PMK-2 Signaling Pathway Mediates The Defense Response Against Bacillus Thuringiensis Crystal Protein Cry6A In C.elegans

Parasporal crystal proteins produced by Bacillus thuringiensis(Bt)exhibit diversely insecticidal activity,therefore are widely used as biological pesticide.Recent reports show that there are many Bt crystal proteins are toxic to nematode.Among these nematicidal crystal proteins,Cry5 B and Cry6 A are two most typical crystal proteins.Cry6 A shows no sequence similarity and a distinct structure than Cry5 B,a well-documented nematicidal crystal protein.Moreover,Cry6 A also has distant evolution relationship from Cry5 B and other crystal protein.Cry6 A does not contain the three conserved domains in the Cry5 B subfamily and other Bt crystal proteins.Previous studies have shown that Cry6 A exhibits a different nematicidal action mode than Cry5 B.Therefore,Cry6 A has great advantage in solving the insect resistance to Bt toxins,and it is a highly promising insecticidal crystal proteins.In this study,Caenorhabditis elegans,a model nematode,was used to study the defense responses of nematode to Cry6 A crystal protein toxin.In C.elegans,three genes(pmk-1,pmk-2 and pmk-3)with homology to mammalian p38 MAPK are in a single operon,and they locate nematode chromosome IV.PMK-1 and PMK-2 share a 62% amino acid sequence identity.PMK-1 regulates innate immunity in the intestine cell of C.elegans,and is activated by a MAPK signaling cassette TIR-1-NSY-1-SEK-1.However PMK-2 protein is found nearly exclusively in the nervous system of C.elegans,because the miR-58/80-82 family switches off the expression of PMK-2 in non-neuronal.Although PMK-1 and PMK-2 are highly homologous,their biofunctions are likely to be very different.Our previous study indicated that expression level of PMK-2 protein is found increased when C.elegans exposed to Cry6 A toxin.Hence,we proposed that PMK-2probably mediates the defense response of C.elegans against Cry6 A intoxication.PMK-1 is activated by the upstream kinases of p38 MAPK SEK-1and NSY-1.Due to pmk-1and pmk-2 locate in a single operon,therefore we hypothesized that PMK-2 shares the same activation pathway with PMK-1.Our research data showed: 1.Cry6 A crystal toxin protein can induce significant increase on the gene transcription level of pmk-2 in the C.elegans;2.The sensitivity of C.elegans to Cry6 A crystal protein was significantly enhanced when pmk-2 was silenced by RNAi technique;3.Western blot experiment indicated that Cry6 A crystal protein can induce phosphorylation of PMK-1 and PMK-2 when C.elegans was intoxicated by Cry6 A toxin,then activates the PMK signaling pathway;4.Two upstream kinase of PMK NSY-1 and SEK-1 mutants,sek-1(km4)and nsy-1(ag3),exhibited more hypersensitivity to Cry6 A toxin than wild-type N2.In addition,Cry6 A could not induce the phosphorylation of PMK-2 protein in the two mutants.This result indicates that,the PMK-2,like PMK-1,is also activated by the upstream kinase NSY-1 and SEK-1in phosphorylation cascades.In conclusion,this study proved that PMK-2signal pathway plays a significant role in the defense response of C.elegans against Cry6 A crystal protein toxin.