Mechanisms Of Cry1Ac Resistance In The LF256 Strain And Characterization Of The APN Gene Family Of Helicoverpa Armigera

The cotton bollworm Helicoverpa armigera(Hubner)is one of the most damaging pests in a variety of crops including cotton.A suite of physiological,behavioral,and ecological characteristics enables this insect to develop resistance readily to most insecticides.Resistance to insecticides by the cotton bollworm eventually led to severe outbreaks in the northern China in 1990s,resulting in tremendous economic losses.Transgenic cotton producing Bacillus thuringiensis(Bt)insecticidal protein Cry 1 Ac has been adopted to control this pest in China since 1997,and the adoption rate of Bt cotton increased to 96%in 2015.The planting of Bt cotton for a long period of time and at high adoption rate has been posed strong selection pressure for evolution of Cry 1 Ac resistance in H.armigera.Bioassay data revealed that the percentage of resistant individuals increased from 0.93%in 2010 to 5.5%in 2013 in field populations from six provinces of northern China.Evolution of pest resistance threatens the benefits of planting Bt cotton,therefore the research focused on the genetic and molecular mechanisms of Bt resistance in H.armigera is essential for the establishment of early detection technology and adaptive resistance management strategy.Resistance to Cry1Ac was characterized in a strain(LF256)of H.armigera,which was originated from a field-captured male moth in Langfang,Hebei province of China in 2009.The second-site noncomplementation(SSNC)for Cry 1 Ac resistance was revealed between the cadherin gene(HaCad)and an independently segregating locus of LF256.Furthermore,QTL mapping showed that a novel gene in chromosome 15 was linked with the high level resistance to Cry1Ac in LF256 strain,and also the MAPK-MEK signaling pathway mediated by FGFR may involve in the recessive resistance in LF256 strain.A family of 10 aminopeptidase N genes(APN)of H.armigera was cloned,sequenced and analyzed.A number of splice isoforms with exon skipping were detected in HaAPN1 and HaAPN3 Further study revealed that the formation of circular RNAs may result in the complex transcripts of the two HaAPN genes.The results of the present research is of great significance to understand interactions between different Bt resistance genes,and also to provide important basis to further study the biological functions of the noncoding circular RNAs derived from the APN genes of H.armigera.1.Second-site non-complementation between the LF256 and a strain with cadherin mutation confers resistance to CrylAc in Helicoverpa armigeraThe F1 screen based on the complementation test for allelism has been a key tool for monitoring resistance to Bt crops,isolating resistant strains from field populations.It can detect recessive alleles at the cadherin locus,as well as nonrecessive alleles at any locus based on the survival of the F1 progeny,which was produced by pairing individually the field-captured moth and the resistant SCD-rl moth that is homozygous for the recessive resistance allele r1 at the cadherin locus.As part of an F1 screen,a field-captured male(#256)from Langfang was paried with a female from the resistant strain SCD-r1.Survival of their F1 offspring at the diagnostic concentration was 85.4%,suggesting the male had two resistance alleles at the cadherin locus or he was homozygous for dominant resistance at another locus.Sequencing of the cDNA in the F1 survivors revealed one new cadherin allele from the field-captured male with a 12-bp deletion at 2403 bp and insertions of 4 bp and 7 bp at 4247 bp and 4257 bp of the cDNA sequence,respectively,yielding a premature stop codon in the region coding for the eleventh cadherin repeat(CR11).Here we name this mutant cadherin allele r18.The other cadherin allele from the field-captured male lacks deletions and insertions.Nonetheless,to represent its hypothesized role in resistance,we tentatively name it rx,we establish the strain with homozygous rxrx through family selection and treatment with Cry1Ac,and we named this strain LF256.Compared with SCD,resistance to Cryl Ac in LF256 was autosomal,recessive,and 220-fold relative to susceptible strain SCD,and also,LF256 had>67-fold cross-resistance to CrylAa and 34-fold cross-resistance to CrylAb but lacking cross resistance to Cry2Ab.We conducted complementation tests by crossing LF256 with strain SCD-rl,in which resistance to CrylAc is conferred by the recessive r1 allele at the cadherin locus.The resulting F1 offspring were resistant,suggesting that resistance to Cryl Ac in LF256 is also conferred by resistance alleles at this locus.However,the HaCad amino acid sequence had no conserved changes in LF256 compared with SCD.Moreover,Cry1Ac resistance was not genetically linked with the HaCad locus in LF256.In addition,LF256 and SCD were similar in levels of HaCad transcript,cadherin protein,and binding of Cry 1 Ac to cadherin.Overall,the results imply that LF256 strain has a recessive resistance gene which segregated with HaCad independently,and this gene fails to genetically complementary with HaCad indicating SSNC occurs between the two genes.Although SSNC has received little or no previous attention in the resistance management literature,it is well documented in model organisms and should be considered when interpreting results of the widely used F1 screen for resistance.2.Genetic mapping and mechanism of resistance to CrylAc in LF256 strain After repeated selection with Cry1Ac toxin for 4 years,the resistance levels to CrylA toxins were increased in the selected LF256 than the original LF256 strains.Compared with the susceptible SCD strain,resistance levels in the selected LF256 strain increased to 702-fold for Cry1Ac,213-fold for Cry1Aa,56-fold for Cry1Ab and 9.5-fold for Cry2Ab.Dominance levels of CrylAc resistance were changed from recessive in LF256 to non-recessive in the selected LF256.The increase of the resistance levels and the change of the dominance levels indicated a new resistance gene was selected for besides the original recessive resistance gene during the repetitive exposure to Cry1Ac,and this selected strain was named as LF256-P.A QTL approach based on bulk segregant analysis(BSA)was deleveloped to map the loci containing major resistance genes in the LF256-P strain.Genetic mapping results revealed one region(4.4 Mb-11.9 Mb)in chromosome 15 and two regions(2.3 Mb-3.3 Mb,6.7 Mb-9.4 Mb)in chromosome 9 associated with resistance to CrylAc in LF256-P.Eleven SNP markers(nine in the candidate regions and another two outside the candidate regions)were sequenced for each individual from the two mapping families to verify their linkage with resistance,and the results were consistent with that obtained from the BSA approach.In order to clarify which resistance gene derived from the initial LF256 strain,a marker-assisted selection method was used to separate resistance genes in the two chromosomes.Two strains with different resistance alleles were established:LF256-9R(resistance allele derived from Chr09)and LF256-15R(resistance allele derived from Chr15).Compared with SCD,LF256-9R has 50-fold non-recessive resistance to CrylAc and 6.4-fold cross-resistance to Cry2Ab.LF256-15R has 160-fold recessive resistance to Cry1Ac,but no cross-resistance to Cry2Ab.According to different resistance patterns of the two strains,it was concluded that the resistant allele in the chromosome 15 was originated from the initial LF256 strain and the resistant allele in the chromosome 9 was newly selected.The results of complementation tests of the two resistance strains with the SCD-rl strain(with mutated HaCad)also support the above conclusion.For consistency with previous studies,LF256-15R was renamed as LF256.Several genes including ABCC2,MAP4K4 and ABCC3 within the candidate region in chromosome 15 were further studied in the LF256 and SCD strains.The amino acid sequences of these genes in LF256 were intact and without any conserved amino acid substitutions.Further,the transcript levels of the receptor genes were not significantly decreased in LF256 compared with SCD.Overall,these results indicated a novel gene in chromosome 15 is responsible for the high level resistance in the LF256 strain.The RNA-seq analysis and the bioassays with inhibitor suggest that the MAPK-MEK signaling pathway mediated by FGFR may be involved in Cry1Ac resistance of LF256.The identification of the resistance gene of LF256 is of great significance for the improvement of the present mechanic models of Bt toxins.3.Circular RNAs are involved in the novel splicing isoforms of Aminopeptidases N genes of H.armigeraAminopeptidases N(APN),a group of membrane-bound aminopeptidases with non-stringent substrate specificity,is widely known as putative receptors of Bacillus thuringiensis(Bt)Cry toxins.The cDNAs of three APN genes(HaAPN1,HaAPN2 and HaAPN3)located in the same APN gene cluster of H.armigera were cloned and sequenced.Twenty and thirteen novel splicing isoforms with exon skipping were detected in HaAPNl and HaANP3 respectively.Further study identified sixteen and seven circular RNAs in HaAPNl and HaAPN3 respectively.Neither novel splicing isoforms nor circular RNAa were detected in HaAPN2.Different from the conventional GT/AG splicing signal,short repeat sequences in the same direction were involved in the splicing of the linear and circular isoforms of HaAPNl and HaAPN3.It is suggested that the circular RNAs may be related to formation of the novel splicing isoforms of HaAPN1 and HaAPN3.4.Structural,evolutionary and expressional analyses of the ten aminopeptidases N genes in H.armigeraA number of studies indicated that lepidopteran APN 1 genes were putative Bt receptors and involved in resistance to Bt Cry toxins in several insects.In several lepidopterans including Bombyx mori,Danaus plexippus,Spodoptera frugiperda and Plutella xylostella,about 10 APN genes were located in a single chromosome(homologous to Chr9 of B.mori)as a gene cluster.According to syteny and sequence similarity of APNs among lepidopteran insects,it is indicated that these APN genes arose by gene duplication events prior to the most recent common ancestor of Lepidoptera.Ten ANP genes(from HaAPN1 to HaAPN10)were annotated in the genome of H.armigera.Expressional analysis of the 10 APN genes showed that the mRNA of the 10 APN genes was expressed at similar levels throughout larval development,while nearly no expression in eggs,pupa and adults.Most APN genes were highly expressed in the midgut except for the HaAPN7,-9,-10.Similar to HaAPNl and HaAPN3,novel splicing isoforms resulted from post-transcription modification were also found in HaAPN4,-6,-8,and-9,indicating this phenomenon may be common in the APN family of H.armigera.