Linda J. Gahan

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

Evolution of resistance by pests is the main threat to long-term insect control by transgenic crops that produce Bacillus thuringiensis (Bt) toxins. Because inheritance of resistance to the Bt toxins in transgenic crops is typically recessive, DNA-based screening for resistance alleles in heterozygotes is potentially much more efficient than detection of resistant homozygotes with bioassays. Such screening, however, requires knowledge of the resistance alleles in field populations of pests that are associated with survival on Bt crops. Here we report that field populations of pink bollworm (Pectinophora gossypiella), a major cotton pest, harbored three mutant alleles of a cadherin-encoding gene linked with resistance to Bt toxin Cry1Ac and survival on transgenic Bt cotton. Each of the three resistance alleles has a deletion expected to eliminate at least eight amino acids upstream of the putative toxin-binding region of the cadherin protein. Larvae with two resistance alleles in any combination were resistant, whereas those with one or none were susceptible to Cry1Ac. Together with previous evidence, the results reported here identify the cadherin gene as a leading target for DNA-based screening of resistance to Bt crops in lepidopteran pests.

An ABC Transporter Mutation Is Correlated with Insect Resistance to Bacillus thuringiensis 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.

Efficacy of genetically modified Bt toxins against insects with different genetic mechanisms of resistance

Transgenic crops that produce Bacillus thuringiensis (Bt) toxins are grown widely for pest control, but insect adaptation can reduce their efficacy. The genetically modified Bt toxins Cry1AbMod and Cry1AcMod were designed to counter insect resistance to native Bt toxins Cry1Ab and Cry1Ac. Previous results suggested that the modified toxins would be effective only if resistance was linked with mutations in genes encoding toxin-binding cadherin proteins. Here we report evidence from five major crop pests refuting this hypothesis. Relative to native toxins, the potency of modified toxins was >350-fold higher against resistant strains of Plutella xylostella and Ostrinia nubilalis in which resistance was not linked with cadherin mutations. Conversely, the modified toxins provided little or no advantage against some resistant strains of three other pests with altered cadherin. Independent of the presence of cadherin mutations, the relative potency of the modified toxins was generally higher against the most resistant strains.

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

Insecticidal toxins from Bacillus thuringiensis (Bt) are widely used to control pest insects, but evolution of resistance threatens their continued efficacy. The most common type of Bt resistance (‘Mode 1’) is characterized by recessive inheritance, > 500‐fold resistance to at least one Cry1A toxin, negligible cross‐resistance to Cry1C, and reduced binding of Bt toxins to midgut membrane target sites. Mutations affecting a Cry1A‐binding midgut cadherin protein are linked to laboratory‐selected Mode 1 resistance in Heliothis virescens and Pectinophora gossypiella. Here we show that field‐evolved Mode 1 resistance in the diamondback moth, Plutella xylostella, has a different genetic basis, indicating that screening for resistance in the field should not be restricted to a previously proposed DNA‐based search for cadherin mutations.

Genetic Basis of Resistance to Cry1Ac and Cry2Aa in Heliothis virescens (Lepidoptera: Noctuidae)

Abstract The development of pest resistance to transgenic crop plants producing insecticidal toxins from Bacillus thuringiensis Berliner (Bt) poses a major threat to their sustainable use in agriculture. “Pyramiding” two toxins with different modes of actions in the same plant is now being used to delay the evolution of resistance in the insects, but this strategy could fail if a single gene in a pest confers resistance to both toxins. The CP73 strain of the cotton pest Heliothis virescens (F.) is resistant to both Cry1Ac and Cry2Aa toxins from Bt. We explored the genetic basis of resistance in this strain with a backcross, split-family design. The gene with the largest effect on Cry1Ac resistance in CP73 (BtR-5) maps to linkage group 10 of H. virescens and thus differs from the previously described linkage group 9 BtR-4 resistance found in the YHD2 strain, involving mutation of the gene encoding a 12-domain cadherin-like binding target of the Cry1A toxins. Neither BtR-4 nor BtR-5 seems to confer significant resistance to Cry2Aa. A majority of the linkage groups studied in one backcross family made a small positive contribution to resistance for both toxins. Thus, the Cry2Aa resistance in CP73 is not caused by either of the two major Cry1Ac resistance-conferring genes but instead probably has a quantitative genetic basis.

A Polymerase Chain Reaction Screen of Field Populations of Heliothis virescens for a Retrotransposon Insertion Conferring Resistance to Bacillus thuringiensis Toxin

Abstract The evolution of pest resistance to transgenic crop plants producing insecticidal toxins from Bacillus thuringiensis (Bt) Berliner poses a continuing threat to their sustainable use in agriculture. One component of the U.S.-wide resistance management plan for Bt cotton, Gossypium hirsutum L., involves monitoring the frequency of resistance alleles in field populations. However, existing methods are expensive and may not detect recessive resistance alleles until their frequencies are too high for countermeasures to be effective; therefore, more sensitive methods are needed. The first Bt resistance-causing mutation described at the molecular level was a retrotransposon insertion into the gene encoding a 12-cadherin-domain protein expressed in the midgut of larval Heliothis virescens (F.). We report the first large-scale screen for this mutation using a polymerase chain reaction (PCR)-based approach on >7,000 field-collected individuals. The specific insertion was not detected in any of these samples, nor was it detected in three progeny-tested, field-caught males thought to carry a Bt resistance gene. Unlike the targets of many chemical insecticides where a limited number of resistance-causing mutations compatible with viability can occur; a very large number of such mutations seem possible for the 12-cadherin-domain gene. However, even if these mutations are viable in the laboratory, they may not threaten the effectiveness of transgenic crops because of a high fitness cost in the field. The challenge remains to detect the subset of possible resistance-conferring alleles that are still rare but are viable in the field and increasing due to selection by Bt cotton. This situation will complicate PCR-based Bt resistance monitoring strategies.

Cloning of a putative juvenile hormone-responsive storage protein gene from the tobacco budworm, Heliothis virescens

A cDNA clone with 78% amino acid identity to a basic juvenile hormone (JH)-suppressible hemolymph protein from the cabbage looper, Trichoplusia ni, was isolated from the tobacco budworm, Heliothis virescens. This clone was obtained upon screening a cDNA library derived from larval fat body of a pesticide resistant strain of H. virescens with a cDNA probe for Drosophila melanogaster glutathione S-transferase. By comparison with other insect storage proteins, this clone was predicted to be part of an approximately 2,300 nucleotide (nt) cDNA, of which 691 nt were isolated and sequenced. The partial cDNA clone hybridizes to a RNA of approximately 2,370 nt in H. virescens. Treatment with a juvenoid (2-[1-methyl-2-(4-phenoxyphenoxy)ethoxy] pyridine; pyriproxifen) leads to a decrease in RNA levels of this putative hemolymph storage protein in early fifth stadium larvae of H. virescens, prior to commitment. In contrast, treatment in late fifth stadium (after commitment to pupal development) leads to an increase in the RNA level of this JH-responsive gene. This is the first report of both induction and suppression of storage protein RNA levels in the same stadium. We have given this gene the designation Hv-SP4 (H. virescens, storage protein 4; accession no. U48594). Genetic segregation analysis of restriction fragment length polymorphisms (RFLPs) defined by Hv-SP4 has shown that it is the product of a single-copy, Mendelian, autosomal gene.