Synergistic Effects Of Two Different Bt Receptors On Cry1A And Cry1F Resistance In Plutella Xylostella

Diamondback moth(DBM),Plutella xylostella,is one of the most serious pests of cruciferous vegetables worldwide.The economic loss and control cost caused by P.xylostella have reached $40-50 billion each year.Due to the overuse of insecticides and the biological characteristics of P.xylostella,DBM has developed resistance to almost all chemical insecticides.For the wide use of Bt sprays,P.xylostella has the same resistance problem to Bt and itwas the first pest reported to be resistant to Bt sprays in the field.The interaction between the Bt toxin and the receptors on midgut epithelial cells of lepidopteran larvae is the key process for its toxicity.Accordingly,the loss-of-function mutation of Bt receptor has become the most important resistance mechanism so far.In lepidopteran insects,it is proved that the change of two receptors cadherin and ABC transporter C2/C3 is involved in Bt resistance independently or synergistically and the resistance level is different.The Bt resistance mechanism of P.xylostella is not clear compared with other lepidopteran insects,although the related research started earlier.The critical questions such as types of Bt receptors,interaction between different Bt receptors and cross-resistance between Cryl A and Cry1F in P.xylostella remain to be clarified.In this paper,three single-gene(cadherin gene PxCad,PxABCC2 and PxABCC3)knockout strains and two double-gene(PxABCC2+PxCad,PxABCC2+PxABCC3)knockout strains of P.xylostella were constructed utilizing CRISPR/Cas9 gene editing technology.By detecting the change of sensitivity to Bt toxin in the single-gene knockout strain,the role of these Bt receptors in CrylAb,Cry1Ac and Cry1Fa resistance could be determined.By detecting the change of sensitivity to Bt toxin in the double-gene knockout strain,the question that different receptors where or not have a synergistic effect in Bt resistance could be clarified.Furthermore,Xenopus oocytes system were used to express PxABCC2 and PxABCC3 in vitro,and function of these two receptors in cell membrane perforation of CrylAc was determined based on the cation current recorded by a two-electrode voltage clamp system.These results provide strong evidence for understanding the roles of the two Bt receptors and clarifying the resistance mechanisms to Bt toxin in DBM.1.Synergistic effect of knocking out cadherin and ABCC2 genes on CrylAb resistance in P.xylostellaIn Heliothis virescens and Helicoverpa armigera,mutations in cadherin have been found to cause high level resistance to CrylA toxins.However,the role of cadherin in mode of action of Bt in P.xylostella is still controversial and lack of reverse genetics evidence.In this study,a homozygous PxCad knockout strain Cad-KO was established by CRISPR/Cas9 mediated frameshift mutation technology on susceptible strain WH-10.The bioassay results showed that the Cad-KO was more sensitive to Cry1Fa than WH-10,and the sensitivity to CrylAb and Cry1Ac in Cad-KO were decreased to 2.2-fold and 1.1-fold,respectively.To explore whether PxCad and PxABCC2 have a synergistic effect in Bt toxicity,CRISPR/Cas9 technology was used to knock out PxABCC2 in WH-10 and Cad-KO to establish a PxABCC2 frameshift mutant knockout strain(C2-FM-KO)and double-gene knockout strain Cad/C2KO.The PxABCC2 knockout strain C2-FM-KO was more sensitive to CrylAb,Cry1Ac and Cry1Fa(1.9-to 4.9-fold).Compared with the WH-10,Cad/C2-KO with PxCad and PxABCC2 double-gene knockout produced 3.7-fold and 3.4-fold low level resistance to Cry1Ac and Cry1Fa respectively,meanwhile the resistance to Cry1Ab of Cad/C2-KO was up to 101-fold.Genetic linkage analysis indicated that the resistance of Cad/C2-KO strain to CrylAb was recessive and related to the knockout of PxCad and PxABCC2,and the genotyping result of the backcross offspring implied that PxABCC2 was dominant in the synergistic effect.The above results indicate that PxCad is involed in the toxicity process of Cry1 toxins to varying degrees.PxCad plays a minor role in themechanisms of Cry1Ac and Cry1Fa.However,in the case of CrylAb there is a significant synergistic effect between PxCad and PxABCC2.2.Synergistic effect of knocking out ABCC2 and ABCC3 genes on CrylA and Cry1F resistance in P.xylostellaAbout the role of PxABCC2 and PxABCC3 in resistance to Cry1Ac in P.xylostella,the existing results are not consistent.One of the early studies found that a mutant allele of PxABCC2 was linked to CrylAc resistance,and the transgenic Drosophila melanogaster expressing PxABCC2 gained sensitivity to Cry1Ac subsequently.Recently,CRISPR/Cas9mediated frameshift mutation technology(single sgRNA+Cas9)to perform reverse genetic verification was used to test function of the two Bt receptor genes in two studies,but the results are different.In one of the two studies,knocking out PxABCC2 or PxABCC3 alone could produce about 400-fold resistance to Cry1Ac.Another study indicated that knocking out these two genes alone has little effect on the toxicity of Cry1Ac(<4-fold),while knocking out two genes together can get>8000-fold resistance.In this study,CRISPR/Cas9-mediated gene deletion technology(double sgRNA+Cas9)was used to introduce mutation into WH10 susceptible strain.Two single-gene knockout strains(C2-KO and C3-KO)and one doublegene knockout strain(C2C3-KO)are established,respectively.In these three strains,21.2kb fragment from exon 1 to exon 26 of PxABCC2 was deleted in C2-KO,20.9 kb fragment from exon 3 to exon 24 of PxABCC3 was knocked out in C3-KO,while 43.5 kb of PxABCC2 and PxABCC3 was deleted in C2C3-KO strain.Compared with the susceptible strain WH-10,the two single-gene knockout strains C2-KO and C3-KO only have 1.0-to 2.2-fold resistance to Cry1Ab and 2.1-to 2.9-fold resistance to Cryl Ac.Otherwise the double-gene knockout strain C2C3-KO has>13587-and 10320-fold resistance to Cry1Ab and Cry 1 Ac respectively.The results confirm that simultaneous mutation of two receptor genes has an extremely significant synergistic effect on Cry1Ab and Cry1Ac resistance.This study also indicates that single-gene knockout had no significant effect on Cry 1 Fa,but knocking out both genes could cause 380-fold resistance to Cry1Fa either.CrylAc resistance in the double-gene knockout strain was recessive and genetically linked with the PxABCC2/PxABCC3 loci.Thus,the possible off-target effect caused by gene editing is excluded.In this study,gene editing technology with the completely deletion of the genomic DNA fragments of target gene is employed to avoid other interference effect of frameshift mutation.Therefore,the result is more powerful to support the hypothesis that the functional redundancy in mediating toxicity of Cry1Ac occurs between PxABCC2 and PxABCC3.The result also indicates that simultaneous mutation of PxABCC2 and PxABCC3 can lead to cross-resistance to CrylA and Cry1F in P.xylostella for the first time.3.The functional expression of PxABCC2 and PxABCC3 in Xenopus oocytesBased on the pore-forming model of Bt toxin,protoxin is activated by protease in the insect midgut to become activated toxin,then interacts with the receptor on BBMV to oligomerize,after that the oligomers insert into the cell membrane to form cation selective pores and the balance of osmotic pressure is destroye which could lead to the cell lysis finally.At present,many studies have adopted insect cells to express potential Bt receptor genes and determine whether it is a functional receptor based on the cytotoxicity of Bt toxin.In this study,PxABCC2 and PxABCC3 were expressed in Xenopus oocytes,and the inward cation current changes of the cell membrane induced by Cry1Ac were observed and recorded using a twoelectrode voltage clamp system directly.Under the continuous stimulation of 3 nM Cry1Ac toxin,an increasing inward cation current over time could be observed in the PxABCC2 expressed oocytes,and the maximum current was about 3 μA at 80 s.Under the continuous stimulation of 10 nM Cry1Ac toxin,the maximum current was about 8 μA at 80 s.However,in the PxABCC3 expressed oocytes,the obvious current change could not be observed under the continuous stimulation of 300 nM Cry1Ac toxin.The above results indicate that the heterologous expression of PxABCC2 on the Xenopus oocyte membrane can independently interact with Cry1Ac to form a cation channel,while the ability of PxABCC3 interact with Cry1Ac independently in vitro to form cationic channels is very weak.Here,the effects of expressing PxABCC2 and PxABCC3 on the pore-forming activity of Cry1Ac on the cell membrane were explored by the Xenopus oocyte expression system and electrophysiological techniques for the first time,revealing that the significant difference of the cation channels generated by the interaction of the two receptors with Cry1Ac in vitro.The result provides an important clue for the analysis of functional redundancy of PxABCC2 and PxABCC3 as Cry1Ac receptor in the future.