Proteolytic Activation of Bacillus thuringiensis Cry2Ab through a Belt-and-Braces Approach.
Proteolytic processing of Bacillus thuringiensis (Bt) crystal toxins by insect midgut proteases plays an essential role in their insecticidal toxicities against target insects. In the present study, proteolysis of Bt crystal toxin Cry2Ab by Plutella xylostella L. midgut proteases (PxMJ) was evaluated. Both trypsin and chymotrypsin were identified involving the proteolytic activation of Cry2Ab and cleaving Cry2Ab at Arg(139) and Leu(144), respectively. Three Cry2Ab mutants (R139A, L144A, and R139A-L144A) were constructed by replacing residues Arg(139), Leu(144), and Arg(139)-Leu(144) with alanine. Proteolysis assays revealed that mutants R139A and L144A but not R139A-L144A could be cleaved into 50 kDa activated toxins by PxMJ. Bioassays showed that mutants R139A and L144A were highly toxic against P. xylostella larvae, while mutant R139A-L144A was almost non-insecticidal. Those results demonstrated that proteolysis by PxMJ was associated with the toxicity of Cry2Ab against P. xylostella. It also revealed that either trypsin or chymotrypsin was enough to activate Cry2Ab protoxin. This characteristic was regarded as a belt-and-braces approach and might contribute to the control of resistance development in target insects. Our studies characterized the proteolytic processing of Cry2Ab and provided new insight into the activation of this Bt toxin.
State Key Laboratory of Cellular Stress Biology, Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University , Xiamen, Fujian 361005, People's Republic of China.
Lian
L
Xu
Agricultural Bio-Resources Research Institute, Fujian Academy of Agricultural Sciences , Fuzhou, Fujian 350003, People's Republic of China.
Zhi-Zhen
ZZ
Pan
State Key Laboratory of Cellular Stress Biology, Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University , Xiamen, Fujian 361005, People's Republic of China.
Jing
J
Zhang
Agricultural Bio-Resources Research Institute, Fujian Academy of Agricultural Sciences , Fuzhou, Fujian 350003, People's Republic of China.
Bo
B
Liu
Agricultural Bio-Resources Research Institute, Fujian Academy of Agricultural Sciences , Fuzhou, Fujian 350003, People's Republic of China.
Yu-Jing
YJ
Zhu
State Key Laboratory of Cellular Stress Biology, Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University , Xiamen, Fujian 361005, People's Republic of China.
Qing-Xi
QX
Chen
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics* / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D007306:Insecticides / Q000378:metabolism; D007814:Larva / Q000201:enzymology; D009036:Moths / Q000201:enzymology; D010447:Peptide Hydrolases / Q000378:metabolism; D010572:Pest Control, Biological; D011487:Protein Conformation; D059748:Proteolysis; D014357:Trypsin / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin
Bacillus thuringiensis; Cry2Ab; cleavage site; insecticidal activity; proteolysis