Alternative Splicing and Highly Variable Cadherin Transcripts Associated with Field-Evolved Resistance of Pink Bollworm to Bt Cotton in India

Abstract

Evolution of resistance by insect pests can reduce the benefits of insecticidal proteins from Bacillus thuringiensis (Bt) that are used extensively in sprays and transgenic crops. Despite considerable knowledge of the genes conferring insect resistance to Bt toxins in laboratory-selected strains and in field populations exposed to Bt sprays, understanding of the genetic basis of field-evolved resistance to Bt crops remains limited. In particular, previous work has not identified the genes conferring resistance in any cases where field-evolved resistance has reduced the efficacy of a Bt crop. Here we report that mutations in a gene encoding a cadherin protein that binds Bt toxin Cry1Ac are associated with field-evolved resistance of pink bollworm (Pectinophora gossypiella) in India to Cry1Ac produced by transgenic cotton. We conducted laboratory bioassays that confirmed previously reported resistance to Cry1Ac in pink bollworm from the state of Gujarat, where Bt cotton producing Cry1Ac has been grown extensively. Analysis of DNA from 436 pink bollworm from seven populations in India detected none of the four cadherin resistance alleles previously reported to be linked with resistance to Cry1Ac in laboratory-selected strains of pink bollworm from Arizona. However, DNA sequencing of pink bollworm derived from resistant and susceptible field populations in India revealed eight novel, severely disrupted cadherin alleles associated with resistance to Cry1Ac. For these eight alleles, analysis of complementary DNA (cDNA) revealed a total of 19 transcript isoforms, each containing a premature stop codon, a deletion of at least 99 base pairs, or both. Seven of the eight disrupted alleles each produced two or more different transcript isoforms, which implicates alternative splicing of messenger RNA (mRNA). This represents the first example of alternative splicing associated with field-evolved resistance that reduced the efficacy of a Bt crop.

Analysis of resistance to Cry1Ac in field-collected pink bollworm,Pectinophora gossypiella(Lepidoptera:Gelechiidae), populations

“…23 Broadly, our results agree with the findings of Fabrick et al 23 with respect to demonstration of Cry1Ac resistance in bioassays and BBMV binding studies in populations sourced from Bollgard fields in the states of Gujarat, Maharashtra and Madhya Pradesh. Further our studies have demonstrated Cry1Ac-resistance in populations sourced from non-Bt fields in these 3 states.…”

“…[15][16][17][18][19] Field-resistance to Cry1Ac expressing Bt cotton (Bollgard Ò ) was reported in PBW populations from Gujarat state (western India) in 2010 and 2011. [20][21][22] Molecular basis of resistance in populations of PBW, sourced from Gujarat state, was examined by Fabrick et al (2014). 23 Their results revealed reduced binding of Cry1Ac to cadherin.…”

“…[20][21][22] Molecular basis of resistance in populations of PBW, sourced from Gujarat state, was examined by Fabrick et al (2014). 23 Their results revealed reduced binding of Cry1Ac to cadherin. Further, they reported 8 novel disrupted alleles of cadherin in these resistant population.…”

“…Further, they reported 8 novel disrupted alleles of cadherin in these resistant population. 23 The common message from these studies is that planting of conventional cotton refuge should receive top most priority in India in order to delay development of resistance in cotton bollworms to Bt crops.…”

Hybridizing transgenic Bt cotton with non-Bt cotton counters resistance in pink bollworm

“…Data reported here for a laboratory-selected resistant strain (AQ47) of pink bollworm derived from the Yangtze River Valley also demonstrate autosomal recessive inheritance of resistance to Cry1Ac in bolls of Bt cotton, F 1 hybrid cotton, and F 2 hybrid cotton (SI Appendix, Table S18). In addition, preliminary data indicate that in several strains of pink bollworm established recently from populations in the Yangtze River Valley, resistance to Cry1Ac is conferred by mutations in the same cadherin gene that is associated with recessive resistance to Cry1Ac in pink bollworm from Arizona and India (32)(33)(34)(35)(36). We used the data from China reported here and the extensive, previously published empirical data on pink bollworm tested in greenhouse or field experiments to estimate the relative fitness of each genotype on Bt cotton and non-Bt cotton (25) (SI Appendix, Tables S12 and S18).…”