X-tox: a new family of putative antimicrobial protein specific to Jean-Michel Escoubas, Pierre-Alain Girard, Anne Nathalie Volkoff, Y. Boublik, A. Cousserans, Emmanuelle d’Alençon, K. Mita, P. Taillez, Michel Brehélin

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Jean-Michel Escoubas, Pierre-Alain Girard, Anne Nathalie Volkoff, Y. Boublik, A. Cousserans, et al.. X-tox: a new family of putative antimicrobial protein specific to Lepidoptera. 7. International Workshop on the Molecular Biology and Genetics of the Lepidoptera, Aug 2006, Kolympari, Greece. ￿10.1673/031.007.2901￿. ￿hal-01123360￿

HAL Id: hal-01123360 https://hal.archives-ouvertes.fr/hal-01123360 Submitted on 3 Jun 2020

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Distributed under a Creative Commons Attribution| 4.0 International License Journal of Science | www.insectscience.org ISSN: 1536-2442

Colombia. 6 University of Tennessee, USA. 305-8634, Japan. [email protected] [email protected] A new family of putative antimicrobial proteins, -level phylogenetic hypotheses can be used characterized by imperfectly conserved tandem to explore patterns of divergence and speciation. repeats (up to 11) of cystein-stabilized alpha beta Warningly-colored mimetic butterflies are motifs (CS-αβ), a structural scaffold characteristic interesting models, because their wing patterns of invertebrate defensins and scorpion toxins, was are under strong selection, and might be involved identified through functional genomic approaches in speciation. Here we present an almost in the lepidopteran species Spodoptera complete phylogenetic hypothesis for the frugiperda. Orthologs were also found in ESTs or neotropical mimetic butterfly genus Napeogenes genomic databases from other lepidopteran (20 species), based on mitochondrial genes species (Bombyx mori, Galleria mellonella, (entire cytochrome oxydase I and II) and nuclear Papillo dardanus), but not in other insect orders genes (tektin, 715bp; and elongation factor 1α, or other invertebrate or metazoans, thus 1028bp). Whenever possible, we included several suggesting that this protein family is specific to subspecies of the same species (with different the lepidopteran . In S. frugiperda as well wing patterns). The results showed good as in B. mori, more than one transcript can be congruence between the different genetic regions. related to this protein family. In silico analysis of In most cases individuals of the same species B. mori genome (the sole lepidopteran genome clustered together, supporting the current available to date) suggests that the three mRNA . However, N. larina otaxes and N. encoding X-tox proteins that were characterized larina aethra appeared to be two sister species in this insect are generated through a mechanism rather than two subspecies. A combined evidence of alternative splicing. This mechanism thus topology is presented based on a Bayesian allows the production of different X-tox proteins analysis of all the genes regions. Using the containing five or six CS-αβ motifs. S. frugiperda phylogentic hypothesis we investigate whether X-tox genomic organization is currently under changes in wing pattern were associated with investigation. The role of these proteins in speciation, and whether speciation was lepidopteran immune response was investigated dominantly sympatric or allopatric. Using the by transcriptional analyses with one of the S. phylogenetic hypothesis we explore patterns of frugiperda cDNAs. Results indicate that the diversification, and we investigate whether corresponding gene, spod-11-tox, is expressed changes in altitudinal range and in wing pattern mainly in larval fat body and hemocytes and that were associated with speciation. transcription is enhanced in both immune tissues upon bacterial challenge. Phylogenetic analyses X-tox: a new family of putative show that CS-αβ motifs-containing proteins are antimicrobial protein specific to divided into three clusters. The first, considered as “ancestral” group, comprises molecules from Lepidoptera different invertebrate taxa (odonate insect, J.-M. Escoubas1, P.-A. Girard1, N. Volkoff2, Y. arachnids and mollusks) and even a fungus Boublik3, F. Cousserans2, E. D’Alençon2, K. molecule whereas the second group includes Mita4, P. Taillez1, M. Brehélin1. defensins isolated from neopteran insects. Finally, the third cluster contains all the CS-αβ motifs 1Écologie Microbienne des Insectes et stem from lepidopteran X-tox proteins. According Interactions Hôte-Pathogène, Institut National de to this phylogenetic analysis and to the position of Recherche Agronomique, Université de lepidoptera in insect evolution (one of the latest Montpellier II, CC54, 2 place E. Bataillon, 34095 order in insect speciation), we propose that X-tox Montpellier, France;2Biologie Intégrative et proteins evolved from insect defensins to generate Virologie des Insectes, Institut National de a new family of antimicrobial proteins. The main Recherche Agronomique, Université de feature of this family is the multi-domain Montpellier II, CC101, 2 place E. Bataillon, 34095 organisation who leads to molecular diversity Montpellier, France;3Plateforme Protéines generated through a mechanism of alternative Recombinantes, C.R.B.M., Centre National de la splicing. The full characterization of this new Recherche Scientifique, 34293 Montpellier, protein family (gene organization and expression, France; Laboratoty of Insect Genome, National protein structure and mechanism of action) in Institut of Agrobiological Sciences, Tsukuba, several lepidopteran species should contribute to

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solve the puzzle of the evolutionary history of recognition protein genes (Hemolin, defensins. Immulectin-2, Peptidoglycan recognition protein, Pattern recognition Serine Proteinase, The role of serineprotease β-glucan recognition protein-1, β-glucan recognition protein-2), but down-regulated the homolgue 3 (SPH-3) in Manduca transcription of certain antibacterial effector sexta shown by RNA interference genes (Attacin, Cecropin, Moricin). Our findings G. Felföldi1,2, I. Eleftherianos1, R. H. strongly suggest that SPH-3 serves specific ffrench-Constant3, I. Venekei2, S. E. Reynolds1 immune functions and plays a distinctive role in M. sexta immune pathways. 1Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 Host plant adaptation and 7AY, UK; 2Department of Biochemistry, Eötvös Loránd University, Pázmány sétány 1/C, specialization in the Pieridae Budapest, H-1117, Hungary; 3School of familiy Biosciences, University of Exeter, Cornwall, UK. H. M. Fischer1, C. W. Wheat2, U. Wittstock3, D. G. [email protected] Heckel1, H. Vogel1

Photorhabdus luminescens lives in symbiosis with 1Entomology Dept., MPI for Chemical Ecology, nematodes that invade insects. Following entry D-07745 Jena, Germany; 2 Dept. of Biological and into the insect, the bacteria are released from the Environmental Science, University of Helsinki, nematode gut into the open blood system of the FI-00014 Helsinki, Finland; 3Inst. für insect. Here they secrete factors, which kill the Pharmazeutische Biologie, Technische Universität host and also convert the host tissues into food for Braunschweig, D-38106 Braunschweig, Germany. the replicating bacteria and nematodes. One of [email protected] the secreted proteins is an rtx-like zinc metalloprotease PrtA. Using a proteomic A metabolic diversion of a plant chemical defense approach to determine the natural substrates of caused by an enzyme (Nitrile Specifier Protein, PrtA in host haemolymph, we identified a number NSP) enables a majority of Pieridae species to of Manduca sexta haemolymph proteins that are feed on Brassicaceous plants. The NSP is a newly selectively cleaved when plasma was incubated recruited detoxifying protein in this family. It with purified PrtA. One of the PrtA protein targets shows a specific repeat structure and has no in M. sexta haemolymph included the Serine sequence homology to any protein with a known Protease Homologue Protein 3 (SPH-3). We function. We are trying to unravel the molecular found that SPH-3 is not transcribed in the mechanisms leading to the evolution of this fat-body or haemocytes of naive M. sexta, but only protein and the subsequent adaptation to a plant when insects are exposed to bacteria, including defense system. The nucleotide sequence is Photorhabdus. We used RNA interference known for two model species. By generating (RNAi)-mediated inhibition of expression through cDNA libraries of related species and amplifying injection of double-stranded RNA of SPH-3 into intron and upstream regions, species and allelic M. sexta caterpillars and we achieved reduced differences are being located. Although NSP transcription of SPH-3 in both fat-body and shows no sequence homology to any protein of haemocyte tissues. Knock-down of SPH-3 known function, single domains show similarities dramatically reduced the ability of insects to resist to the so-called major allergen proteins, found in infection to a standard dose of Photorhabdus, as the gut lumen of many insect species. By measured by the rate at which infected insects die. establishing heterologous expression systems for RNAi of SPH-3 was also associated with both proteins we are trying to investigate the decreased levels of phenoloxidase (PO) activity in mode of action of NSP and the function of MA. haemolymph cell-free plasma from insects infected with either a non-pathogenic strain of E. coli or Photorhabdus. The reduction in PO The effect of microorganisms in the activity was also reflected by a reduction in the food on the growth rate and number of melanotic nodules present in tissues of immune system of cabbage looper dissected larvae. Finally, we found that RNAi of (Trichoplusia ni) SPH-3 did not affect the transcription of pattern D. Freitak, D. Heckel, H. Vogel

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