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

Tom Walsh,Juan Ferré,Maissa Chakroun,Bill James,Sharon Downes

doi:10.1038/s41598-018-21012-w

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.

Highlights

Transgenic cotton, Gossypium hirsutum L. varieties expressing Bacillus thuringiensis (Bt) toxins have been grown around the world since 19961
While it was thought that H. armigera posed a greater threat than H. punctigera to the continued effectiveness of Bt proteins, in both species resistance alleles have been detected to all three Bt proteins currently deployed
Pooling the data from the three seasons yields a frequency of Hp9-3784-like Cry1Ac resistance alleles in H. punctigera of 0.033 with 95% credibility intervals of 0.021 and 0.047

Summary

Introduction

Transgenic cotton, Gossypium hirsutum L. varieties expressing Bacillus thuringiensis (Bt) toxins have been grown around the world since 19961. Larvae of H. armigera and H. punctigera typically survived on post-squaring plants of the first generation single toxin Cry1Ac cotton due to poor expression, and for short periods post-squaring can develop to adults on dual-toxin Cry1Ac and Cry2Ab cotton presumably due to sub-optimal expression[17]. Both of these scenarios represent opportunities for resistance to Cry1Ac to be selected.

Methods

Results

Conclusion