#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

An ABC Transporter Mutation Is Correlated with Insect Resistance to Cry1Ac Toxin


Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are commercially successful in reducing pest damage, yet knowledge of resistance mechanisms that threaten their sustainability is incomplete. Insect resistance to the pore-forming Cry1Ac toxin is correlated with the loss of high-affinity, irreversible binding to the mid-gut membrane, but the genetic factors responsible for this change have been elusive. Mutations in a 12-cadherin-domain protein confer some Cry1Ac resistance but do not block this toxin binding in in vitro assays. We sought to identify mutations in other genes that might be responsible for the loss of binding. We employed a map-based cloning approach using a series of backcrosses with 1,060 progeny to identify a resistance gene in the cotton pest Heliothis virescens that segregated independently from the cadherin mutation. We found an inactivating mutation of the ABC transporter ABCC2 that is genetically linked to Cry1Ac resistance and is correlated with loss of Cry1Ac binding to membrane vesicles. ABC proteins are integral membrane proteins with many functions, including export of toxic molecules from the cell, but have not been implicated in the mode of action of Bt toxins before. The reduction in toxin binding due to the inactivating mutation suggests that ABCC2 is involved in membrane integration of the toxin pore. Our findings suggest that ABC proteins may play a key role in the mode of action of Bt toxins and that ABC protein mutations can confer high levels of resistance that could threaten the continued utilization of Bt–expressing crops. However, such mutations may impose a physiological cost on resistant insects, by reducing export of other toxins such as plant secondary compounds from the cell. This weakness could be exploited to manage this mechanism of Bt resistance in the field.


Vyšlo v časopise: An ABC Transporter Mutation Is Correlated with Insect Resistance to Cry1Ac Toxin. PLoS Genet 6(12): e32767. doi:10.1371/journal.pgen.1001248
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1001248

Souhrn

Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are commercially successful in reducing pest damage, yet knowledge of resistance mechanisms that threaten their sustainability is incomplete. Insect resistance to the pore-forming Cry1Ac toxin is correlated with the loss of high-affinity, irreversible binding to the mid-gut membrane, but the genetic factors responsible for this change have been elusive. Mutations in a 12-cadherin-domain protein confer some Cry1Ac resistance but do not block this toxin binding in in vitro assays. We sought to identify mutations in other genes that might be responsible for the loss of binding. We employed a map-based cloning approach using a series of backcrosses with 1,060 progeny to identify a resistance gene in the cotton pest Heliothis virescens that segregated independently from the cadherin mutation. We found an inactivating mutation of the ABC transporter ABCC2 that is genetically linked to Cry1Ac resistance and is correlated with loss of Cry1Ac binding to membrane vesicles. ABC proteins are integral membrane proteins with many functions, including export of toxic molecules from the cell, but have not been implicated in the mode of action of Bt toxins before. The reduction in toxin binding due to the inactivating mutation suggests that ABCC2 is involved in membrane integration of the toxin pore. Our findings suggest that ABC proteins may play a key role in the mode of action of Bt toxins and that ABC protein mutations can confer high levels of resistance that could threaten the continued utilization of Bt–expressing crops. However, such mutations may impose a physiological cost on resistant insects, by reducing export of other toxins such as plant secondary compounds from the cell. This weakness could be exploited to manage this mechanism of Bt resistance in the field.


Zdroje

1. JamesC

2008

Global Status of Commercialized Biotech/GM Crops: 2008.

Ithaca, NY

International Service for the Acquisition of Agri-biotech Applications

2. TabashnikBE

Van RensburgJBJ

CarrièreY

2009

Field-evolved insect resistance to Bt crops: Definition, theory, and data.

J Econ Entomol

102

2011

2025

3. PietrantonioPV

GillSS

1996

Bacillus thuringiensis toxins: Action on the insect midgut.

LehaneMJ

BillingsleyPF

Biology of the Insect Midgut

London

Chapman & Hall

345

372

4. SoberónM

GillSS

BravoA

2009

Signaling versus punching hole: How do Bacillus thuringiensis toxins kill insect midgut cells?

Cell Mol Life Sci

66

1337

1349

5. ZhangXB

CandasM

GrikoNB

TaussigR

BullaLA

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 USA

103

9897

9902

6. PigottCR

EllarDJ

2007

Role of receptors in Bacillus thuringiensis crystal toxin activity.

Microbiol Mol Biol Rev

71

255

281

7. FerréJ

Van RieJ

2002

Biochemistry and genetics of insect resistance to Bacillus thuringiensis.

Annu Rev Entomol

47

501

533

8. TabashnikBE

LiuYB

MalvarT

HeckelDG

MassonL

1998

Insect resistance to Bacillus thuringiensis: uniform or diverse?

Philos Trans R Soc Lond, Ser B: Biol Sci

353

1751

1756

9. TabashnikBE

1994

Evolution of resistance to Bacillus thuringiensis.

Annu Rev Entomol

39

47

97

10. GouldF

AndersonA

ReynoldsA

BumgarnerL

MoarW

1995

Selection and genetic analysis of a Heliothis virescens (Lepidoptera: Noctuidae) strain with high levels of resistance to Bacillus thuringiensis toxins.

J Econ Entomol

88

1545

1559

11. AkhurstRJ

JamesW

BirdLJ

BeardC

2003

Resistance to the Cry1Ac delta-endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae).

J Econ Entomol

96

1290

1299

12. González-CabreraJ

EscricheB

TabashnikBE

FerréJ

2003

Binding of Bacillus thuringiensis toxins in resistant and susceptible strains of pink bollworm (Pectinophora gossypiella).

Insect Biochem Mol Biol

33

929

935

13. CarrièreY

CrowderDW

TabashnikBE

2010

Evolutionary ecology of insect adaptation to Bt crops.

Evolutionary Applications

3

561

573

14. StorerNP

BabcockJM

SchlenzM

MeadeT

ThompsonGD

2010

Discovery and characterization of field resistance to Bt maize: Spodoptera frugiperda (Lepidoptera: Noctuidae) in Puerto Rico.

J Econ Entomol

103

1031

1038

15. XuXJ

YuLY

WuYD

2005

Disruption of a cadherin gene associated with resistance to Cry1Ac delta-endotoxin of Bacillus thuringiensis in Helicoverpa armigera.

Appl Environ Microbiol

71

948

954

16. MorinS

BiggsRW

SistersonMS

ShriverL

Ellers-KirkC

2003

Three cadherin alleles associated with resistance to Bacillus thuringiensis in pink bollworm.

Proc Natl Acad Sci USA

100

5004

5009

17. GahanLJ

GouldF

HeckelDG

2001

Identification of a gene associated with Bt resistance in Heliothis virescens.

Science

293

857

860

18. LeeMK

RajamohanF

GouldF

DeanDH

1995

Resistance to Bacillus thuringiensis CryIA delta-endotoxins in a laboratory-selected Heliothis virescens strain is related to receptor alteration.

Appl Environ Microbiol

61

3836

3842

19. Jurat-FuentesJL

GahanLJ

GouldFL

HeckelDG

AdangMJ

2004

The HevCaLP protein mediates binding specificity of the Cry1A class of Bacillus thuringiensis toxins in Heliothis virescens.

Biochemistry

43

14299

14305

20. TabashnikBE

LiuYB

MalvarT

HeckelDG

MassonL

1997

Global variation in the genetic and biochemical basis of diamondback moth resistance to Bacillus thuringiensis.

Proc Natl Acad Sci USA

94

12780

12785

21. BaxterSW

ZhaoJZ

GahanLJ

SheltonAM

TabashnikBE

2005

Novel genetic basis of field-evolved resistance to Bt toxins in Plutella xylostella.

Insect Mol Biol

14

327

334

22. BravoA

GómezI

CondeJ

Muñoz-GarayC

SánchezJ

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 Biomemb

1667

38

46

23. HeckelDG

GahanLC

GouldF

AndersonA

1997

Identification of a linkage group with a major effect on resistance to Bacillus thuringiensis Cry1Ac endotoxin in the tobacco budworm (Lepidoptera: Noctuidae).

J Econ Entomol

90

75

86

24. Jurat-FuentesJL

GouldFL

AdangMJ

2002

Altered glycosylation of 63- and 68-kilodalton microvillar proteins in Heliothis virescens correlates with reduced Cry1 toxin binding, decreased pore formation, and increased resistance to Bacillus thuringiensis Cry1 toxins.

Appl Environ Microbiol

68

5711

5717

25. GahanLJ

GouldF

LópezJD

MicinskiS

HeckelDG

2007

A polymerase chain reaction screen of field populations of Heliothis virescens for a retrotransposon insertion conferring resistance to Bacillus thuringiensis toxin.

J Econ Entomol

100

187

194

26. GahanLJ

MaYT

CobleMLM

GouldF

MoarWJ

2005

Genetic basis of resistance to Cry1Ac and Cry2Aa in Heliothis virescens (Lepidoptera: Noctuidae).

J Econ Entomol

98

1357

1368

27. YasukochiY

AshakumaryLA

BabaK

YoshidoA

SaharaK

2006

A second-generation integrated map of the silkworm reveals synteny and conserved gene order between lepidopteran insects.

Genetics

173

1319

1328

28. YoshidoA

BandoH

YasukochiY

SaharaK

2005

The Bombyx mori karyotype and the assignment of linkage groups.

Genetics

170

675

685

29. AllerSG

YuJ

WardA

WengY

ChittaboinaS

2009

Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding.

Science

323

1718

1722

30. BellierA

ChenCS

KaoCY

CinarHN

AroianRV

2009

Hypoxia and the hypoxic response pathway protect against pore-forming toxins in C. elegans.

PLoS Pathog

5

e1000689

doi:10.1371/journal.ppat.1000689

31. ChenCS

BellierA

KaoCY

YangYL

ChenHD

2010

WWP-1 Is a novel modulator of the DAF-2 insulin-like signaling network involved in pore-forming toxin cellular defenses in Caenorhabditis elegans.

PLoS ONE

5

e9494

doi:10.1371/journal.pone.0009494

32. KnowlesBH

EllarDJ

1987

Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis delta-endotoxins with different insect specificity.

Biochim Biophys Acta

924

509

518

33. SchwartzJL

GarneauL

SavariaD

MassonL

BrousseauR

1993

Lepidoperan-specific crystal toxins from Bacillus thuringiensis form cation-selective and anion-selective channels in planar lipid bilayers.

J Membr Biol

132

53

62

34. SlatinSL

AbramsCK

EnglishL

1990

Delta-endotoxins form cation-selective channels in planar lipid bilayers.

Biochem Biophys Res Commun

169

765

772

35. BravoA

GillSS

SoberónM

2007

Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control.

Toxicon

49

423

435

36. ChenJ

HuaG

Jurat-FuentesJL

AbdullahMA

AdangMJ

2007

Synergism of Bacillus thuringiensis toxins by a fragment of a toxin-binding cadherin.

Proc Natl Acad Sci USA

104

13901

13906

37. PengDH

XuXH

YeWX

YuZN

SunM

2010

Helicoverpa armigera cadherin fragment enhances Cry1Ac insecticidal activity by facilitating toxin-oligomer formation.

Appl Microbiol Biotechnol

85

1033

1040

38. SoberónM

Pardo-LópezL

LópezI

GómezI

TabashnikBE

2007

Engineering modified Bt toxins to counter insect resistance.

Science

318

1640

1642

39. Muñoz-GarayC

PortugalL

Pardo-LópezL

Jiménez-JuárezN

ArenasI

2009

Characterization of the mechanism of action of the genetically modified Cry1AbMod toxin that is active against Cry1Ab-resistant insects.

Biochim Biophys Acta

1788

2229

2237

40. NagamatsuY

TodaS

KoikeT

MiyoshiY

ShigematsuS

1998

Cloning, sequencing, and expression of the Bombyx mori receptor for Bacillus thuringiensis insecticidal CryIA(a) toxin.

Biosci Biotechnol Biochem

62

727

734

41. SangadalaS

WaltersFS

EnglishLH

AdangMJ

1994

A mixture of Manduca sexta aminopeptidase and phosphatase enhances Bacillus thuringiensis insecticidal CryIA(c) toxin binding and (Rb+-K+)-Rb-86 efflux in vitro.

J Biol Chem

269

10088

10092

42. KnightPJK

CrickmoreN

EllarDJ

1994

The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N.

Mol Microbiol

11

429

436

43. Jurat-FuentesJL

AdangMJ

2004

Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae.

Eur J Biochem

271

3127

3135

44. ArenasI

BravoA

SoberónM

GómezI

2010

Role of alkaline phosphatase from Manduca sexta in the mechanism of action of Bacillus thuringiensis Cry1Ab toxin.

J Biol Chem

285

12497

12503

45. PandianGN

IshikawaT

TogashiM

ShitomiY

HaginoyaK

2008

Bombyx mori midgut membrane protein P252, which binds to Bacillus thuringiensis Cry1A, is a chlorophyllide-binding protein, and the resulting complex has antimicrobial activity.

Appl Environ Microbiol

74

1324

1331

46. ValaitisAP

JenkinsJL

LeeMK

DeanDH

GarnerKJ

2001

Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity.

Arch Insect Biochem Physiol

46

186

200

47. ZhuangMB

OlteanDI

GómezI

PullikuthAK

SoberónM

2002

Heliothis virescens and Manduca sexta lipid rafts are involved in Cry1A toxin binding to the midgut epithelium and subsequent pore formation.

J Biol Chem

277

13863

13872

48. ValaitisAP

2008

Bacillus thuringiensis pore-forming toxins trigger massive shedding of GPI-anchored aminopeptidase N from gypsy moth midgut epithelial cells.

Insect Biochem Mol Biol

38

611

618

49. MassonL

LunYJ

MazzaA

BrousseauR

AdangMJ

1995

The CryIA(c) receptor purified from Manduca sexta displays multiple specificities.

J Biol Chem

270

20309

20315

50. LuoK

SangadalaS

MassonL

MazzaA

BrousseauR

1997

The Heliothis virescens 170kDa aminopeptidase functions as Receptor A by mediating specific Bacillus thuringiensis Cry1a delta-endotoxin binding and pore formation.

Insect Biochem Mol Biol

27

735

743

51. LeeMK

JenkinsJL

YouTH

CurtissA

SonJJ

2001

Mutations at the arginine residues in alpha 8 loop of Bacillus thuringiensis delta-endotoxin Cry1Ac affect toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N.

FEBS Lett

497

108

112

52. JenkinsJL

LeeMK

SangadalaS

AdangMJ

DeanDH

1999

Binding of Bacillus thuringiensis Cry1Ac toxin to Manduca sexta aminopeptidase N receptor is not directly related to toxicity.

FEBS Lett

462

373

376

53. RajagopalR

SivakumarS

AgrawalN

MalhotraP

BhatnagarRK

2002

Silencing of midgut aminopeptidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiensis toxin receptor.

J Biol Chem

277

46849

46851

54. YangYL

ZhuYC

OtteaJ

HussenederC

LeonardBR

2010

Molecular characterization and RNA interference of three midgut aminopeptidase N isozymes from Bacillus thuringiensis-susceptible and -resistant strains of sugarcane borer, Diatraea saccharalis.

Insect Biochem Mol Biol

40

592

603

55. CandasM

LosevaO

OppertB

KosarajuP

BullaLA

2003

Insect resistance to Bacillus thuringiensis - Alterations in the indianmeal moth larval gut proteome.

Mol Cell Proteomics

2

19

28

56. KrishnamoorthyM

Jurat-FuentesJL

McNallRJ

AndachtT

AdangMJ

2007

Identification of novel CrylAc binding proteins in midgut membranes from Heliothis virescens using proteomic analyses.

Insect Biochem Mol Biol

37

189

201

57. VadlamudiRK

JiTH

BullaLA

1993

A specific binding protein from Manduca sexta for the insecticidal toxin of Bacillus thuringiensis subsp Berliner.

J Biol Chem

268

12334

12340

58. NagamatsuY

TodaS

YamaguchiF

OgoM

KogureM

1998

Identification of Bombyx mori midgut receptor for Bacillus thuringiensis insecticidal CryIA(a) toxin.

Biosci Biotechnol Biochem

62

718

726

59. BaxterSW

ZhaoJZ

SheltonAM

VogelH

HeckelDG

2008

Genetic mapping of Bt-toxin binding proteins in a Cry1A-toxin resistant strain of diamondback moth Plutella xylostella.

Insect Biochem Mol Biol

38

125

135

60. WangP

ZhaoJZ

Rodrigo-SimónA

KainW

JanmaatAF

2007

Mechanism of resistance to Bacillus thuringiensis toxin Cry1Ac in a greenhouse population of the cabbage looper, Trichoplusia ni.

Appl Environ Microbiol

73

1199

1207

61. MahonRJ

OlsenKM

DownesS

AddisonS

2007

Frequency of alleles conferring resistance to the Bt toxins Cry1Ac and Cry2Ab in Australian populations of Helicoverpa armigera (Lepidoptera: noctuidae).

J Econ Entomol

100

1844

1853

62. DownesS

ParkerT

MahonR

2010

Incipient resistance of Helicoverpa punctigera to the Cry2Ab Bt toxin in Bollgard II (R) cotton.

PLoS ONE

5

e12567

doi:10.1371/journal.pone.0012567

63. CacciaS

Hernández-RodríguezCS

MahonRJ

DownesS

JamesW

2010

Binding site alteration is responsible for field-isolated resistance to Bacillus thuringiensis Cry2A insecticidal proteins in two Helicoverpa species.

PLoS ONE

5

e9975

doi:10.1371/journal.pone.0009975

64. OlsenKM

DalyJC

2000

Plant-toxin interactions in transgenic Bt cotton and their effect on mortality of Helicoverpa armigera (Lepidoptera: Noctuidae).

J Econ Entomol

93

1293

1299

65. CarrièreY

Ellers-KirkC

BiggsR

HigginsonDM

DennehyTJ

2004

Effects of gossypol on fitness costs associated with resistance to Bt cotton in pink bollworm.

J Econ Entomol

97

1710

1718

66. JoynerK

GouldF

1985

Developmental consequences of cannibalism in Heliothis zea (Lepidoptera: Noctuidae).

Ann Entomol Soc Am

78

24

28

67. RobinsonR

1971

Lepidoptera Genetics.

Oxford

Pergamon Press

68. YasukochiY

1998

A dense genetic map of the silkworm, Bombyx mori, covering all chromosomes based on 1018 molecular markers.

Genetics

150

1513

1525

69. MitaK

KasaharaM

SasakiS

NagayasuY

YamadaT

2004

The genome sequence of silkworm, Bombyx mori.

DNA Res

11

27

35

70. XiaQY

ZhouZY

LuC

ChengDJ

DaiFY

2004

A draft sequence for the genome of the domesticated silkworm (Bombyx mori).

Science

306

1937

1940

71. YamamotoK

NohataJ

Kadono-OkudaK

NarukawaJ

SasanumaM

2008

A BAC-based integrated linkage m0ap of the silkworm Bombyx mori.

Genome Biology

9

R21

72. XiaQY

WangJ

ZhouZY

LiRQ

FanW

2008

The genome of a lepidopteran model insect, the silkworm Bombyx mori.

Insect Biochem Mol Biol

38

1036

1045

73. WangJ

XiaQY

HeXM

DaiMT

RuanJ

2005

SilkDB: a knowledgebase for silkworm biology and genomics.

Nucleic Acids Res

33

D399

D402

74. ShimomuraM

MinamiH

SuetsuguY

OhyanagiH

SatohC

2009

KAIKObase: An integrated silkworm genome database and data mining tool.

BMC Genomics

10

486

75. LanderES

GreenP

AbrahamsonJ

BarlowA

DalyMJ

1987

MAPMAKER: An interactive computer package for constructing primary genetic linkage maps of experimental and natural populations.

Genomics

1

174

181

76. XiaQY

ChengDJ

DuanJ

WangGH

ChengTC

2007

Microarray-based gene expression profiles in multiple tissues of the domesticated silkworm, Bombyx mori.

Genome Biology

8

R162

77. WolfersbergerMG

LuethyP

MaurerP

PrentiP

SacchiVF

1987

Preparation and partial characterisation of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae).

Comp Biochem Physiol

86A

301

308

78. LeeMK

MilneRE

GeAZ

DeanDH

1992

Location of a Bombyx mori receptor binding region on a Bacillus thuringiensis delta-endotoxin.

J Biol Chem

267

3115

3121

79. WuCC

ProestouD

CarterD

NicholsonE

SantosF

2009

Construction and sequence sampling of deep-coverage, large-insert BAC libraries for three model lepidopteran species.

BMC Genomics

10

283

80. KallL

KroghA

SonnhammerELL

2007

Advantages of combined transmembrane topology and signal peptide prediction - the Phobius web server.

Nucleic Acids Res

35

W429

W432

81. KallL

KroghA

SonnhammerELL

2004

A combined transmembrane topology and signal peptide prediction method.

J Mol Biol

338

1027

1036

82. GaudetR

WileyDC

2001

Structure of the ABC ATPase domain of human TAP1, the transporter associated with antigen processing.

EMBO J

20

4964

4972

Štítky
Genetika Reprodukčná medicína

Článok vyšiel v časopise

PLOS Genetics


2010 Číslo 12
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#