Molecular Mechanisms Of MAPK Signaling Pathway Regulating Plutella Xylostella (L.) Resistance To Bacillus Thuringiensis Cry1Ac Toxin

The diamondback moth,Plutella xylostella(L.),is a cosmopolitan pest of cruciferous crops.P.xylostella can rapidly develop resistance to different kinds of insecticides on account of its high fecundity and short generation time,which makes the pest controlling more difficult.The entomopathogenic Gram-positive bacterium,Bacillus thuringiensis(Bt),produces diverse insecticidal crystal proteins(ICP)during sporulation,which is widely used to control agricultural pest due to its highly specific activity and environmental safety.Transgenic crops harboring Bt toxin genes(Bt crops)have been successful insecticidal biotechnology.However,massive use of Bt pesticides and Bt crops represents high and persistent selection pressure for insect resistance evolution.Given the environmental and economic importance of Bt pesticides and Bt crops,unraveling molecular resistance mechanisms is important for sustainable utilization of Bt and integrated pest manage.P.xylostella is the first insect which has been reported to have field-evolved resistance to Bt pesticides.Furthermore,the whole genome sequencing of P.xylostella has been obtained and released.Consequently,P.xylostella becomes an excellent model to reveal the molecular resistance mechanisms to Bt.Our prior work revealed that Px MAPK4K4,an upstream gene of MAPK signaling pathway inside of the multigenic resistance locus to Bt(Bt R-1),trans-regulates expression of many resistance genes and causes resistance to Cry1 Ac in P.xylostella.These resistance genes are multiple ABC transporter genes(Px ABCC1-3)inside of Bt R-1,Pxm ALP and Px ABCG1 outside of Bt R-1.MAPKs function as four tiered signaling cascades(MAP4K-MAP3K-MAP2K-MAPK).To date,which kinases of MAPK signaling pathway activated by MAPK4K4 and participating in regulating receptor genes are still unknown.In this study,we make further investigation of the role of MAPK signaling pathway in Bt resistance via a series of techniques of omics,biochemistry and molecular biology.We identified and cloned the MAPK signaling pathway genes in P.xylostella at the whole genome level,and detected the difference in transcription level,protein level and phosphorylation level of MAPK signaling pathway genes between Bt susceptible and resistant strains.Our results demonstrate that MAPK signaling pathway can trans-regulate multiple resistance genes through a complex signal cascade activated by phosphorylation,resulting in high resistance to Bt Cry1 Ac in P.xylostella.These findings are of theoretical and practical significance for further understanding the molecular mechanisms of MAPK signaling pathway mediating Cry1 Ac resistance and the development of integrated management of insect resistance.The main conclusions are as follows:1.The annotation of P.xylostella MAPK pathway genesWe confirmed two MAP4 K genes,seven MAP3 K genes,four MAP2 K genes and four MAPK genes in P.xylostella genome database via a series of bioinformatic analysis.All the 17 genes constitute the basic architecture of MAPK four tiered signaling cascades.Furthermore,we analyzed the MAPK pathway of Bombyx mori,Manduca sexta,Danaus plexippus,Acyrthosiphon pisum,Rhodnius prolixus,Tribolium castaneum,Apis mellifera,Nasonia vitripennis,Pediculus Humanus corporis and Tetranychus urticae.After assessed the distribution of MAPK cascade genes,selection pressure and phylogenetic analysis among different species,we found MAPK pathway is evolutionarily conserved among different arthropod species.2.The quantitative analysis of MAPK genes transcription level and protein level in Bt susceptible and resistant P.xylostella strainsWe measured m RNA level of MAPKs in dissected midgut of the susceptible strain and four resistant strains by qPCR.A large proportion of MAPK genes showed upregulation in resistant strains.Given that phosphorylation is a hallmark of signaling transduction in MAPK pathway,we detected the level of total protein and phosphorylated protein of p38?JNK and ERK in different strains by Western Blot.The relative abundance of p38,JNK and ERK increased in resistant strains while the phosphorylated proteins dramatically increased in resistant strains.These results indicated that MAPK pathway is mainly activated by phosphorylation in Bt resistant strains.3.Study on the role of p38,JNK and ERK pathway in regulating Cry1 Ac resistance genes by inhibition assayTo investigate the role of MAPK pathway in P.xylostella resistance to Cry1 Ac,we detected the transcription level of resistance genes in NIL-R after treatment with specific MAPK inhibitor.The result revealed that in regulating Px ABCC1 and Px ABCC2,p38 and ERK pathways cooperate with each other.In regulating Px ABCC3,p38 pathway plays dominant role with assistance of JNK pathway.For Pxm ALP,p38 pathway plays dominant role with assistance of ERK pathway.Regarding Px ABCG1,p38 and JNK pathways cooperate with each other to regulate this gene.Our findings indicate p38,JNK and ERK pathways mediate resistance to Cry1 Ac by regulating resistance genes.4.Phosphoproteomic studyWe used large-scale quantitative phosphoproteomic experiment to detecte the difference of phosphorylated proteins in susceptible strain DBM1Ac-S and high resistant strain NIL-R.Finally we identified phosphorylation levels of some MAPK pathway kinases and other phosphorylated proteins are different between DBM1Ac-S and NIL-R.The phosphorylation levels of MAP3K7,TAO,MAP2K6,p38 and ERK are upregulated in NIL-R with significant difference,while the phosphorylation levels of phosphatase(PP1,Protein Phosphatase 1 and DUSP13,Dual Specificity Protein Phosphatase 13)and scaffold protein JIP3(JNK-Interacting Protein 3)are also dramatically changed in NIL-R.Moreover,we also detected the phosphorylation levels of some membrane functional receptors such as ABC transporter superfamily(especially ABCC and ABCG)and amino acid transporters are changed in NIL-R.These results suggest the difference in phosphorylation level of MAPK signaling pathway is tightly linked to the Bt Cry1 Ac resistance.5.RNAi assay of MAPK pathway kinasesWe performed RNAi in NIL-R larvae to further investigate the role of MAPK pathway kinases in regulating Cry1 Ac resistance genes.Our findings indicated that Raf,MAP2K1 and ERK constitute the ERK pathway to regulate resistance genes.MAP2K6 and p38 belong to p38 pathway to mediate the expression of resistance genes.Meanwhile,MAP3K7 can regulate resistance genes via p38 and JNK pathways and MAP4K4 can regulate resistance genes via p38,JNK and ERK pathways.These results elucidate how the MAPK signaling pathway regulates Cry1 Ac resistance genes through four tiered signaling cascades.In conclusion,we dissect the components of MAPK cascade in P.xylostella and demonstrate that MAPK pathway plays a crucial role in Cry1 Ac resistance by trans-regulating Bt Cry1 Ac resistance genes.This study further clarifies the molecular mechanism of Bt Cry1 Ac resistance mediated by trans-regulation of MAPK signaling pathway.