Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol

We identify a cytochrome P450 gene (CYP6AE14) from cotton bollworm (Helicoverpa armigera), which permits this herbivore to tolerate otherwise inhibitory concentrations of the cotton metabolite, gossypol. CYP6AE14 is highly expressed in the midgut and its expression correlates with larval growth when gossypol is included in the diet. When larvae are fed plant material expressing double-stranded RNA (dsRNA) specific to CYP6AE14, levels of this transcript in the midgut decrease and larval growth is retarded. Both effects are more dramatic in the presence of gossypol. As a glutathione-S-transferase gene (GST1) is silenced in GST1 dsRNA–expressing plants, feeding insects plant material expressing dsRNA may be a general strategy to trigger RNA interference and could find applications in entomological research and field control of insect pests.

[1]  Shivakundan Singh Tej,et al.  Elucidation of the Small RNA Component of the Transcriptome , 2005, Science.

[2]  Andrew Fire,et al.  The rde-1 Gene, RNA Interference, and Transposon Silencing in C. elegans , 1999, Cell.

[3]  L. Puckhaber,et al.  Terpenoid aldehyde formation and lysigenous gland storage sites in cotton: variant with mature glands but suppressed levels of terpenoid aldehydes. , 2004, Phytochemistry.

[4]  A. Aravin,et al.  Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline , 2001, Current Biology.

[5]  A. Hobbs,et al.  Isolation and characterization of two cytochrome P450 cDNA clones for CYP6B6 and CYP6B7 from Helicoverpa armigera (Hubner): possible involvement of CYP6B7 in pyrethroid resistance. , 1998, Insect biochemistry and molecular biology.

[6]  Olivier Voinnet,et al.  Systemic signalling in gene silencing , 1997, Nature.

[7]  L. Du,et al.  Effect of cotton cultivar on development and reproduction of Aphis gossypii (Homoptera: Aphididae) and its predator Propylaea japonica (Coleoptera: Coccinellidae). , 2004, Journal of economic entomology.

[8]  A. Hobbs,et al.  Isolation and characterisation of a cytochrome b5 cDNA clone from Helicoverpa armigera (Hubner): possible involvement of cytochrome b5 in cytochrome P450 CYP6B7 activity towards pyrethroids. , 1999, Insect biochemistry and molecular biology.

[9]  J. Carrington,et al.  Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. , 2001, Plant physiology.

[10]  P. Waterhouse,et al.  Construct design for efficient, effective and high-throughput gene silencing in plants. , 2001, The Plant journal : for cell and molecular biology.

[11]  D. Hickey,et al.  Genomic organization and expression of a trypsin gene from the spruce budworm, Choristoneura fumiferana. , 1995, Insect biochemistry and molecular biology.

[12]  M. Berenbaum,et al.  Plant allelochemicals differentially regulate Helicoverpa zea cytochrome P450 genes , 2002, Insect molecular biology.

[13]  M. Berenbaum,et al.  CYP6B cytochrome P450 monooxygenases from Papilio canadensis and Papilio glaucus: potential contributions of sequence divergence to host plant associations , 2002, Insect molecular biology.

[14]  J. Gatehouse.  Plant resistance towards insect herbivores: a dynamic interaction. , 2002, The New phytologist.

[15]  W. Liang,et al.  Expression pattern of (+)-δ-cadinene synthase genes and biosynthesis of sesquiterpene aldehydes in plants of Gossypium arboreum L. , 2000, Planta.

[16]  I. Fridovich,et al.  Superoxide radical inhibits catalase. , 1982, The Journal of biological chemistry.

[17]  Craig P. Hunter,et al.  Systemic RNAi in C. elegans Requires the Putative Transmembrane Protein SID-1 , 2002, Science.

[18]  O. Terenius,et al.  Hemolin gene silencing by ds‐RNA injected into Cecropia pupae is lethal to next generation embryos , 2002, Insect molecular biology.

[19]  J. Gershenzon,et al.  Successful herbivore attack due to metabolic diversion of a plant chemical defense. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[20]  R. Hussey,et al.  Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene , 2006, Proceedings of the National Academy of Sciences.

[21]  D. Baulcombe,et al.  An SNF2 Protein Associated with Nuclear RNA Silencing and the Spread of a Silencing Signal between Cells in Arabidopsis[W][OA] , 2007, The Plant Cell Online.

[22]  A. Fire,et al.  Specific interference by ingested dsRNA , 1998, Nature.

[23]  S. Mazumdar-Leighton,et al.  Identification of six chymotrypsin cDNAs from larval midguts of Helicoverpa zea and Agrotis ipsilon feeding on the soybean (Kunitz) trypsin inhibitor. , 2001, Insect biochemistry and molecular biology.

[24]  R. ffrench-Constant,et al.  Prior infection of Manduca sexta with non-pathogenic Escherichia coli elicits immunity to pathogenic Photorhabdus luminescens: roles of immune-related proteins shown by RNA interference. , 2006, Insect biochemistry and molecular biology.

[25]  X. Miao,et al.  Preliminary studies on differential defense responses induced during plant communication , 2005, Cell Research.

[26]  A. Fire,et al.  Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. , 2001, Gene.

[27]  A. Hobbs,et al.  Isolation and sequence analysis of a cDNA clone for a pyrethroid inducible cytochrome P450 from Helicoverpa armigera. , 1995, Insect biochemistry and molecular biology.

[28]  H. Vaucheret,et al.  Systemic acquired silencing: transgene‐specific post‐transcriptional silencing is transmitted by grafting from silenced stocks to non‐silenced scions , 1997, The EMBO journal.

[29]  C. Mello,et al.  RNAi in C. elegans: Soaking in the Genome Sequence , 1998, Science.

[30]  E. Hodgson,et al.  Pesticide-metabolizing enzymes. , 1995, Toxicology letters.

[31]  D. Heckel,et al.  Identification of a Gene Associated with Bt Resistance in Heliothis virescens , 2001, Science.

[32]  X. Chen,et al.  Molecular cloning and functional identification of (+)-delta-cadinene-8-hydroxylase, a cytochrome P450 mono-oxygenase (CYP706B1) of cotton sesquiterpene biosynthesis. , 2001, The Plant journal : for cell and molecular biology.

[33]  M J Beller,et al.  The "tip of the iceberg" sign. , 1998, Radiology.

[34]  May R. Berenbaum,et al.  Jasmonate and salicylate induce expression of herbivore cytochrome P450 genes , 2002, Nature.

[35]  Timothy J. Dennehy,et al.  Long-term regional suppression of pink bollworm by Bacillus thuringiensis cotton , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[36]  M. Berenbaum,et al.  Characterization and evolution of furanocoumarin‐inducible cytochrome P450s in the parsnip webworm, Depressaria pastinacella , 2004, Insect molecular biology.

[37]  A. Shelton,et al.  Economic, ecological, food safety, and social consequences of the deployment of bt transgenic plants. , 2002, Annual review of entomology.

[38]  R. Newcomb,et al.  RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double‐stranded RNA feeding , 2006, Insect molecular biology.

[39]  B. J. Shaw,et al.  "Possible involvement of the long terminal repeat of transposable element 17.6 in regulating expression of an insecticide resistance-associated P450 gene in Drosophila.". , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[40]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[41]  Adam M. Gustafson,et al.  Genetic and Functional Diversification of Small RNA Pathways in Plants , 2004, PLoS biology.

[42]  Nicolas Bouché,et al.  An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs , 2006, The EMBO journal.

[43]  S. Clough,et al.  Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. , 1998, The Plant journal : for cell and molecular biology.

[44]  N. Chua,et al.  Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance , 2006, Nature Biotechnology.

[45]  J. Hull,et al.  Targeted disruption of genes in the Bombyx mori sex pheromone biosynthetic pathway. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[46]  R. Feyereisen,et al.  Cytochrome P450 monooxygenases and insecticide resistance in insects. , 1998, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[47]  Z. Wen,et al.  Insect cytochromes P450: diversity, insecticide resistance and tolerance to plant toxins. , 1998, Comparative biochemistry and physiology. Part C, Pharmacology, toxicology & endocrinology.

[48]  Juan D. López,et al.  Effect of Racemic and (+)- and (−)-Gossypol on the Survival and Development of Helicoverpa zea Larvae , 2006, Journal of Chemical Ecology.

[49]  M. Berenbaum,et al.  Molecular analysis of CYP321A1, a novel cytochrome P450 involved in metabolism of plant allelochemicals (furanocoumarins) and insecticides (cypermethrin) in Helicoverpa zea. , 2004, Gene.

[50]  J. Diaz‐ruiz,et al.  Double-Stranded RNA-Mediated Interference with Plant Virus Infection , 2001, Journal of Virology.

[51]  Z. Wen,et al.  Cytochromes P450 of insects: the tip of the iceberg. , 2001, Pest management science.