Download Insectos resistentes: el reto de las plantas transgénicas

Management of insect
resistance to Bacillus
thuringiensis toxins
Juan Ferré (University of Valencia, Spain)
Insect species that have evolved Bt
resistance in the laboratory
Lepidoptera
Ö Plodia interpunctella
Ö Heliothis virescens
Ö Helicoverpa armigera
Ö Pectinophora gossypiella
Ö Spodoptera exigua
Ö Spodoptera littoralis
Ö Trichoplusia ni
Ö Ostrinia nubilalis
Ö Plutella xylostella
Coleoptera
Ö Leptinotarsa decemlineata
Ö Chrysomela scripta
Diptera
Ö Culex quinquefasciatus
Insect species that have evolved Bt
resistance in the laboratory
Lepidoptera
Coleoptera
Ö Plodia interpunctella
Ö Leptinotarsa decemlineata
Ö Heliothis virescens
Ö Chrysomela scripta
Ö Helicoverpa armigera
Cotton pests
Ö Pectinophora gossypiella
Diptera
Ö Spodoptera exigua
Ö Culex quinquefasciatus
Ö Spodoptera littoralis
Ö Trichoplusia ni
Corn pest
Ö Ostrinia nubilalis
Ö Plutella xylostella
Insect species that have evolved Bt
resistance in the field
Lepidoptera
Ö Plodia interpunctella
Ö Heliothis virescens
Ö Helicoverpa armigera
Ö Pectinophora gossypiella
Ö Spodoptera exigua
Ö Spodoptera littoralis
Ö Trichoplusia ni
Ö Ostrinia nubilalis
Ö Plutella xylostella
Coleoptera
Ö Leptinotarsa decemlineata
Ö Chrysomela scripta
Diptera
Ö Culex quinquefasciatus
Global area of Bt crops
Global area of Bt
crops (million Ha)
24
21
18
15
12
9
6
3
0
1995 1996 1997 1998 1999 2000 2001 2002 2003 2004
Year
Any type of Bt plants
Only insect resistance
Bt/herbicide res. plants
Strategies for resistance
management in Bt crops
ÖHigh expression of the Bt transgene to ensure that all
heterozygotes are killed.
ÖTemporal rotation of cultivars expressing different Bt
genes.
ÖExpression, in the same plant, of more than one Bt gene
(“pyramided” plants).
ÖUse of refuges with nontransformed plants (to permit a
certain part of the population to escape selection).
Ö Application of spatial mosaics of cultivars expressing
different Bt genes.
Strategies for resistance
management in Bt crops
ÖHigh expression of the Bt transgene to ensure that all
heterozygotes are killed.
ÖTemporal rotation of cultivars expressing different Bt
genes.
ÖExpression, in the same plant, of more than one Bt gene
(“pyramided” plants).
ÖUse of refuges with nontransformed plants (to permit a
certain part of the population to escape selection).
Ö Application of spatial mosaics of cultivars expressing
different Bt genes.
The high-dose/refuge strategy:
Requirements
Ö Inheritance of resistance recessive or partially
recessive.
ÖToxin concentration in plants high enough to kill
all heterozygotes.
ÖRandom mating between resistant and susceptible
insects.
Ö Low initial frequency of the resistance allele.
Strategies for resistance
management in Bt crops
ÖHigh expression of the Bt transgene to ensure that all
heterozygotes are killed.
ÖTemporal rotation of cultivars expressing different Bt
genes.
ÖExpression, in the same plant, of more than one Bt gene
(“pyramided” plants).
ÖUse of refuges with nontransformed plants (to permit a
certain part of the population to escape selection).
Ö Application of spatial mosaics of cultivars expressing
different Bt genes.
The combination of two toxins strategy:
Requirements
Ö Resistance to one toxin must not confere crossresistance to the other toxin (i.e., different modes of
action of the two toxins).
Ö Lower fitness associated to resistance alleles in
the absence of selection pressure (to permit reversion
of resistance).
Ö Low initial frequency of the resistance alleles.
Bacillus thuringiensis: mode of action
ingestion
proteolytic activation
solubilisation
receptor binding
PM crossing
pore formation
Binding site model in P. xylostella
Cry1Aa Cry1Ab
Cry1Ac
Cry1F
Cry1J
Cry1B
Cry1C
Alteration of the shared receptor was associated to resistance to
the 5 toxins in strains NO-QA and PEN
Biochemical basis of crossresistance in Lepidoptera
ÖThe pattern found in Plutella xylostella seems also
to apply to most lepidopterans:
Herrero, González-Cabrera, Tabashnik and Ferré (2001). Shared binding sites in
Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A toxins. Appl. Environ.
Microbiol. 67: 5729-5734.
Hernández and Ferré (2005). Common receptor for Bacillus thuringiensis toxins
Cry1Ac, Cry1Fa, and Cry1Ja in Helicoverpa armigera, Helicoverpa zea, and
Spodoptera exigua. Appl. Environ. Microbiol. 71: 5627-5629.
Cross-resistance to Cry1Aa, Cry1Ab, Cry1F and Cry1J was also
found in Cry1Ac-selected H. virescens
Mechanisms of resistance to Bt
1) Reduced binding
Plodia interpunctella (3 strains)
Plutella xylostella (8 strains)
Heliothis virescens (3 strains)
Pectinophora gossypiella (1 strain)
Helicoverpa armigera (1 strain)
Trichoplusia ni (1 strain)
Ostrinia nubilalis (1 strain)
2) Altered proteolytic processing
Plodia interpunctella (1 strain)
Heliothis virescens (2 strains)
Plutella xylostella (1 strain)
Ostrinia nubilalis (1 strain)
3) Faster cell repair/replacement
Heliothis virescens (1 strain)
4) Elevated immune response
High levels of resistance but
narrow spectrum
Low levels of resistance but
broad spectrum
Ephestia kuehniella (1 strain)
Helicoverpa armigera (1 strain)
5) Toxin sequestering by esterases
Helicoverpa armigera (1 strain)
Plutella xylostella (1 strain)
Classification of Bt resistance
Ö
An approach to classify the different cases of resistance to Bt toxins
was proposed in 1998 by Tabashnik et al. (Phil. Trans. R. Soc. Lond.
B 353: 1751-6).
Ö
“Mode 1” resistance is characterized by:
1.
2.
3.
4.
Extremely high resistance to at least one Cry1A toxin
Recessive inheritance
Little or no cross-resistance to Cry1C
Reduced binding to at least one Cry1A toxin
Ö
“Type I” binding site alteration: when binding of only one Cry1A toxin is affected
Ö
“Type II” binding site alteration: when the alteration affects binding of the 3
Cry1A toxins
Ö Different mechanisms
Ö Different genes or alleles
Candidate genes for Bt resistance
Ö
Trypsin-like proteinase
Ö
Chymotrypsin-like proteinase?
Ö
Cadherin
Ö
Aminopeptidase N
Ö
Alkaline phosphatase
Ö
Enzymes in the glycosylation pathway of membrane glycoproteins or
glycolipids
Ö
Esterase genes?
Ö
Genes involved in the immune response?
Genetic evidence as resistance genes
Ö
Trypsin-like proteinase (in insects)
Ö
Chymotrypsin-like proteinase?
Ö
Cadherin (in insects)
Ö
Aminopeptidase N
Ö
Alkaline phosphatase
Ö
Enzymes in the glycosylation pathway of membrane glycoproteins or
glycolipids (in C. elegans)
Ö
Esterase genes?
Ö
Genes involved in the immune response?
Genetic evidence for involvement of
trypsin proteinases in Bt resistance
Ö Reduction of a trypsin-like proteinase has been linked to
resistance in P. interpunctella:
Oppert, Kramer, Beeman, Johnson and McGaughey (1997). Proteinase-mediated
insect resistance to Bt toxins. J. Biol. Chem. 272: 23473-76.
Ö Decreased expression of a trypsin-like proteinase gene
has been observed in Ostrinia nubilalis:
Li, Oppert, Higgins, Huang, Buschman, Gao and Zhu (2005). Characterization of
cDNAs encoding three trypsin-like proteinases and mRNA quantitative analysis
in Bt-resistant and –susceptible strains of Ostrinia nubilalis. IBMB 35:847-60
Genetic evidence for involvement of
cadherin receptors in Mode 1 resistance
Ö Genes for cadherin-like proteins have been linked to
resistance in H. virescens, P. gossypiella and H. armigera:
Gahan, Gould and Heckel (2001). Identification of a gene associated with Bt resistance in
Heliothis virescens. Science 293: 857-60.
Morin et al. (2003). Three cadherin alleles associated with resistance to Bacillus thuringiensis
in pink bollworm. PNAS 100: 5004-09.
Xu, Yu and Wu (2005). Disruption of a cadherin gene associated with resistance to Cry1Ac δendotoxin of Bt in Helicoverpa armigera. Appl. Environ. Microbiol. 71: 948-54
Ö Lack of binding is not been linked to the cadherin gene in
P. xylostella:
Baxter, Zhao, Gahan, Shelton, Tabashnik and Heckel (2005) Novel genetic basis of fieldevolved resistance to Bt toxins in P. xylostella. Insect Mol. Biol. 14:327-34
Appl. to resistance management
1. Type of inheritance
2. Type of resist. mechanism
3. Gene identification
Autosomal/sex-linked
Recessive/dominant
Monogenic/polygenic
Broad/narrow spectrum
Protoxin/toxin effective
Biochemical
characterisation
Linkage analysis
Identification of mutation
Dev. of DNA-based
probes
Appl. to resistance management
1. Type of inheritance
Autosomal/sex-linked
Recessive/dominant
Monogenic/polygenic
2. Type of resist. mechanism
Broad/narrow spectrum
Protoxin/toxin effective
Biochemical characterisation
3. Gene identification
Linkage analysis
Identification of mutation
Dev. of DNA-based probes
Appl. to resistance management
1. Type of inheritance
Autosomal/sex-linked
Recessive/dominant
Monogenic/polygenic
2. Type of resist. mechanism
Broad/narrow spectrum
Protoxin/toxin effective
Biochemical characterisation
3. Gene identification
Linkage analysis
Identification of mutation
Dev. of DNA-based probes
Conclusion
Bt resistance may be conferred by a
variety of mechanisms. The challenge is
to determine those playing a major role
in the insect population under study to
apply the appropriate measures