| Gram-positive soil bacteria Bacillus thuringiensis(Bt)can produce parasporal crystal proteins(Cry proteins)during sporing period which are highly toxic to agricultural pests,therefore it has been developed into the main microbial pesticides to control insects and nematodes.So far,the insecticidal mechanism of Bt is mainly concentrated on Cry proteins which are the main virulence factors,and hosts mainly depend on innate immunity to defense against them.However,as a pathogen,how Bt activates expression of its own genes to break-through the host immune defenses for better infection has not been discovered.There is a bottleneck of agricultural pests in the practical research,as a model organism,Caenorhabditis elegans has a clear genetic background and a sound mutant library which are convenient for us to study the specific infecting process of Bt.We will extend the mechanism of the interaction between Bt and nematodes to other hosts,and provide a certain amount of important theoretical guidance for the development of more efficient poison activity of Bt.First,it was found that Bt infecting cause immunosuppression.By comparing the RNA-seq data of N2 after different treatments of Bt(BMB171 / Cry5B)and toxin(Cry5B),we found that compared with the toxin,Bt could downregulate some immune effectors like lysozymes(lys-7 etc),antibacterial peptides(abf-2 etc),lectins(clec-55 etc)and unnamed defense factors(F01D5.5 etc).q RT-PCR was used to verify 30 effectors which were downregulated obviously,and 29 were consistent with the expectation.Through pathway enrichment and gene clustering analysis,it was found that the downregulated immune effectors are mostly downstream of the DAF-2 Insulin like immune signaling pathway,the ZIP-2 immune defense pathway and the ELT-2 transcription factor.Then specific pathways and transcription factor for Bt to inhibit nematodes’ immunity had been verified: For insulin like immune signaling pathway,Bt activated DAF-2 by inhibiting the upstream antagonist INS-18,in turn to cause delocalization of nuclear DAF-16 and inhibit downstream immune effectors like lys-7 etc;regarding the ZIP-2/irg-1defense pathway,Bt downregulated the expression of immune genes such as irg-1 by inhibiting the expression of the transcription factor ZIP-2;Bt could also downregulate lys-1,spp-8 and other immune effectors by inhibiting the nuclear localization of transcription factor ELT-2.Finally we found that global regulatory factors Plc R and Lux S were involved in regulating this process.To explore the regulatory mechanism in Bt when it suppressed nematodes’ immunity we draw the conclusion.Bt activating DAF-2 Insulin like pathway to inhibit nematodes’ immunity was regulated by Plc R,plc R mutation weakened the ability of Bt to activate DAF-2 and inhibit DAF-16 and downstream immune effectors;Bt inhibiting ZIP-2 immune defense pathway and Elt-2 transcription factor was regulated by Lux S,lux S mutation weakened the ability of Bt to inhibit the expression of ZIP-2 and ELT-2,and their downstream immune effectors like irg-1 and lys-1.In this study we went deep into the infection process of Bt,and revealed that Bt could inhibit the immune defenses to better poison nematodes in the infecting process from the perspective of pathogenic bacteria,which more,global regulatory factors participated in regulating this process.This work.brokethrough the limitation that on the past research the insecticidal mechanism was mainly focused on Cry proteins,and provided some ideas about a new perspective for the interactions between other pathogens and hosts.In the application of Bt,the discovery of new immune-suppressors can be prepared for the development of more efficient,rapid biopesticides and new genetically modified(GM)targets.At the same time,these signaling pathways are conserved in many insects and plant parasitic nematodes,targeting specific pests conservative signaling pathway can enhance the insecticidal activity of Bt and reduce the development of drug resistance of pests. |
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