Study On The Insecticidal Activity And Mechanism Of Bt Proteins Against Yellow Peach Moth,conogethes Punctiferalis(guenée)(lepidoptera: Crambidae)

Transgenic maize expressing Bt insecticidal proteins have been commercialized in some countries since 1996,and the estimated acreage is increasing gradually.However,the evolution of resistance within target pest populations poses a challenge to Bt maize's long-term use and success.Although Bt maize has not yet been commercially planted in China,the Ministry of Agriculture and Rural Affairs of the People's Republic of China issued safety certificates for several Bt maize events in2020-2021.Developing different events of Bt maize is a significant step ahead in the commercial deployment of genetically modified maize in China.Therefore,prior to the broad commercialization of Bt maize in China,resistant management strategies are essential to prolong and delay the development of resistance to target pests.Yellow peach moth(YPM)Conogethes punctiferalis is a polyphagous pest of global significance in agricultural plants in South,Southeast and East Asian countries and Australia and Papua New Guinea.In more recent years,with climate change and the adjustment of cropping systems in China,the damage of the YPM in many cropping regions has been rapidly increasing remarkably in the Huang-Huai-Hai Summer Maize Region and Southwest Hilly Maize Region.Therefore,YPM is becoming a significant pest that seriously threatens maize production.The larvae have an endophytic habitat that can bore into the maize ear during the reproductive stage,resulting in difficulty implementing conventional chemical insecticide control and not so insufficient.Chemical pesticides have remained the most prevalent method of YPM management thus far.However,their effectiveness decreases because of their negative impacts on non-target pests,beneficial organisms,human health,and the environment.Therefore,using the Bt as a spray or transgenic crop is an alternative to chemical insecticides to manage critical agricultural pests,including YPM.The insecticidal activities of Bt proteins against YPM have gotten little attention.In this work,we conducted diet-overlay bioassays to assess the insecticidal effects of Cry1Ab,Cry1Ac,Cry1Fa,Cry1Ah,Cry1Ie,Cry2Aa,and Vip3Aa19,as well as the interaction between Cry1-Class,Cry2Aa,and Vip3Aa19.The results showed that the LC₅₀ values varied from 1.08 to 178.12 ng/cm²(protein/diet).Cry1Ab and Cry1Ac had lower LC₅₀ and L_C90values,followed by Cry1Fa and Cry1Ah,while Cry1Ie,Cry2Aa,and Vip3Aa19 have less in toxicity.Combinations of Cry2Aa with Cry1Ac,Cry1Ie,and Cry1Ab showed antagonism in YPM bioassays,but Cry2Aa with Cry1Fa and Cry1Ah demonstrated synergism.When Vip3Aa19 and Cry proteins were coupled,all combinations interacted positively,with differences in synergistic factors(SF).Particularly,Cry1Ah and Vip3Aa19 protein combinations in three different ratios yielded SF values of 5.20,5.63,and 8.98,respectively.Therefore,we performed the binding assays to investigate the binding affinity and synergism of Cry1Ah and Vip3Aa19 proteins with ABC transporter subfamily C receptors ABCC1,ABCC2 and ABCC3 proteins from the midgut of YPM larva.The selected ABCC1,ABCC2 and ABCC3 proteins were synthesized using a PCR-based Accurate Synthesis(PAS)system and were successfully expressed and purified in the E.coli system.The purified proteins were further qualitatively analyzed on SDS-PAGE gel electrophoresis and Western blotting method.We used SPR and pull-down assays to confirm the binding affinity of Cry1Ah or Vip3Aa19 to ABCC1,ABCC2,and ABCC3 proteins.Both assays revealed that Cry1Ah could interact with ABCC1,ABCC2,and ABCC3,whereas Vip3Aa19only interacts with ABCC1 and ABCC3,but not with ABCC2.Hence,when compared to Vip3Aa19protein,Cry1Ah had a higher binding affinity for ABCC1,ABCC2,and ABCC3 in both methods.Furthermore,the competitive binding assay between Cry1Ah and Vip3Aa19 protein with ABC transporter subfamily C receptors resulted in the final eluted protein samples displaying vibrant blue bands of Cry1Ah and very faint bands of Vip3Aa19.It suggested that Cry and Vip proteins could deliver a synergistic effect after cleaving the midgut proteases.Hence,the combination of Cry1Ah and Vip3Aa19 proteins presented a higher synergistic value in our diet overlay bioassay results.Therefore,the diet overlay bioassay results correlate with our protein-protein interaction findings,indicating that the Cry1Ah and Vip3Aa19 combination does not compete for interacting with midgut receptors and thus provides strong synergism against YPM.These data can be used to develop novel pyramided transgenic crops with appropriate candidates and resistance management strategies.In order to use Bt maize against YPM,the baseline sensitivity of the local populations in the targeted areas must be established.The results of a diet-overlay bioassay revealed that all fourteen YPM populations in China are extremely vulnerable to Cry1Ab.Over the two years of collection,the LC₅₀values ranged from 0.35 to 2.38 ng/cm²,with a sevenfold difference between the most vulnerable and most tolerant populations.The upper limit of six pooled population LC₉₉ estimates yielded>99%larval mortality for representative eight populations collected in 2020 and was recognized as diagnostic concentrations for monitoring susceptibility in YPM populations in China.Hence,we evaluated the laboratory selection of resistance in YPM to Cry1Ab using the diet-overlay bioassay method.Although the resistant ratio was generally low,YPM potentially could evolve resistance to Cry1Ab protein.The possibility of resistance development by target pests emphasizes the importance of implementing resistance management measures for delaying the establishment of pest resistance to Bt crops.