The Study On The Mechanism Of Action Of The Detoxification Enzymes Towards Four Classes Of Compounds In Plutella Xylostella

The diamondback moth,Plutella xylostella(L.)(Lepidoptera: Plutellidae),is one of the important pests attacking cruciferous crops.It has such characters: wide distribution,short generations,rapid reproduction and fast resistance development.Meanwhile,P.xylostella has caused massive economic losses to the vegetable industry annually.Due to the irrational use of insecticides,P.xylostella has developed different levels of resistance against almost all the commercial types of insecticides.Therefore,exploring the resistance mechanism of P.xylostella is imminent.The detoxification enzymes-mediated metabolic resistance play an important role in the resistance mechanism of P.xylostella against insecticides.However,the study on the detoxification mechanism mediated by detoxification enzymes is still unclear.Therefore,determining the detoxification metabolic mechanism of the detoxification enzymes towards different compounds is important to solve the resistance problems of P.xylostella.This study investigated the detoxification metabolic function of P.xylostella against cantharidin(CTD)through detoxification enzyme activity analysis.Additionally,the present study combined biological method and molecular simulation to illustrate the detoxification metabolic mechanism of glutathione S-transferases in P.xylostella(PxGSTs)towards tolfenpyrad(TFP).Meanwhile,the interactions and the key amino acid in binding mode between PxGSTs and the compounds were uncovered by joint use of homology modeling,molecular-dynamics(MD)simulations,per-residue free-energy decomposition and computational alanine scanning(CAS)through using the S-hexyl glutathione(GTX,a GST inhibitor)as the molecular probe.Additionally,the metabolic mechanism of one representative P.xylostella carboxylesterase(PxEst-6)towards pyrethroids(bifenthrin,cyfluthrin,cypermethrin,and ?-cyhalothrin)was illustrated by the combination use of 3D structural modeling,molecular docking,molecular dynamics(MD)simulations and biological experiments.The main results are as follows:(1)The detoxification metabolism the detoxification enzyme systems towards CTD in P.xylostellaCurrently,threr are a larger number of literatures on the detoxicification metabolism of P.xylostella against biopescides Nevertheless,few reports have been reported the detoxified metabolism of detoxification enzyme towards CTD.Results from detecting the activity of serine/threonine phosphatases(PSPs)and detoxification enzymes(glutathione S-transferases,carboxylesterases and cytochrome P450)showed that the sublethal concentration of CTD could inhibit the activity of PSPs in vivo and the inhibition activity displayed a trend from decline to gradually recover with time goes by.Meanwhile,the activity of detoxification enzymes in P.xylostella showed a trend of firstly decrease and then increase(the increased activity of P450 was the most significant).In addition,the increasing trend of detoxification enzyme activity was congruent with the recovery trend of PSPs activity.These results indicate that the detoxification enzymes enable detoxify and metabolize CTD in P.xylostella and restore the activity of PSPs.These results fill the gap of the detoxification metabolic function of the detoxification enzyme system towards CTD of P.xylostella.(2)The function of PxGSTs in detoxifying and metabolizing TFPTo date,little information is available about the function of insect GST in the detoxification metabolism towards TFP.This study uses P.xylostella as the research subject,RT-q PCR showed PxGSTs(PxGST ?,PxGST? and PxGST?)significantly up-regulated after being exposured to TFP.In vitro inhibition assay and metabolism assay exhibited that TFP could inhibit PxGSTs,and PxGSTs could metabolize TFP in vitro.Among the PxGSTs,PxGST? has the strongest metabolic capacity.The molecular docking of TFP and PxGST?revealed that the H-bond provided by the sidechains of Tyr107 and Tyr162 were key to the detoxification of TFP by PxGST? binding to TFP.Further tests using mutant PxGST?proteins at the sites of Tyr107(PxGST?Y107A)and Tyr162(PxGST?Y162A)corroborated that the individual replacement of Tyr107 and Tyr162 could greatly weaken the binding and metabolic abilities to TFP.These results decipher the interactions between PxGSTs and TFP and provide a new methodfor the design and optimization of the new TFP-type insecticides.(3)The interaction between PxGSTs and the inhibitor S-Hexyl glutathione(GTX)Insect GSTs are involved in metabolic resistance against many kinds of insecticides.GTX(a GST inhibitor)can be used as a molecular probe to reveal the binding mode between insect GSTs and insecticides.Through the use of homology modeling and molecular docking,the 3D model of PxGST?-GTX-complex was constructed.Meanwhile,the stability of the PxGST?-GTX complex were generated using molecular-dynamics(MD)simulations.The results revealed the formation and stability of the PxGST?-GTX complex are mainly driven by the H-bond interactions provided by GSH-protein contacts and hydro-phobic interactions derived from S-hexyl-protein contacts.Lys43 and Arg99 were determined that are key sites involved in the PxGST?-GTX interaction by computational alanine scanning(CAS)and site-directed mutagenesis.Meanwhile,combining the analysis of key site in the interaction between TFP and PxGST? showed that Tyr107 maybe the key residue for the inhibition of compounds on PxGST?.This result provides a structural biological reference for revealing the inhibition molecular mechanism of existing insecticides against GST and provides a theoretical basis and a solution for the evaluation of the interaction of insecticides with GSTs in the development of new insecticides.(4)The metabolic mechanism of carboxylesterase PxEst-6 towards pyrethroid in P.xylostellaInsect carboxylesterases are reported that involved in the pyrethroid resistance.In this study,RT-q PCR showed that PxEst-6 was highly expressed in the midgut and cuticles of the third instar larvae.Exposure to pyrethroid(bifenthrin,cyfluthrin,cypermethrin,and?-cyhalothrin)insecticides resulted in a rapid PxEst-6 up-regulation.Metabolic assays indicate that PxEst-6 has the capacity to metabolize these pyrethroid insecticides.The combination of molecular docking,binding mode analyses and alanine mutations demonstrated that His451,Lys458 and Gln431 were key residues of PxEst-6 for metabolizing pyrethroids,and the acetate groups derived from pyrethroids were key sites for being metabolized by PxEst-6.H451-and K458-derived hydrogen bond(H-bond)interactions with the pyrethroid acetate groups and the polar interactions with the pyrethroid acetate group provided by the Q431 sidechain were crucial to the pyrethroids' metabolism by PxEst-6.These findings reveal the molecular mechanism of PxEst-6 in metabolizing pyrethroid insecticides.