Molecular Mechanism Of Detoxification And Metabolic Adaptation Of Frankliniella Occidentalis To Defense Response Of Kidney Bean Plant Induced By Jasmonic Acid

Frankliniella occidentalis,western flower thrips(WFT),is a worldwide import insect pest.It seriously threatens the production of important economic vegetable and flower crops because of its feeding,oviposition and transmission of plant viruses.WFT has a wide range of hosts,strong concealment,high fecundity,which cause the thrips frequently outbreak.Chemical control can lead a significant increase in resistance.The development and utilization of plant induced resistance and RNA interference(RNAi)technology is a very ecological and potential method in pest control research.Spraying jasmonic acid(JA)can induce the resistance of plants.Therefore,the kidney bean plants sprayed with JA and deionized water were chosen as the host plant.Firstly,the function of exogenous JA inducing the defense response of kidney bean plants to WFT were revealed.Then,the key genes for detoxification and metabolic adaptation of WFT to JA-induced defense response of kidney bean plants were screened by transcriptomics and metabolomics analysis methods.Finally,the molecular mechanisms involved in the regulation of WFT detoxification and metabolic adaptation were studied by RNAi methods.It will provide a theoretical basis for the green prevention and control of WFT.The results were as follows:1.The induction of Jasmonic acid on defense response of kidney bean plantExogenous JA spraying on kidney bean plants could induce the increase of the activity of enzymes.Exogenous JA spraying not only induced the increase of JA biosynthetic enzymes(Lipooxygenase,LOX;Allene oxide synthase,AOS;12-oxy-phytodienate reductase,OPR),and the expression levels of JA pathway marker genes(PvLOX,PvAOS and PvOPR2).,but also could increase the activity of downstream defense enzymes(Peroxidase,POD;Polyphenol oxidase,PPO;Trypsin inhibitor,TI).JA induction could increase the contents of secondary metabolites(Total phenols,flavonoids,tannins,lignin and alkaloids)in kidney bean plants.After WFT feeding the kidney bean leaves pretreated with JA,there was more significant upregulation of the expression levels of JA pathway marker genes and contents of secondary metabolites though JA enzyme activities were disturbed.The metabolites had a big change after JA induction by Ultra High-Performance Liquid Chromatography AB Triple TOF 6600 mass spectrometer system(UHPLC-Q-TOF)analysis.27 metabolites of kidney bean leaves were significantly upregulated and 5 metabolites were significantly downregulated by JA induction.Among these significantly upregulated metabolites,succinic acid,bexarotene,bufogenin,nicotinamide and vinpocetine,had a certain degree of biological toxicity and antioxidant effects.2.Jasmonic acid induced defense response of kidney bean plants to resistance of western flowerJA-induced kidney bean plants inhibited the preference of WFT female adults in feeding and oviposition,and the feeding preference of second-instar larvae.The longevities of female(32.66 d)and male(24.37d)adults feeding kidney bean plants induced by JA were significantly shortened compared to those fed on leaves of the control group(236.72 d and 26.94 d);The average number of eggs laid per female was 24.29 thrips,which was significantly lower than that of the control group(53.72 thrips).The population parameters of WFT reared on JA-treated kidney bean leaves were significantly affected.the values of rm and R0 decresed significantly.3.The adaptation of western flower thrips to defense response of kidney bean plants induced by jasmonic acid by transcriptional analysisRNAseq was used to determine and analyze the differential gene expression profiles among F0generation thrips fed by deionized water-treated kidney bean plants(CKF0T),F1-generation thrips(CKF1T),F0-generation thrips fed by JA-treated kidney bean plants(JAF0T),and F1-generation thrips(JAF1T).The results showed that 1069 genes were significantly up-regulated and 84 genes were significantly down-regulated in JAF0T group compared with CKF0T group.Compared with CKF1T group,2832 genes were significantly up-regulated and only 24 genes were significantly down-regulated in JAF1T group fter 1 generation of adaptation.Compared with JAF1T group,99 genes were significantly up-regulated and 425 genes were significantly down-regulated in JAF0T group.These indicated that the mRNA transcription level of WFT was significantly affected in the process of adapting to JA-induced defense response.There were 789 identical differentially expressed genes between JAF0T_vs_CKF0T comparison group and JAF1T_vs_CKF1T comparison group.There were 34 identical differential genes between JAF0T_vs_JAF1T comparison group and JAF0T_vs_CKF0T comparison group.There were 242 identical differential genes in the JAF0T_vs_JAF1T comparison group and the JAF1T_vs_CKF1T comparison group.These showed that there were multiple common genes of WFT involved in regulating the adaptability in the process of responding to or adapting to JA-induced kidney bean plant defense response.KEGG pathway enrichment analysis showed that the differentially expressed genes in the JAF0T_vs_CKF0T and JAF1T_vs_CKF1T comparison groups were enriched in metabolic pathways,nicotinate and nicotinamide metabolism,biosynthesis of secondary metabolites,metabolism of xenobiotics by cytochrome P450 and drug metabolism-cytochrome P450 and other pathways.The differentially expressed genes of JAF0T_vs_JAF1T were mainly enriched in starch and sucrose metabolism,caffeine metabolism,glycolysis/gluconeogenesis related to digestion metabolism.These results indicated that WFT mainly adapted to defense response of kidney bean plants induced by JA through regulating the transcription levels of detoxification and digestive metabolism genes.4.The adaptation of western flower thrips to defense response of kidney bean plants induce by jasmonic acid by metabolism analysisUHPLC-Q-TOF MS was used to analyze the differential metabolites of F0 generation WFT feeding kidney bean plants treated by deionized water,F0 generation WFT feeding kidney bean plants treated by JA,F1 generation WFT feeding bean plants treated by deionized water,and F1 generation WFT feeding bean plants treated by JA.The results showed that a total of 1348 metabolites were identified in the four groups of experimental samples,in which 842 metabolites were identified in the positive ion mode(Pos)and 506 metabolites were identified in the negative ion mode(Neg).Principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)showed that the metabolite profiles of JAF0M_vs_ControlF0M,JAF1M_vs_ControlF1M and JAF0M_vs_JAF1M were clear,which could be completely distinguished,and there was no overfitting phenomenon.Specifically,55 different metabolites in the positive ion mode and 36 different metabolites in the negative ion mode were identified between JAF0M_vs_ControlF0M.These metabolites were mainly involved in mineral absorption,β-alanine metabolism,and pyrimidine metabolism.38 different metabolites in the positive ion mode and 22 different metabolites in the negative ion mode were identified between JAF1M_vs_ControlF1M.These different metabolites were mainly involved in purine metabolism,fatty acid degradation,and protein digestion and absorption pathways.106 different metabolites in the positive ion mode and 69 different metabolites in the negative ion mode were identified between JAF0M_vs_JAF1M.These different metabolites were mainly involved in ABC transporters,synaptic vesicle cycle and mTOR signaling pathway.5.The regulatory function of detoxification and metabolism genes in western flower thrips adaptation to the defense response of kidney bean plants induced by jasmonic acidBased on the results of differential expression gene by transcriptome analysis,8 genes enriching in the metabolic pathway of exogenous substances in cytochrome P450 were screened,and 6 genes of P450 enzyme and 2 carboxylesterase genes that were not enriched in this metabolic pathway but closely related to detoxification metabolism and with large differences were cloned and identified too.The results showed that CDS sequences of 16 genes and their encoded protein sequences had their own unique signature sequences of functional sites,signature sequences,and characteristic conserved domains shared with similar genes of other species.Phylogenetic analysis also showed that each gene was clustered with similar genes of other species.They were named as FoGSTd1,FoGSTe1,FoGSTe2,FoGSTs2,FoUGT2C1,FoUGT2B30,FoUGT2B2,FoADH3,FoCYP6K1,FoCYP6d4,FoCYP6a1,FoCYP4c1,FoCYP4g15,FoCYP4v2,FoCarEl,and FoCarE2,respectively.And these 16 genes were expressed in different degrees throughout the life cycle of WFT.In order to further analyze the regulatory function of these detoxification metabolism-related genes in WFT adapting to JA-induced defense response of kidney bean plants.The mRNA expression levels of these 16 genes in WFT were analyzed by RT-qPCR during their adaptation to JA-induced kidney bean plant defense response.The results showed that the mRNA transcription levels of FoGSTd1 and FoGSTs2 in GSTs family,FoADH3 of alcohol dehydrogenase,FoCYP4g15 and FoCYP4v2 in P450 family,and FoCarE1 of carboxylesterase were significantly up-regulated in multiple periods during the adaptation of thrips to JA-induced defense response in kidney bean plants.The other 10 genes were only significantly highly expressed at some time points,and the expression trend was up-regulated at some time,but the differences weren’t significant.Among the significantly up-regulated genes,the overall up-regulation folds of FoGSTd1,FoGSTs2,FoCYP4g15 and FoCYP4v2 were highest.These four genes by RNAi showed that the mRNA expression levels of FoGSTd1,FoGSTs2,FoCYP4g15 and FoCYP4v2 were significantly downregulated after feeding dsRNA solutions of 100,300 and 600 ng/μL for 48 h,but the downregulation degree varied with dsRNA concentrations and genes.On this basis,WFT female adults feeding 600 ng/μL dsRNA was chosen to analyze their survival rate during adaptation to JA-induced kidney bean defense response.It was found that JA-induced treatment of kidney bean plants could significantly reduce the survival rate of WFT.WFT survival rate of feeding JA-treated kidney bean plants decreased n more significantly when coupled with RNAi down-regulation of the mRNA expression levels of FoGSTs2,FoCYP4g15 and FoCYP4v2.The above results showed that exogenous JA-treated kidney bean plants combined with RNAi downregulating key genes in detoxification and metabolic adaptation could effectively weaken the antidefense of WFT.6.The regulatory function of digestive enzyme genes of western flower thrips in their adaptation to the defense response of jasmine induced kidney bean plantsFour differential genes of digestive enzymes were selected among three comparative groups of JAF0T_vs_CKF0T,and JAF1T_vs_CKF1T,and JAF0T_vs_JAF1T,based on the results of transcriptome gene differential expression profile.Four genes were cloned and identified.These four genes had signal peptides and their own conserved domains.The three genes had their own signature sequences except FoαAmy.Phylogenetic tree analysis showed that these four genes had the closest genetic relationship with Thrips palmi in Thysanoptera,and also had high homology with similar genes of other species.Combined with the annotation information of the transcriptome and reference genome,they were named FoαAmy,FoSep,FoTry and FoEG1,respectively.The expression patterns at different developmental stages showed that each gene was expressed throughout the life cycle of WFT except that the low expression level of FoSep in the adult stage.The different expression levels of FoαAmy,FoEG1,FoSep and FoTry were detected by RTqPCR in F0 and F1 generation of WFT adapting to defense response of kidney bean plant induced by JA.The results showed that the degrees of these four genes involved in regulating the adaptation of WFT to JA-induced defense response were different.The upregulations of the expression levels of FoαAmy and FoEG1 were more obvious.The results of the RNAi showed that the survival rate of WFT was significantly lower in the JA-treated kidney bean plants fed by WFT downregulating the expression level of FoαAmy than that of the other treatment groups.While there was no significant difference in survival rate between Control+dsFoEG1 group and JA+dsFoEG1 group.The results of feeding damage area on showed that the feeding scar areas of Control+HS group and Control+dseGFP group were the largest,while they were second at the treatments of Control+dsFoαAmy and Control+dsFoEG1.The feeding scar areas of JA+dsFoαAmy group and JA+dsFoEG1 group were the smallest after feeding the target gene dsRNA,especially JA+dsFoEG1.The above results showed that FoαAmy and FoEG1 could participate in the regulation WFT’s counter-adaptation to JA-induced defense response at varying degrees.In summary,this research had clarified that exogenous JA could induce a defensive response in kidney bean plants to enhance the resistance to WFT.And,WFT adapt to defense responses of kidney bean plant induced by JA through regulating changes in its own transcription and metabolism levels.Further,downregulation of the transcription levels of FoGSTs2,FoCYP4g15,FoCYP4v2,FoαAmy and FoEG1 mRNA,key genes for detoxification and digestion and metabolism of WFT,by RNAi could weaken the anti-defense of WFT to JA-induced defense response at varying degrees.The results can provide a new idea for improving the effectiveness of WFT prevention by using induced resistance combining with RNAi technology,and it also can provide a basis for further study on the mechanism of interaction between WFT and plant and screening target genes for WFT prevention and control.