Functional Study Of SeABCC2 And SeVipR Genes In Mediating Bt Toxicity In Spodoptera Exigua

The Spodoptera exigua,one of the representative species of Spodoptera,is a kind of serious agricultural pests with worldwide distribution.It hosts more than 170 kinds of plants,including cruciferous vegetables of gramineae and grass plants,which are usually harmful to corn,cotton,peanut,soybean and vegetables.In recent years,the damage of S.exigua has increased significantly in North China,seriously affecting the planting of economic crops such as corn,peanuts,soybeans and vegetables.Since 1997,Bt cotton(expressing Cry1Ac protein)has been commercialized to control the damage caused by H.armigera and P.gossypiella in China.But because of the low toxicity of Cry1Ac protein expressed in Bt cotton to the Spodoptera pests,so S.exigua and S.litura,as the secondary pests in cotton field,become more harmful.Meanwhile,S.frugiperda has rapidly expanded to 21 provinces in China since it invaded Yunnan in January 2019 and becomes the seriously threaten to corn in China.Bt corn with effective control to S.frugiperda has been planted in USA,Brazil and other countries for nearly 20 years,but S.frugiperda has developed resistance to Bt corn in Puerto Rico,Brazil and Argentina et al.Therefore,further study on the resistance mechanism of S.exigua to Bt toxin has great significance for the resistance management of Spodoptera pests,especially S.frugiperda,and it can also provide references for the development and application of transgenic Bt maize in China.At present,studies on mode of action and resistance mechanism of Bt toxin in S.exigua mainly focus on functional identification of Bt receptors,and the related genes including SeABCC2,SeABCC3,SeCadl and SeAPN1.Previous studies usually used RNAi and in vitro expression techniques to verify the function of Bt receptors in the process of Bt action,but the conclusions of different studies are inconsistent or even opposite.In this study CRISPR/Cas9 gene editing technology were used to knock out five candidate Bt receptor genes in a susceptible strain(WH-S)of S.exigua,and five homozygous knockout strains named SeAPN1-KO,SeCad1-KO,SeABCC1-KO,SeABCC2-KO and SeABCC3-KO were constructed.The role of these five genes in mediating Bt toxicity was evaluated based on the bioassay results in vivo.It is confirmed that S.exigua with the knockout of SeABCC2 shows resistance to both Cry1Fa and Cry1Ac,while the knockouts of other four candidate genes had no significant effects on Bt susceptibility.It is speculated that SeABCC2 gene plays an important role in the process of toxicity of Cry1Fa and Cry1Ac toxins.Further,four strains with SeABCC2 indel mutants at different positions were constructed with the CRISPR/Cas9 technology and the phenotype and inheritance of resistance in these four mutant strains were compared.The results indicate that the different mutants of SeABCC2 could have a different degree of resistance dominance.In vitro cytotoxicity measurements using baculovirus-insect cell expression system indicated that SeABCC2 played an important role in mediating Cry1Ac toxicity while SeABCC3 played no or limited effect.In addition,CRISPR/Cas9 gene editing technology was used to verify the role of Vip3Aa toxin resistance-related gene SeVipR,providing a reference for the future study on the mode of action of Vip3Aa toxin.1.Effects of the knockout of five Bt receptor candidate genes on Bt toxin sensitivity in S.exiguaIn several lepidopterans,Cadherin,ABCC2 and APN1 have been identified as the functional receptors or binding proteins.Here we used CRISPR-mediated knockouts to evaluate the role of five genes encoding candidate Bt toxin receptors in S.exigua.We compared susceptibility to Bt toxins Cry1Fa,Cry1Ac and Cry1Ca between the parent susceptible strain and each of five homozygous knockout strains of the candidate genes(SeAPN1,SeCad1,SeABCC1,SeABCC2 or SeABCC3).The results from the 15 pairwise comparisons reveal that SeABCC2-KO has 240-fold and>470-fold resistance to Cry1Fa and Cry1Ac,respectively,and SeCadl-KO has 3-fold resistance to both Cry1Fa and CrylAc,it means SeABCC2 has a major role and SeCadl a minor role in mediating toxicity of Cry1Fa and Cry1Ac.The knockout of SeAPN1,SeABCC1,or SeABCC3 had no effect on the toxicity of Cry1Fa and Cry1Ac.In addition,the results imply little or no role in toxicity of Cry1Ca for the five candidate receptor genes.2.The phenotype and inheritance of resistance to Cry1Ac and Cry1Fa in four SeABCC2 indel mutants at different positionsThe above study indicates that the loss of function mutation of SeABCC2 could resulte high level resistance to Cry1Fa and Cry1Ac,suggesting that SeABCC2 play a key role in mediating the sensitivity of S.exigua to Cry1Fa and Cry1Ac.It has been found that mutations in the HaCad gene in the extracellular region of H.armigera lead to a high level of recessive resistance to Cry1Ac,while a deletion mutation in the intracellular region leads to a moderate level of incomplete dominant resistance to Cry1Ac.In order to explore the influence of mutations at different sites of SeABCC2 gene on the phenotype and inheritance of resistance to Bt toxin Cry1Fa and CrylAc,CRISPR/Cas9 gene editing technology was used to establish four SeABCC2 gene mutant strains,C2-N,C2-M,C2-C with the mutation sites in the N-terminal,middle and C-terminal of protein,respectively,and C2-NULL with complete deletion of coding sequence).All four SeABCC2 mutant knockout strains showed high level of resistance to Cry1Fa and Cry1Ac,but no significant susceptibility change in Cry1Ca.It was interesting that the resistance of C2-N to Cry1Fa and Cry1Ac is incomplete dominant,while the other three knockout strains are all incomplete recessive.Recessive phenotypes are generally generated by functional loss mutations of genes,while dominant phenotypes are generated by functional gain mutations of genes.The resistance of C2-M,C2-C and C2-NULL was not completely recessive,suggesting that the mutations of SeABCC2 of the three knockout strains may cause the loss of all functions of the SeABCC2 protein due to structural damage.However,the mutation of SeABCC2 gene in C2-N strain may result in the structural change of SeABCC2 protein and obtained unknown new functions,resulting in incomplete dominance of resistance.The above results also suggest that specific resistance genes of pest may be adapted to different selection pressure scenarios under field conditions by evolving different mutation types.3.The heterologous expression and functional identification of SeABCC2 and SeABCC3 in S.exiguaIn Helicoverpa armigera,HaABCC2 and HaABCC3 genes were found as two ABC transporter proteins with functional redundancy in mediating toxicity of CrylAc.The CRISPR-mediated knockouts of HaABCC2 and HaABCC3 together caused>15,000-fold resistance to Bt toxin Cry1Ac,whereas knocking out either HaABCC2 or HaABCC3 alone had little or no effect.But in the similar knockout experiment in S.exigua,only the SeABCC2 knockout strain showed high levels resistance to Cry1Ac toxin,but the SeABCC3 knockout strain showed no significant change in the sensitivity of Cry1Ac toxin,suggesting that the ABCC3 gene was not involved in the action of Cry1Ac toxin.In order to verify the role of ABC transporter genes in mediating Bt toxicity in S.exigua.SeABCC2,SeABCC3 and three SeABCC2 mutant proteins(C2-N,C2-M,C2-C)were expressed in Sf9 cells using Bac-to-Bac baculovirus expression system.Cytotoxicity assay showed that the LC50 of Cry1Ac to those Sf9 cells expressing SeABCC2-WT was 0.048 nM(95%CL:0.018 nM-0.150 nM),otherwise the LC50 of Cry1Ac to those Sf9 cells expressing SeABCC3-WT was 11.0 nM(95%CL:6.430 nM-18.884 nM),there is 229-fold difference.So the in vitro results about SeABCC2,SeABCC3 are consistent with the in vivo results,and it is confirmed again that SeABCC2 play an important role in mediating Bt toxicity in S.exigua.Compared with Sf9 cells expressing SeABCC2-WT,those cells expressing C2-N,C2-M mutants had low sensitivity to Cry1Ac,it is suggested that the N-terminal and middle position mutation could result in the loss of function of the entire SeABCC2 protein.The cytotoxicity results of these two ABCC2 mutants are also consistent with the in vivo results.However,the sensitivity of Sf9 cells expressing C2-C mutant did not change significantly,this result needs to be explored further.4.Effect of knockout of SeVipR in S.exigua on the susceptibility to Bt toxin Vip3AaVip3Aa is another Bt toxin with high toxicity to insects of Noctuidae.It is secreted extracellularly during the vegetative growth stage of Bacillus thuringiensis,so Vip3Aa is different with Cry toxin in protein structure and mode of action and there is no cross resistance between the two toxins.HaVipR gene has been identified play an important role in the resistance to Vip3Aa in H.armigera.In the present study,a VipR gene from transcriptome based on its similarity to HaVipR gene was identified and named as SeVipR.SeVipR was knockouted utilizing the CRISPR/Cas9 system and a homozygous knockout strain named SeVipR-KO was set up.Toxicity response of the susceptibility strain(WH-S)and knockout strain(SeVipR-KO)to Vip3Aa and Cry1Ac showed that SeVipR-KO exhibited significant resistance to Vip3Aa(>235-fold)compared with WH-S,but no resistance to Cry1Ac.Genetic analysis indicated that resistance in SeVipR-KO is nearly completely recessive and tightly linked with the SeVipR locus.These results imply that SeVipR is involved the resistance to Vip3Aa in S.exigua and do not have cross-resistance with Cry1Ac.