Mechanism Of Pink Bollworm Resistance To Transgenic Bt Cotton Mediated By Cadherin Alleles R17 And R18

Transgenic cotton producing insecticidal proteins from Bacillus thuringiensis?Bt cotton?has effectively suppressed major targeted pests including pink bollworm and cotton bollworm since its commercialization in 1997 in China.However,vast plantings of Bt cotton caused continuous selection pressure to targeted pests,especially for the pink bollworm,which feeds almost exclusively on cotton.Resistance monitoring data in recent years has revealed a significant increase in resistance ratio to Bt cotton in several field populations of pink bollworm in the Yangtze River Valley,posing a threat to the sustainable use of Bt cotton.Ahead of solving this problem,how the resistance level of pink bollworm to Bt cotton develops and the mechanism of resistance in pink bollworm to Bt cotton remain to be further studied.In this study,we monitored the susceptibility of pink bollworm to Cry1Ac by diet bioassays as well as resistance allele frequency by DNA screening methods using both laboratory strains and field populations of pink bollworm,and established two resistant strains of pink bollworm from field populations in the Yangtze River Valley.Relevant resistant alleles were cloned and identified,and the inheritance of resistant alleles was analysed.The mechanism of resistance was finally revealed at genic,genetic and cellular levels.In addition,DNA-based detection methods were established for two resistant alleles.Main results of this study are listed as below:1. The susceptibility of pink bollworm field populations to Cry1Ac was determined by incorporate Bt toxin-diet bioassays,and results indicate that the susceptibility of pink bollworm to Bt cotton has recovered compared with previous bioassay data in 2008-2010.The annual average LC₅₀ values of pink bollworm to Cry1Ac ranged from 0.23 to 0.27?g/m L,and the annual average resistance ratios?RR?ranged from 3.2 to 4.5 folds?mean=3.8?,showing a crest in 2014.2. DNA screening results indicate that resistance level of pink bollworm remains low to Bt cotton in the Yangtze River Valley. The resistance allele frequency of 7 cadherin resistance alleles?r1,r2,r3,r13,r14,r15,r16?were detected by PCR-based DNA screening method in pink bollworm from the Yangtze River Valley in 2012-2015.The total resistance allele frequencies were 0.0105,0.0044,0.0023,0.0046,respectively,in 2012-2015,with a magnitude range of 10^-3?10^-2.The resistance allele frequency showed a significant decrease in 2013-2015 compared with that in 2012.Among 194 resistant alleles detected in 175 resistant individuals,r1 accounted for 71%of all the resistant alleles detected,indicating r1 a dominant resistant allele among the 7 cadherin resistant alleles in the Yangtze River Valley.All the resistant alleles were detected except r3.3. Two Cry1Ac-resistant pink bollworm strains?TM6 and AQ17?were screened and established from field populations in the Yangtze River Valley using F1 detection and F2detection methods.The two strains showed a 281.6-fold and 278.5-fold resistance to Bt Cry1Ac toxin,respectively.Resistance ratio of TM6 and AQ17 was only 0.8-fold and 1.7-fold to Bt toxin Cry2Ab,respectively,indicating no cross-resistance between Cry1Ac and Cry2Ab in pink bollworm.Both two resistant strains were not susceptible to Bt toxin Vip3Aa.Results of reciprocal cross tests showed that the inheritance of resistance alleles r17 and r18 were autosomal recessive.Backcross experiments showed that r17 and r18were closely linked to Cry1Ac resistance.4. Sequence analysis showed that Cry1Ac resistance of r17,r18 were both mediated by the deletion mutation in Cadherin Repeat region. The full lengths of c DNAs of r17 and r18 were 5206 bp and 4051 bp,respectively,resulting in a 2 bp and 1157 bp deletion,respectively,compared with wildtype s Pg Cad1.Non-3-fold base pair deletions entailed a frameshift in subsequent sequence translations and introduced pre-mature stop codons.Therefore,cadherin protein translations were terminated prematurely,resulting in severe truncation of cadherin and incomplete protein structure.Pg Cad1 of r17?r18 encode only211 AA and 1278 AA,respectively.Protein domain analysis indicated that both of them lacked the structures of partial cadherin repeats region,the membrane-proximal region,transmembrane and intracellular regions.DNA deletion and substitution in the Pg Cad1g DNA of r17 and r18 entails DNA deletion or mis-splicing of m RNA.5. Functional validation of r17 and r18 was performed in an insect cell line Tn H5.Sub-cellular localization showed that fusion proteins including susceptible cadherin protein?s Pg Cad1-EGFP?mainly located on the cell membrane;while fusion proteins including resistant cadherin protein?r17Pg Cad1-EGFP and r18Pg Cad1-EGFP?located on the endoplasmic reticulum instead of cell membrane.This confirms that the absence of transmembrane region in cadherin protein caused failure to anchor on the cell membrane.The cytotoxicity assay showed that Tn H5 cells expressing r17Pg Cad1-EGFP and r18Pg Cad1-EGFP fusion proteins were not susceptible to 40?g/m L Cry1Ac treatment,and there was no swelling,floating or rupture symptoms,which appeared in Tn H5 cells expressing wild type s Pg Cad1-EGFP fusion proteins to 10?g/m L Cry1Ac treatment.This proves r17 and r18 were resistant to Cry1Ac.6. Cotton boll bioassays were deployed to investigate the growth and development of both resistant and susceptible pink bollworm strains on three cotton varieties.Results demonstrated that TM6 and AQ17 pink bollworm individuals could survive and complete life cycle on both GK19 and CV163 cotton bolls,indicating that pink bollworm resistance to Cry1Ac was associated with field resistance.However,TM6 and AQ17 strains showed some disadvantages compared with survival on Simian3 cotton bolls,such as lower survival,prolonged larval developmental period,reduced pupa weight and decreased fertility.7. Molecular detection methods of r17 and r18 in pink bollworm were established.Allele-specific primers were designed according to sequence differences between r17,r18and the wild type,to detect these 2 resistance alleles and to discrimate genotypes from field populations of pink bollworm.Results above reveals the annual changes of pink bollworm susceptibility to Cry1Ac as well as resistance allele frequency to Bt cotton in the Yangtze River Valley of China,clarifies the mechanism of pink bollworm resistance to Cry1Ac mediated by r17 and r18cadherin mutation,and enriches resistance monitoring methods of pink bollworm to Bt cotton.This study provides a scientific reference for reasonable resistant management tactics of bollworm resistance to Bt cotton.