of 17 72 Received in revised form considerable debate. This problem is fuelledPROOF by a lack of understanding of the genes expressed in the 18 2 April 2009 73 insect midgut and their physiological roles. The poplar leaf beetle, Chrysomela tremulae, is an important Accepted 2 April 2009 74 19 model for understanding the mode of action of, and resistance to, coleopteran-specific Bt toxins and 20 currently shows the only known naturally occurring case of resistance to Cry3A toxins. Moreover it 75 Keywords: 21 belongs to the same family as the corn rootworm, Diabrotica virgifera, an economically important beetle 76 Insect midgut 77 22 Chitin deacetylase pest and target of hybrid corn expressing Cry3 toxins. Pyrosequencing is a fast and efficient way of 23 Cellulase defining the transcriptome of specific insect tissues such as the larval midgut. Here we use 454 based 78 24 Endopolygalacturonase pyrosequencing to sample the larval midgut transcriptome of C. tremulae. We identify candidate genes of 79 25 Chrysomela putative Bt receptors including transcripts encoding cadherin-like proteins, aminopeptidase N and 80 Bt resistance 26 alkaline phosphatase. We also describe a wealth of new transcripts predicting rapidly evolving gene 81 families involved in plant tissue digestion, which have no homologs in the genome of the stored product 27 82 pest the Red Flour beetle, Tribolium castaneum. 28 83 Ó 2009 Published by Elsevier Ltd. 29 84 30 85 31 86 32 87 33 1. Introduction (Bowen et al.,1998; ffrench-Constant et al., 2007). However, despite 88 34 extensive recent study, the precise role and nature of different Bt 89 35 Traditionally the major target site for small molecule insecticides receptors remains a subject of considerable debate (Pigott and Ellar, 90 36 has been the insect nervous system (Buckingham et al., 2005; Dong, 2007; Soberon et al., 2009). This problem is fuelled by a lack of 91 37 2007; ffrench-Constant et al., 2004; Millar and Denholm, 2007; understanding of the genes expressed in the insect midgut and their 92 38 Raymond-Delpech et al., 2005). However with the advent of Bt respective roles in digestion, response to invading micro-organ- 93 39 toxins, and Bt transformed insect resistant crops, the midgut has isms, homeostatic regulation, and the production and remodeling 94 40 become the primary focus for the development of novel insecticidal of the peritrophic membrane. We aim to address this problem by 95 41 proteins (Pigott and Ellar, 2007). Interest in insect midgut physi- describing the midgut transcriptome of representatives of key 96 42 ology has been strengthened by concerns over emerging Bt resis- insect pest groups such as leaf feeding beetles and Lepidoptera. 97 43 tance and by ongoing research into a growing number of candidate Beetles represent approximately half of all insect species recor- 98 44 Bt receptors and how these may become altered in Bt resistant ded to date and within this group the chrysomelids are one of the 99 45 insects (Pigott and Ellar, 2007). Identifying and understanding largest families (Hunt et al., 2007), many of which are important 100 46 midgut receptors will also be important in the study of new toxins defoliating and/or root damaging pests such as the corn rootworm 101 47 proposed as alternatives to toxinsUNCORRECTED from Bacillus, such as the Toxin Diabrotica virgifera (Gray et al., 2009). With the recent sequencing of 102 48 complexes (Tc’s) from Photorhabdus and Xenorhabdus bacteria the complete genome of the Red Flour Beetle, Tribolium castaneum, 103 49 we are now uniquely poised to understand more about the geno- 104 50 mics and transcriptomics of beetles. Moreover, given the rather 105 Abbreviations: EST, expressed sequence tag; PM, peritrophic membrane; unusual lifestyle of T. castaneum in feeding exclusively on stored 106 51 Bt, Bacillus thuringiensis toxin; SNP, single nucleotide polymorphism. 52 * Corresponding author. Tel.: þ44 1326 371 852; fax: þ44 1326 253 638. products, comparisons with the commoner lifestyle of leaf-, root- or 107 53 E-mail address: [email protected] (Y. Pauchet). bark-feeding are a priority. The poplar leaf beetle, Chrysomela 108 54 109 0965-1748/$ – see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.ibmb.2009.04.001 Please cite this article in press as: Pauchet, Y., et al., Pyrosequencing of the midgut transcriptome of the poplar leaf beetle Chrysomela..., Insect Biochemistry and Molecular Biology (2009), doi:10.1016/j.ibmb.2009.04.001 ARTICLE IN PRESS IB2039_proof 16 April 2009 2/11 2 Y. Pauchet et al. / Insect Biochemistry and Molecular Biology xxx (2009) 1–11 110 tremulae, is an important model for understanding Bt receptors and was further purified by using the RNeasy MinElute Clean up Kit 175 111 Bt resistance, and this is currently the only known beetle for which (Qiagen) following the manufacturer’s protocol and eluted in 20 ml 176 112 Cry3A resistance alleles have been found in the field (Genissel et al., of RNA storage solution (Ambion). 177 113 2003a). A Cry3A resistant C. tremulae strain was selected on Bt 178 114 transformed poplar trees expressing the Cry3A toxin (Genissel et al., 2.2. Midgut cDNA library preparation 179 115 2003b). This strain was derived from an isofemale line established 180 116 from field-caught insects that generated F2 offspring that survived Full-length, enriched, cDNAs were generated from 2 mg of total 181 117 on this Bt poplar clone (Augustin et al., 2004). Resistance to Cry3A in RNA using the SMART PCR cDNA synthesis kit (BD Clontech) 182 118 C. tremulae is under control of a single, completely recessive, following the manufacturer’s protocol. Reverse transcription was 183 119 autosomal trait (Augustin et al., 2004), suggesting that changes in performed with the PrimeScript reverse transcription enzyme 184 120 a single receptor, or other gene product, may be involved in resis- (Takara) for 60 min at 42 C and 90 min at 50 C. In order to prevent 185 121 tance. Resistance to Cry3A in the related Chrysomela scripta can be over-representation of the most common transcripts, the resulting 186 122 overcome using Cyt1Aa (Federici and Bauer, 1998), a protein from double-stranded cDNAs were normalized using the Kamchatka 187 123 a different class of Bt toxins, suggesting some potential specificity in crab duplex-specific nuclease method (Trimmer cDNA normaliza- 188 124 the nature of the resistance mechanism. In the Lepidoptera, Bt tion kit, Evrogen) (Zhulidov et al., 2004). The resulting normalized 189 125 resistance has been associated with changes in midgut associated cDNA midgut library was used for 454 pyrosequencing (Margulies 190 126 aminopeptidase N, cadherin-like proteins, alkaline phosphatase et al., 2005). 191 127 and, via work in model nematodes, midgut glycolipids (Pigott and 192 128 Ellar, 2007). However the molecular basis of Cry3A resistance in 2.3. Pyrosequencing, sequence preprocessing and assembly 193 129 beetles has not been reported to date. 194 130 Pyrosequencing is a fast and efficient way of defining the For 454 pyrosequencing, a cDNA aliquot was sent to the Advanced 195 131 transcriptome of specific insect tissues or cells and has recently Genomics facility at the University of Liverpool (http://www.liv.ac. 196 132 been used with great effect to define the transcriptome of immune uk/agf). A single full plate run was performed on the 454 GS-FLX 197 133 related tissues in Manduca sexta larvae (Zou et al., 2008).