Study On The Target Of PMK-2 Signaling Pathway Mediating The Immune Stress Response Of Nematode To Bacillus Thuringiensis Crystal Protein

Parasporal crystal proteins produced by Bacillus thuringiensis exhibit diverse insecticidal activities.Among which,Cry5 B and Cry6 A are two most typical nematicidal crystal proteins.Cry6 A shows distinct structure with Cry5 B,a well-documented nematicidal crystal protein.Some studies have preliminarily demonstrated that Cry6 A have different action mode with Cry5 B,suggesting that the nematode probably take a different immune stress defense response to Cry6 A toxin.In model nematode Caenorhabditis elegans,three genes(pmk-1,pmk-2 and pmk-3)with homology to mammalian p38 MAPK are in a single operon.PMK-2is highly homologous to PMK-1 and is mainly expressed in nematode nerve cells.Our previous results show that PMK-2 is associated with defensive responses to Cry6 A crystal protein toxin in C.elegans,and is important for nematode survival.Based on the previous results,this study will screen the target genes regulated by the PMK-2 signaling pathway,and elucidate the molecular mechanism of PMK-2 signal pathway in the defense responses of nematode to Cry6 A crystal protein.PMK-1 can be activated by the upstream kinases NSY-1 and SEK-1.This study firstly demonstrated that Cry6 A could not induce sek-1,nsy-1mutation through western blotting.Phosphorylation of PMK-1 and PMK-2 proteins in vivo indicated that PMK-2 and PMK-1 share the common upstream kinases SEK-1 and NSY-1.Previous studies have shown that C.elegans regulates the expression of ttm-1 and ttm-2 target genes through the PMK-1 pathway when subjected to Cry5 B toxin.In this experiment,the sensitivity of ttm-1 and ttm-2 mutants to Cry6 A were found to be unchanged when ttm-2 was silenced by RNAi technique.This indicates that PMK-2 does not share downstream targets TTM-1 and TTM-2 with PMK-1.This experiment also used RNA-seq technology to compare thetranscriptome of wild type C.elegans with or without Cry6 A toxin,and discussed some ways of PMK-2 regulation.Eleven differential genes were screened and their transcriptional levels were identified by qRT-PCR.It was found that in the process of defense against Cry6Aa2 toxin,the nematode activates heat shock protein(HSP-70)to improve its stress response;Some energy-related pathways,such as fat metabolism,electron transport,also have corresponding changes in the process of Cry6Aa2 to provide sufficient energy for nematodes;at the same time,five proteins related to the innate immune response were also activated during the defense against Cry6Aa2.The expression of these energy-related proteins can provide energy to support natural immunity against toxin attacks,which may be derived from the glycolytic pathway linking the Krebs cycle,and the arginine kinase pathway unique to invertebrates.The activated MAPK by crystal protein toxin might target the genes related to energy production,therefore meet the energy demand for innate immunity.In summary,this study enriches the diversity of p38 MAPK signal transduction pathways,which helps us to understand the diversity of innate immune mechanisms of nematode under natural conditions.