Identifiction And Detection Of Mutated Cadherin Alleles Associated With Cry1Ac Resistance In Helicoverpa Armigera (H(?)bner)

Cotton bollworm. Helicoverpa armigera (Hubner) (Lepidioptera: Noctuidae) is one of the most important agricultural pests in the old world including China. The serious outbreaks of cotton bollworm in the cotton areas in China caused tremendous economic losses. Since 1980s, the massive use of chemical insecticides resulted in serious resistance problem in cotton bollworm. The resistance of cotton bollworm, especially to pyrethroids was one of the reasons for the outbreaks of cotton bollworm in Northern China during 1990s. Cotton bollworm has been controlled effectively since the Bt cotton was planted in China. But continuous expression of Bt toxin in the whole development time of cotton plant and more than ten-year planting of Bt cotton will impose the cotton bollworm under a strong selection pressure by Bt toxin and increase the risk of resistance of cotton bollorm to Bt cotton.Under selection in the lab, many strains of cotton bollworm developed resistance to Bt toxins. Evolution of Bt resistance by cotton bollworm in the field will affect the longevity of Bt cotton. The studies on the mechanisms of Bt resistance could provide guidance on the management of cotton bollworm. Cadherin is one of the important receptors in Bt action and the mutation of cadherin may cause the resistance of pests to Bt toxin. So far, at least in 3 pests the mutations of cadherin were associated with the resistance to Bt toxin. Particularly, disruption of the cadherin gene (Ha_BtR) by a premature stop codon was associated with a high level of Cry1Ac resistance in H. armigera. In this study, the mutations of cadherin involved in Cry1Ac resistance were researched in both the laboratory-selected GYBT strain and field-collected populations of H. armigera. The study will facilitate understanding of the molecular mechanism of Cry1Ac action in cotton bollworm and prediction of the mechanism of resistance of cotton bollworm to Cry1Ac. The work will also provide technical support on Bt resistance monitoring and resistance management in cotton bollworm.1. Identification and detection of a mutated cadherin allele in the laboratory-selected GYBT strain of H. armigeraThe genomic organization of Ha_BtR was compared from both the susceptible and resistant strains and a deletion was found to be responsible for a truncated cadherin protein in the resistant GYBT strain. Determination of the genomic DNA sequences of Ha_BtR gene showed that the wild type Ha_BtR coding sequence (Genbank No:DQ523166) is comprised of 34 exons. A deletion between Exon 8 and Exon 25 was found to be responsible for a truncated cadherin (Genbank No:DQ523167) in the Cry1Ac-resistant GYBT strain of H. armigera. A DNA-based detection method specific to the r1 allele was developed. This study will facilitate the monitoring of cadherin mutant frequency in field populations of H. armigera.2. Identification and detection of Ha_BtR mutations in field populations of H. armigeraA deletion mutation allele (r1) of a cadherin gene (Ha_BtR) was previously identified as genetically linked with Cry1Ac resistance in a laboratory-selected strain of H.armigera. Using a biphasic screen strategy, we successfully trapped two new cadherin alleles (r2 and r3) associated with Cry1Ac resistance from a field population of H.armigera collected from Anyang, Henan province of China in 2005. The r2 and r3 alleles, respectively, were created by inserting the long terminal repeat of a retrotransposon (designated HaRT1) and the intact HaRT1 retrotransposon at the same position in exon 8 of Ha_BtR, which results in a truncated cadherin containing only two ectodomain repeats in the N terminus of Ha_BtR. This is the first time that the Bt resistance alleles of a target insect of Bt crops have been successfully detected in the open field. This study also demonstrated that bollworm larvae carrying two resistance alleles can complete development on Bt cotton. The cadherin locus should be an important target for intensive DNA-based screening of field populations of H.armigera.Using an F1 screen method,10 candidate and 4 putative candidate single-pair families were selected for Ha_BtR mutation analyses from a field population of H. armigera collected from Langfang, Hebei Province of China in 2009. Through cloning and sequencing of cadherin alleles, at least 5 new mutated alleles of Ha_BtR were found compared with the wild cadherin.117#:an insertion resulting in 30 animo acid insertion at 754aa or premature stop at 751aa; 239#:705bp deletion caused 235 animo acid deletion in the EC11-CYT domain; 234#:alternative splicing caused deletion of the EC2-6 domain; 256#:4 animo acid YTIT deletion; 124#:165bp deletion resulting in deletion of 55 animo acids in the CYT domain. These five mutations associated with Cry1Ac resistance can impair the normal fuction of cadherin. Cadherin alleles with amino acid substitutions were also found to be associated with Cry1Ac resistance. The role of the animo acid substitutions should be investigated in future. In conclusion, the mutation diversity of Ha_BtR will complicate resistance allele frequency monitoring in H. armigera.3. Construction of a pair of near-isogenic strains with and without Ha_BtR r1 allele and its cross resistance patternThe r1 allele of Ha_BtR was introgressed into a susceptible SCD strain by crossing the GYBT strain to the SCD strain, followed by repeated backcrossing to the SCD strain and molecular marker assisted family selection. The introgressed strain (designated as SCD-r1, carrying homozygous r1 allele) obtained 438-fold resistance to Cry1Ac,> 41-fold resistance to Cry1Aa and 31-fold resistance Cry1Ab compared with the SCD strain; however, there was no significant difference in susceptibility to Cry2Aa between the integrated and parent strains. It confirms that the loss of function mutation of Ha_BtR alone can confer medium to high levels of resistance to the three Cry1A toxins in H. armigera. Reciprocal crosses between the SCD and SCD-r1 strains showed that resistance to CrylAc in the SCD-r1 strain was completely recessive.Modified toxin Cry1AcMod, CrylAbMod and the protoxin CrylAcPro and Cry1AbPro countered the resistance in the resistant SCD-r1 strain. Resistance to Cry1AcMod, Cry1AbMod, Cry1AcPro and Cry1AbPro was significantly reduced when compared with resistance to the activated Cry1Ac and Cry1Ab respectively. These modified toxins which can counter cadherin-based resistance may become the candidate genes engineering into the transgenic cotton.4. Susceptibility monitoring of field populations of H. armigera to Cry1AcUsing the toxin overlay method, susceptibilities of H. armigera populations from main cotton areas of China were monitored during 2006-2008. Limited variation (less than 6-fold) in Cry1Ac susceptibility was found in the 12 field populations sampled during 2006-2008. Ten populations (LC50:0.063～0.162μg cm-2) collected from Yellow River cotton area showed less susceptibility than that of two populations (LC50:0.027～0.049μg cm-52) collected from Xinjiang cotton area. Although there are no signifianct changes in Cry1Ac susceptibility in the field populations of H. armigera colleted in this study, we should keep an eye on resistance evolution of H. armigera to Bt cotton in China. Development of effective techniques for resistance gene frequency detection should be a priority for Bt resistance research in H. armigera.