Research On The Genetics And Molecular Basis Of The Cry1Ah Resistance In Ostrinia Furnacalis

Although transgenic crops producing insecticidal proteins have been effective to manage lepidopteran pests for more than 20 years,evidences from laboratory and/or field show that evolution of insect-resistance is the major threat to transgenic Bacillus thuringiensis?Bt?crops including Cry1Ah-Maize targeting the Asian corn borer?ACB?Ostrinia furnacalis,one of the most important pests on maize.Understanding genetics of resistant trait and its molecular mechanisms are fundamental for governing and establishing proper strategy with suitable tactics.This study was conducted to determine the evolution and the inheritance of Cry1Ah resistance and to identify the molecular basis of the resistance.We evaluated maternal effects,sex linkage and effective dominance with a Cry1Ah resistant strain?ACB-AhR?selected from the susceptible strain?ACB-BtS?.In addition,back-crosses were performed to estimate the number of loci affecting the inheritance.The identification of Cry1Ah-binding proteins from brush border membrane vesicles?BBMV?of these populations was performed by an improved pull-down assay that includes coupling Cry1Ah to NHS-activated sepharose combined with liquid chromatography-tandem mass spectrometry?LC-MS/MS?."Omics"analyses were also performed to examine the difference between ACB-AhR and ACB-BtS strains of ACB at both transcriptional and translational levels.Laboratory selection with Bt-Cry1Ah toxin incorporated in artificial diet had evolved 200-fold resistance after 48 generations of exposure.This resistant colony ACB-AhR readily consumed and survived on Cry1Ah-expressing Bt-maize.Cross-resistance analysis showed high cross-resistance to Cry1F?464-fold?,moderate cross-resistance to Cry1Ab?28.38-fold?,Cry1Ac?22.11-fold?and no cross-resistance to Cry1Ie toxin.This ACB-AhR cross-resistant phenotype is different from ACB-Cry1Fa resistant population that showed no cross resistance to Cry1Ah,suggesting that different mechanisms of resistance were selected in these two populations.Bioassays of reciprocal F₁ crosses-progeny suggested autosomal inheritance of Cry1Ah resistance with no maternal effects.The dominance of resistance increased as concentration decreased.In Cry1Ah-maize tissues the progeny of reciprocal F₁ crosses behaved as functionally recessive.Progenies analysis from backcrosses?F₁×resistant strain?suggested polygenic contribution to Cry1Ah-resistance in ACB-AhR.Pull-down analyses data showed that Cry1Ah bound to alkaline phosphatase?ALP?,cadherin-like ?CAD?,actin,aminopeptidase-N?APN?,prophenoloxidase?proPO?,serine proteinase inhibitor?SPI?,immulectin,and V-ATPase and to other proteins which were not previously characterized as Cry-binding proteins from BBMV samples of ACB-BtS.Analysis Cry1Ah binding to BBMV proteins from ACB-AhR revealed that this toxin did not bind to ALP among few other gut proteins suggesting that these proteins may correlate with the resistant phenotype of this population.In addition,we analyzed the expression in both ACB-AhR and ACB-BtS strains of 12 selected representative genes coding for some of Cry1Ah binding proteins identified.ACB-AhR showed increased gene expression levels of proPO?7.5 fold?,ALP?6.2 fold?and APN?1.4 fold?in comparison to ACB-BtS strain.In contrast,the cad gene showed decreased expression in ACB-AhR strain?0.7 fold?compared with ACB-BtS strain."Omics"analyses performed to examine the difference between ACB-AhR and ACB-BtS revealed that a total of 7,007 mRNAs and 182 proteins were expressed between ACB-AhR and ACB-BtS and 90 transcripts had simultaneous transcription and translation profiles.Down-regulated genes associated with Cry1Ah resistance included APN,ABCC3,DIMBOA-induced cytochrome P450,ALP,glutathione S-transferase?GST?,CAD,and V-ATPase.Whereas,anti-stress genes,such as heat shock protein 70 and carboxylesterase were up-regulated in ACB-AhR,displaying that higher proportion of genes/proteins related to resistance was down-regulated compared to up-regulated.Kyoto Encyclopedia of Genes and Genomes?KEGG?analysis mapped 578 and 29 differentially expressed genes?DEGs?and proteins,to 27 and 10 pathways,respectively?P?0.05?.Furthermore,Real-time quantitative PCR?RT-qPCR?results based on relative expression levels of randomly selected genes confirmed the"omics"response.The DEGs results are valuable for further clarifying Cry1Ah-mediated resistance.These results indicated that ACB would potentially evolve resistance to Cry1Ah maize after seven days of infestation.Cry1Ah resistance in ACB was autosomal,no maternal effects and polygenic as well as functional recessive,which was moderate cross-resistance to Cry1Ab and Cry1Ac,asymmetric cross-resistance to Cry1F,but no cross-resistance to Cry1Ie.The differences in the susceptibility to Cry1Ah toxin in the ACB-AhR strain may be associated with reduced ALP and CAD binding sites.Cry1Ah-resistance in ACB was involved in metabolic and catalytic response,with down-regulations of detoxifying genes and bingding genes,but up-regulations of anti-stress genes.