Caenorhabditis Elegans AMPK Mediated Defense Against Bacillus Thuringiensis Cry5Ba Toxin

Bacillus thuringiensis(Bt) is used as micropesticide worldwide, because it can produce insecticidal crystal(Cry) protein during the sporulation phase. The molecular mechanism how the pests defend against Bt insecticidal Crystal protein, is not well understood. Recent research have shown that there is some relation between energy metabolism and pests’ defense response against Bt insecticidal Crystal protein. Adenosine(AMP)-activated protein kinase(AMPK) is highly conserved in all cells, its main biological function is the cell’s energy regulator. It is unclear whether AMPK and pests’ defense response have some relationship. In our study, using the insecticidal Crystal protein Cry5 Ba and Caenorhabditis elegans as a model, we found that the AMPK mediated the C elegans defense against Bt insecticidal Crystal protein Cry5 Ba. Our results are as following:1 The AMPK defend against insecticidal Crystal protein Cry5 Ba produced by Bacillus thuringiensis in Caenorhabditis elegans(1) AMPK is activated by the Bt Crystal protein Cry5BaBy comparing the transcriptomes when C. elegans fed with the Cry5Ba(Data not shown), we found that the α2 subunit of the Adenosine 5-monophosphate(AMP)-activated protein kinase(AMPK) was upregulated. Quantitative RT-PCR analyses indicated that the transcript of gene aak-2 increased when C.elegans N2 were fed with Cry5Ba; Therefore we measured the concentrations of AMP and ATP by LC-MS, the ratio of AMP/ATP increased 7 times when C.elegans N2 were fed with Cry5 Ba. Western Blotting experiment confirmed that Thr172 of α2 subunit of AMPK was phosphorylated. These showed that Cry5 Ba activate AMPK via the Thr-172 phosphorylation of α2 subunit in C.elegans.(2) AMPK is required for the defense against toxin Cry5BaWe test the growth inhibition rate and the survival assay in different doses of Cry5 Ba using the wild type C.elegans N2 and the null alleles mutants of AMPK, aak-1(tm1944), aak-2(ok524), aakb-1(tm2658), and aakg-4(tm5269). The mutant aak-2(ok524) showed hypersensitivity to Cry5Ba; The AICAR-activating AMPK worms showed resistance to toxin Cry5 Ba, and the resistance to toxin Cry5 Ba disappeared in null mutant worm aak-2(ok524); The transgenic experiments indicated that intestinal AAK-2 can be sufficient to rescue the hypersensitivity of mutant aak-2(ok524) to toxin Cry5 Ba, neither muscle nor neuronal AAK-2 was necessary or sufficient. Our results indicated that the AMPK is required for defense against toxin Cry5 Ba.(3) Several defense responses against Cry5 Ba are regulated by AMPK pathwayUsing the transgenic worm strain TJ356(Isdaf-16::gfp), we confirmed that the Cry5 Ba can induce the transcription factor DAF-16 nuclear translocation via AMPK pathway. We also found that AMPK also regulate several defense responses besides transcription factor DAF-16. We also confirmed that the Cry5 Ba can induce autophagy via AMPK in C. elegans. These results indicated that AMPK can regulate several defense responses against Cry5 Ba.(4) The crystal inclusions Cry5 Ba tiggers feeding cessation behaviorThe pump frequency and intaked Bt spores per worm of C. elegans N2, that fed with crystal inclusions Cry5 Ba produced by B. thuringiensis, reduced dramatically than those fed with Bt. However, when Cry5Ba-receptor deficient C. elegans bre-5(ye5) fed with the Cry5 Ba, the pump frequency and intaked Bt spores per worm were no difference with those fed with Bt. In a word, the crystal inclusions Cry5 Ba can tigger feeding cessation behavior in C. elegans.2. Isolation of the novel novel nematicidal bacteriumIn almost ten years of C. elegans research, we have sometimes observed that almost all worms die on NGM agar plates under standard culture conditions. To find potential reasons, we tried to isolate pathogens from C. elegans cadavers.The root-knot nematodes(RKNs)(Meloidogyne spp.) are an important agricultural pest of many crops. It is capable of infecting almost all crops. The management of RKNs is more difficult than that of other pests because RKNs are obligate parasites of the roots of plants and most of the life cycle of RKNs is spent as parasites in the host roots. Bacterial control of RKNs is a potential management strategy that avoids the potential environmental problems associated with chemical control of RKNs. The current means for RKNs management rely on chemical nematicides, which are often ineffective and environmentally hazardous. Currently, only several bacteria have been reported to have nematicidal activity, the typical nematicidal bacteria are mainly Bacillus, Pseudomonas and Pasteuria. Identifying novel nematicidal bacteria has become urgent for RKNs biological management and has extremely practical and economical significance.(1) Isolation and identifition of Alcaligenes faecalis ZD02Strain ZD02 was isolated from Caenorhabditis elegans cadavers, had nematicidal activity that was confirmed by the growth assay and life span assay of C. elegans; Subsequently, the fermentation broth of A. faecalis ZD02 was showed toxicity activity against C. elegans and Meloidogyne incognita. The 16 S rRNA gene sequence indicated strain ZD02 belongs to Alcaligenes faecalis.(2) An extracellular serine protease(Esp) protein from A. faecalis ZD02 functions as a nematicidal virulence factorWe sequenced the complete genome of A. faecalis ZD02. By analysing the genome, an extracellular serine protease(Esp) protein was found, and the serine proteinase activity was confirmed. The reverse transcription PCR analysis confirmed that esp gene can transcript in A. faecalis ZD02. By the mortality assays, the Esp protein showed significant toxicity against C. elegans and M. incognita, the median lethal dose(LC50) of C. elegans was 260.45μg/mL, the LC50 of M. incognita J2 s was 211.50μg/m L. Using the C. elegans as model, we found that A. faecalis ZD02 and the Esp protein appear to exert their nematicidal activity at least in part via damaging the intestines of C. elegans.(3) A. faecalis ZD02 is able to inhabit the defense responsesIn C. elegans, QRT-PCR indicated that A. faecalis ZD02 inhabit the transcriptions of typical defense effectors lysozyme and antibiotic peptide, and A. faecalis ZD02 can also inhabit the typical defense responses, that include Insulin-like siganl(daf-2/daf-16) pathway, p38 mitogen-activated protein kinase(p38MAPK) pathway,, c-Jun protein N-terminal kinase(C-Jun MAPK) pathway,, autophagy and unfolded protein response(UPR) response.