Resistance Mechanism Of Several Major Lepidopteran Pests To Bacillus Thuringiensis Protein

Bacillus thuringiensis proteins are important biological tools for controlling major agricultural insect pests such as Helicoverpa armigera,Spodoptera frugiperda and Agrotis ipsilon.Insects have a strong capacity of developing genetic resistance which can restrict the prolonged use of the same insecticidal toxins.Insecticidal Crystal Proteins(Cry)and vegetative insecticidal proteins(Vip)are the two main types of Bt toxins.The insecticidal action of Cry protein and the mechanism of insect resistance to Cry protein are comparatively well known,however,the information about the mode of insecticidal action and mechanism of insect resistance of vegetative insecticidal proteins(Vip)is still limited.In this thesis,the detailed information about the structure,function and mode of action of Vip3 A proteins are carefully reviewed to update the current knowledge and to focus on the future research needs.In this study,the CRISPR/Cas9 mediated genome editing system has been used to study the genetic basis of H.armigera resistance to Cry2 Ab toxin.The result revealed that a 4-nucleotide deletion in the ATP-binding cassette transporter ABCC1 confers >60-fold resistance to Cry2 Ab in H.armigera and the inheritance of this resistance is completely recessive.Scavenger receptor-C(SR-C)which is reported as a receptor of Vip3 A protein has been knocked out in S.frugiperda and A.ipsilon using CRISPR/Cas9 mediated genome editing system to study whether it is involved in the susceptibility to Vip3 A toxin.The bioassay result revealed that the SR-C knockout strain showed 1.36-and 4.70-fold sensitivity to Vip3 A toxin,respectively,which suggested that the SR-C does not serve as a functional receptor of Vip3 A and is not involved in the mode of action Vip3 A in S.frugiperda and A.ipsilon.1 CRISPR/Cas9 mediated knockout of ATP-binding cassette transporter ABCC1 confers resistance to Bacillus thuringiensis toxin Cry2 Ab in Helicoverpa armigeraInsect resistance to Bt toxins poses a significant risk to the cultivation of genetically modified crops expressing Bt proteins.The Insect ATP-binding cassette(ABC)transporter functions as a receptor of Bt toxins and mutations in these transporters are associated with Bt resistance.In this study,CRISPR/Cas9 genome editing tool has been used to knockout the ABC transporter ABCC1 to study the genetic basis of H.armigera resistance to Bt toxin.Here,we successfully created the Ha ABCC1-KO strain which is homozygous for 4-nucleotide deletion in exon 2 of Ha ABCC1.Bioassay results revealed that the Ha ABCC1-KO strain exhibited >60-fold resistance to Cry2 Ab toxin compared with the susceptible XJ strain while no resistance has been recorded for Cry1 Ab,Cry1Ac and Vip3 A.Furthermore,inheritance of H.armigera resistance to Cry2 Ab was autosomal and complete recessive which was significantly associated with 4-nt deletion mutation of ABCC1 in the Ha ABCC1-KO strain.The relative growth of Ha ABCC1-KO and XJ strain also confirmed the role of Ha ABCC1 in the toxicity of Cry2 Ab.This in vivo functional study confirmed that the Ha ABCC1 functions as a receptor of Cry2 Ab and mutation in this receptor was linked to Cry2 Ab resistance in H.armigera.2 Scavenger receptor-C(SR-C)does not mediate the insecticidal activity of Vip3 A against Spodoptera frugiperda and Agrotis ipsilonBacillus thuringiensis vegetative insecticidal proteins are successfully used to control major agricultural pest.The insecticidal mechanisms of Vip3 A toxins are less well known than those of insecticidal crystal proteins.To date,only scavenger receptor-C(SR-C)has been reported as a receptor of Vip3 A toxins.However,the role of SR-C in Vip3 A toxin mode of action is unclear in Spodoptera frugiperda and in other insects.Here,we used CRISPR/Cas9 mediated genome editing system to provide in vivo evidence whether the SR-C is involved in the susceptibility to Vip3 A toxin in S.frugiperda and A.ipsilon.We have created Sf-SR-C-KO strain that is homozygous for 10-nucleotide deletion in exon 1 of Sf-SR-C,and Ai-SR-C-KO strain that is homozygous for 587-nucleotide deletion in exon 4 and exon 5 of Ai-SR-C.The bioassay results suggested that the Sf-SR-C-KO and Ai-SR-C-KO strain showed 1.36-and 4.70-fold sensitivity to Vip3 A toxin compared to their susceptible wild type strain.This result suggest that the SR-C is not serve as a functional receptor of Vip3 A and is not involved in the mode of action Vip3A in S.frugiperda and A.ipsilon.