Isolating, characterising and identifying a Cry1Ac resistance mutation in field populations of Helicoverpa punctigera

Abstract

Transgenic cotton expressing insecticidal proteins from Bacillus thuringiensis (Bt) has been grown in Australia for over 20 years and resistance remains the biggest threat. The native moth, Helicoverpa punctigera is a significant pest of cotton. A genotype causing resistance to Cry1Ac in H. punctigera was isolated from the field and a homozygous line established. The phenotype is recessive and homozygous individuals possess 113 fold resistance to Cry1Ac. Individuals that carry Cry1Ac resistance genes are rare in Australia with a frequency of 0.033 being detected in field populations. RNAseq, RT-PCR and DNA sequencing reveals a single nucleotide polymorphism at a splice site in the cadherin gene as the causal mutation, resulting in the partial transcription of the intron and a premature stop codon. Analysis of Cry1Ac binding to H. punctigera brush border membrane vesicles showed that it is unaffected by the disrupted cadherin gene. This suggests that the major Cry1Ac target is not cadherin but that this molecule plays a key role in resistance and therefore the mode of action. This work adds to our knowledge of resistance mechanisms in H. punctigera and the growing literature around the role of cadherin in the mode of action of Cry1 type Bt proteins.

Transcriptional Analysis of Cotton Bollworm Strains with Different Genetic Mechanisms of Resistance and Their Response to Bacillus thuringiensis Cry1Ac Toxin

“…The first resistance-conferring mutation of cadherin was found in tobacco budworm Heliothis virescens, where the truncation of a cadherin gene caused by a retrotransposon was linked to resistance against Cry1Ac [18]. Subsequently, a variety of cadherin mutations were found in field Bt-resistant populations of Pectinophora gossypiella, H. armigera and H. punctigera [45][46][47]. Knockout of a H. armigera cadherin gene by CRISPR/Cas9 further confirmed the importance of cadherin disruption in Cry1Ac resistance [43].…”

Temporal Exposure to Bt Insecticide Causes Oxidative Stress in Larval Midgut Tissue

“…Cell death is believed to be a result of cell lysis due to the formation of toxin pores [ 24 , 25 , 26 , 27 ]. Binding of the monomer to CaLP, however, is not necessarily a requirement for toxicity, as Helicoverpa armigera CaLP null-mutants remain susceptible to high toxin doses [ 28 ]. Expressing an ABCC2 transgene from P. xylostella in Drosophila melanogaster larval midguts confers susceptibility to the Cry1Ac toxin, despite D. melanogaster lacking a CaLP orthologue [ 29 ].…”

Dominant point mutation in a tetraspanin gene associated with field-evolved resistance of cotton bollworm to transgenic Bt cotton

“…Because such recessive resistance can be suppressed more readily, nonrecessive resistance is more likely to evolve in the field (8). Nonetheless, most research has focused on recessive resistance conferred by mutations that disrupt binding of Bt toxins in the larval midgut to receptors such as cadherin and ATP-binding cassette transporter proteins (12)(13)(14)(15)(16)(17), whereas little is known about the genetic basis of dominant resistance. Moreover, previous efforts to achieve proactive molecular monitoring of Bt resistance have had limited success because the mutations that increase markedly in the field are usually identified after resistance has caused severe control failures (13,14,16).…”