Types And Mechanism Of Combined Action/Induction Between Bt Toxin Proteins Cry1Ac And Plant Defensive Allelochemicals Flavone

Helicoverpa armigera Hubner（Lepidoptera,Noctuidae）,is one of the most polyphagous and destructive insect pests throughout the world.Transgenic Bt crop has been deployed to combat H.armigera since 1997 in China.Transgenic Bt crops can simultaneously produce anti-herbivore allelochemicals and Bt toxin proteins to kill pests.It remains to be unknown how they interact with each other at lethal and sublethal doses and the mechanisms of their interactions when H.armigera ingests these two toxins simultaneously or sequentially.Cytochrome P450 monooxygenases（P450s）has been broadly documented for their function in protecting insect herbivores from toxic phytochemicalsIn this study,we investigated the types and mechanisms of the interactions between the Bt toxin Cry1Ac and the plant allelochemical flavone when these two toxins being simultaneously or sequentially ingested.We futher explored the role of CYP321A5 in the combined and inductive toxicity of Cry1Ac and flavone,and the mechanism of its transcriptional regulation.The specific research results are as follows:1.The combined toxicity of Cry1Ac and flavoneWe calculated three lethal doses((LC₁₀,LC₂₅,LC₅₀)of Cry1Ac and flavone to H.armigera.Simultaneous exposure of H.armigera neonates to lethal doses(LC₂₅)of Cry1Ac and flavone significantly increased the mortality compared to either Cry1Ac or flavone treatment group and their expected additive mortality.This result indicates that Cry1Ac and flavone have synergistic effects.2.The inductive toxicity of Cry1Ac and flavoneIn this study,we found that preexposure to a sublethal dose(LC₁₀)of Cry1Ac could induce the toxicity of flavone,which significantly increased the mortality of group that treated with LC₁₀ dose of Cry1Ac plus LC₅₀ dose of flavone than that of the LC₅₀ dose of flavone or the expected additive mortality of the LC₅₀ dose of flavone and the LC₁₀ dose of Cry1Ac.However,preexposure to the sublethal dose(LC₁₀)of flavone did not significantly induce the toxicity of Cry1Ac to H.armigera.These results suggest that when one of the two toxins reaches its lethal doses in Bt crops,sublethal dose of Cry1Ac induces the toxicity of flavone,whereas sublethal dose of flavone has no impacts on the toxicity of Cry1Ac.3.The mechanism of the combined and inductive toxicity of Cry1Ac and flavoneTranscriptomic comparison revealed a total of 744 differentially expressed genes（DGEs）elicited by Cry1Ac(LC₂₅),a total of 238 DGEs by flavone(LC₂₅),and a total of 1261 DGEs by Cry1Ac(LC₂₅)+ flavone(LC₂₅).Among them 290 genes upregulated only in the combined treatment group,and 289 genes downregulated only in the combined treatment group.A total of 29 P450 genes were differentially expressed among the three treatments.The expression of CYP321A5 was significantly higher in the combined treatment than in either of the two single toxin treatments.Transcriptomic comparison uncovered a total of 1817 DGEs by Cry1Ac(LC₁₀),2820 DGEs by flavone(LC₅₀),and 2578 DGEs by Cry1Ac(LC₁₀)+ flavone(LC₅₀).Among them,131 DGEs were upregulated in the induction treatment while 285 DGEs were downregulated only in the induction treatment.Forty-one P450 genes were differentially expressed among the three toxin treatments.Similarly,the expression of CYP321A5 was significantly higher in the induction treatment than in either of the two single toxin treatments.These DGEs may be responsible for the synergistic/induced toxicity of flavone by Cry1Ac.4.CYP321A5 transcriptional regulation mechanismTo elucidate the transcriptional regutation of H.armigera P450 gene CYP321A5 in synergistical induction by both combination(LC₂₅ + LC₂₅)and induction(LC₁₀ +LC₅₀)of Cry1Ac and flavone,we cloned the promoter region and 5’ untranslated region of CYP321A1（-1380 to +104）.Dual-luciferase assay showed that this promoter was inucible by flavone,but not by Cry1Ac,implied that the cis element responsible for Cry1Ac inducibility may be located upstream of-1380 or disrupted in this allele.Sequence alignme uncovered that XRE-Fla,an essential element responsible for flavone induction of H.zea CYP321A1,was present in this allele,but had three point mutuations that disrupted its enhancer acitivity.In summary,this study should be helpful for understanding of the allelochemicalBt toxin interactions,the management of pest resistance to Bt crops,and development of new transgenic crop.