The identification of functional midgut receptors for pesticidal proteins produced by Bacillus thuringiensis(Bt)is critical for deciphering the molecular mechanism of Bt resistance in insects.Reduced expression of the...The identification of functional midgut receptors for pesticidal proteins produced by Bacillus thuringiensis(Bt)is critical for deciphering the molecular mechanism of Bt resistance in insects.Reduced expression of the PxABCB1 gene was previously found to be associated with Cry1Ac resistance in the diamondback moth,Plutella xylostella(L.).To directly validate the potential receptor role of PxABCB1 and its contribution to Bt Cry1Ac toxicity in P.xylostella,we used CRISPR/Cas9 to generate a homozygous knockout ABCB1KO strain with a 5-bp deletion in exon 3 of its gene.The ABCB1KO strain exhibited a 63-fold resistance to Cry1Ac toxin compared to the parental DBM1Ac-S strain.Intriguingly,the ABCB1KO strain also exhibited significant increases in susceptibility to abamectin and emamectin benzoate.No changes in susceptibility to various other Bt Cry proteins or synthetic insecticides were observed.The knockout strain exhibited no significant fitness costs.Overall,our study indicates that PxABCB1 can protect the insect against avermectin insecticides on one hand,while on the other hand it facilitates the toxic effect of the Bt Cry1Ac toxin.The results of this study will help to inform integrated pest management approaches against this destructive pest.展开更多
The insecticidal Cry proteins produced by the bacterium Bacillus thuringiensis(Bt)are extensively used for pest control in formulated sprays and in genetically modified crops,but resistance to Bt toxins threatens thei...The insecticidal Cry proteins produced by the bacterium Bacillus thuringiensis(Bt)are extensively used for pest control in formulated sprays and in genetically modified crops,but resistance to Bt toxins threatens their sustainable use in agriculture.Understanding the molecular mechanisms involved in Bt pathogenesis is crucial for the development of effective resistance management strategies.Previously,we showed a strong correlation between Cry1Ac resistance in Plutella xylostella(L.)and down-regulation of the glycosylphosphatidylinositol(GPI)-anchored membrane-bound alkaline phosphatase(mALP)and aminopeptidase(APN)and members of the ATP-binding cassette(ABC)transporter subfamily C(ABCC),but we do not yet have a clear understanding of the relative contribution of each midgut receptor type.Here,a P.xylostella strain homozygous for the PxmALP gene knockout was generated using CRISPR/Cas9 and the results showed that this strain had a 294-fold resistance to Cry1Ac toxin and 394-fold cross-resistance to Cry1Ab.Moreover,a triple knockout strain lacking PxmALP,Px-ABCC2,and PxABCC3 exhibited 9,660-fold resistance to Cry1Ac and 5,662-fold cross-resistance to Cry1Ab.These resistance levels surpassed those observed in the previously described double PxABCC2 and PxABCC3 knockout mutant,revealing a functional redundancy between ABC transporters and PxmALP.In addition,the activity of Cry1A toxins against Sf9 cells expressing PxmALP,PxABCC2 or PxABCC3 confirmed that each of these can act as a functional receptor.Our findings are crucial for unraveling the relative role of multiple receptors and the molecular mechanisms underlying Bt resistance in insects.展开更多
Mounting evidence suggests that insect hormones associated with growth and development also participate in pathogen defense.We have discovered a previously undescribed midgut transcriptional control pathway that modul...Mounting evidence suggests that insect hormones associated with growth and development also participate in pathogen defense.We have discovered a previously undescribed midgut transcriptional control pathway that modulates the availability of 20-hydroxyecdysone(20E)in a worldwide insect pest(Plutella xylostella),allowing it to defeat the major virulence factor of an insect pathogen Bacillus thuringiensis(Bt).A reduction of the transcriptional inhibitor(PxDfd)increases the expression of a midgut microRNA(miR-8545),which in turn represses the expression of a newly identified ecdysteroid-degrading glucose dehydrogenase(PxGLD).Downregulation of PxGLD reduces 20E degradation to increase 20E titer and concurrently triggers a transcriptional negative feedback loop to mitigate 20E overproduction.The moderately elevated 20E titer in the midgut activates a MAPK signaling pathway to increase Bt tolerance/resistance.These findings deepen our understanding of the functions attributed to these classical insect hormones and help inform potential future strategies that can be employed to control insect pests.展开更多
基金the Laboratory of Lingnan Modern Agriculture Project,China(NT2021003)National Natural Science Foundation of China(32022074,32221004 and 32172458)Beijing Key Laboratory for Pest Control and Sustainable Cultivation of Vegetables,Chinese Academy of Agricultural Sciences,and the Innovation Program of the Chinese Academy of Agricultural Sciences(CAAS-CSCB-202303)。
文摘The identification of functional midgut receptors for pesticidal proteins produced by Bacillus thuringiensis(Bt)is critical for deciphering the molecular mechanism of Bt resistance in insects.Reduced expression of the PxABCB1 gene was previously found to be associated with Cry1Ac resistance in the diamondback moth,Plutella xylostella(L.).To directly validate the potential receptor role of PxABCB1 and its contribution to Bt Cry1Ac toxicity in P.xylostella,we used CRISPR/Cas9 to generate a homozygous knockout ABCB1KO strain with a 5-bp deletion in exon 3 of its gene.The ABCB1KO strain exhibited a 63-fold resistance to Cry1Ac toxin compared to the parental DBM1Ac-S strain.Intriguingly,the ABCB1KO strain also exhibited significant increases in susceptibility to abamectin and emamectin benzoate.No changes in susceptibility to various other Bt Cry proteins or synthetic insecticides were observed.The knockout strain exhibited no significant fitness costs.Overall,our study indicates that PxABCB1 can protect the insect against avermectin insecticides on one hand,while on the other hand it facilitates the toxic effect of the Bt Cry1Ac toxin.The results of this study will help to inform integrated pest management approaches against this destructive pest.
基金supported by the National Natural Science Foundation of China(32172458,32302354,32221004,32372600)the Beijing Key Laboratory for Pest Control and Sustainable Cultivation of Vegetables and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences(CAAS-CSCB-202303).
文摘The insecticidal Cry proteins produced by the bacterium Bacillus thuringiensis(Bt)are extensively used for pest control in formulated sprays and in genetically modified crops,but resistance to Bt toxins threatens their sustainable use in agriculture.Understanding the molecular mechanisms involved in Bt pathogenesis is crucial for the development of effective resistance management strategies.Previously,we showed a strong correlation between Cry1Ac resistance in Plutella xylostella(L.)and down-regulation of the glycosylphosphatidylinositol(GPI)-anchored membrane-bound alkaline phosphatase(mALP)and aminopeptidase(APN)and members of the ATP-binding cassette(ABC)transporter subfamily C(ABCC),but we do not yet have a clear understanding of the relative contribution of each midgut receptor type.Here,a P.xylostella strain homozygous for the PxmALP gene knockout was generated using CRISPR/Cas9 and the results showed that this strain had a 294-fold resistance to Cry1Ac toxin and 394-fold cross-resistance to Cry1Ab.Moreover,a triple knockout strain lacking PxmALP,Px-ABCC2,and PxABCC3 exhibited 9,660-fold resistance to Cry1Ac and 5,662-fold cross-resistance to Cry1Ab.These resistance levels surpassed those observed in the previously described double PxABCC2 and PxABCC3 knockout mutant,revealing a functional redundancy between ABC transporters and PxmALP.In addition,the activity of Cry1A toxins against Sf9 cells expressing PxmALP,PxABCC2 or PxABCC3 confirmed that each of these can act as a functional receptor.Our findings are crucial for unraveling the relative role of multiple receptors and the molecular mechanisms underlying Bt resistance in insects.
基金supported by the National Natural Science Foundation of China(32221004,32172458,and 32372600)the China Postdoctoral Science Foundation(2023M733828)+1 种基金the earmarked fund for CARS(CARS-23),the Central Public-interest Scientific Institution Basal Research Fund(Y2024XK01)the Beijing Key Laboratory for Pest Control and Sustainable Cultivation of Vegetables,and the Innovation Program of Chinese Academy of Agricultural Sciences(CAAS-CSCB-202303).
文摘Mounting evidence suggests that insect hormones associated with growth and development also participate in pathogen defense.We have discovered a previously undescribed midgut transcriptional control pathway that modulates the availability of 20-hydroxyecdysone(20E)in a worldwide insect pest(Plutella xylostella),allowing it to defeat the major virulence factor of an insect pathogen Bacillus thuringiensis(Bt).A reduction of the transcriptional inhibitor(PxDfd)increases the expression of a midgut microRNA(miR-8545),which in turn represses the expression of a newly identified ecdysteroid-degrading glucose dehydrogenase(PxGLD).Downregulation of PxGLD reduces 20E degradation to increase 20E titer and concurrently triggers a transcriptional negative feedback loop to mitigate 20E overproduction.The moderately elevated 20E titer in the midgut activates a MAPK signaling pathway to increase Bt tolerance/resistance.These findings deepen our understanding of the functions attributed to these classical insect hormones and help inform potential future strategies that can be employed to control insect pests.
