Yajie Guo1,2,3, Yafang Wang3,4, Anthony J O'Donoghue5, Zhenze Jiang5, Rebeca Carballar-Lejarazú6, Guanghong Liang1,2, Xia Hu1,2, Rong Wang1,2, Lei Xu7, Xiong Guan3, Feiping Zhang1,2, Songqing Wu1,2,3. 1. College of Forestry, Fujian Agriculture and Forestry University, Fuzhou, China. 2. Key Laboratory of Integrated Pest Management in Ecological Forests, Fujian Province University, Fujian Agriculture and Forestry University, Fuzhou, China. 3. State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, China. 4. Engineering Research Center of Molecular Diagnostics, Ministry of Education, Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Xiamen, China. 5. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA. 6. Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, USA. 7. Graduate School of Chinese Academy of Agricultural Sciences, Beijing, China.
Abstract
BACKGROUND: Bacillus thuringiensis Cry3 toxins exhibit specific toxicity against several coleopteran larvae. However, owing to its low toxicity to Monochamus alternatus, Cry3A toxin is not useful for managing M. alternatus larvae. Here we assessed the proteolytic activation of Cry3Aa toxin in M. alternatus larval midgut and increased its toxicity by molecular modification. RESULTS: Our results indicated that insufficient processing of Cry3Aa protoxin and non-specific enzymatic digestion of Cry3Aa toxin in the midgut of M. alternatus larvae led to low toxicity. The results of transcriptome analysis, enzymatic assay with fluorogenic substrates, and multiplex substrate profiling by mass spectrometry showed that the main digestive enzymes in M. alternatus larval midgut were trypsin-like proteases that preferentially cleaved peptides with arginine and lysine residues. Consequently, trypsin recognition sites were introduced into the Domain I of Cry3Aa protoxin in the loop regions between α-helix 3 and α-helix 4 to facilitate proteolytic activation. Multiple potential trypsin cleavage sites away from the helix sheet and functional regions in Cry3Aa proteins were also mutated to alanine to prevent non-specific enzymatic digestion. Bioassays indicated that a modified Cry3Aa-T toxin (K65A, K70A, K231A, K468A, and K596A) showed a 9.5-fold (LC50 = 12.3 μg/mL) increase in toxicity to M. alternatus larvae when compared to native Cry3Aa toxin. CONCLUSION: This study highlights an effective way to increase the toxicity of Cry3Aa toxin to M. alternatus, which may be suitable for managing the resistance of transgenic plants to other pests, including some of the most important pests in agriculture.
BACKGROUND:Bacillus thuringiensis Cry3 toxins exhibit specific toxicity against several coleopteran larvae. However, owing to its low toxicity to Monochamus alternatus, Cry3A toxin is not useful for managing M. alternatus larvae. Here we assessed the proteolytic activation of Cry3Aa toxin in M. alternatus larval midgut and increased its toxicity by molecular modification. RESULTS: Our results indicated that insufficient processing of Cry3Aa protoxin and non-specific enzymatic digestion of Cry3Aa toxin in the midgut of M. alternatus larvae led to low toxicity. The results of transcriptome analysis, enzymatic assay with fluorogenic substrates, and multiplex substrate profiling by mass spectrometry showed that the main digestive enzymes in M. alternatus larval midgut were trypsin-like proteases that preferentially cleaved peptides with arginine and lysine residues. Consequently, trypsin recognition sites were introduced into the Domain I of Cry3Aa protoxin in the loop regions between α-helix 3 and α-helix 4 to facilitate proteolytic activation. Multiple potential trypsin cleavage sites away from the helix sheet and functional regions in Cry3Aa proteins were also mutated to alanine to prevent non-specific enzymatic digestion. Bioassays indicated that a modified Cry3Aa-T toxin (K65A, K70A, K231A, K468A, and K596A) showed a 9.5-fold (LC50 = 12.3 μg/mL) increase in toxicity to M. alternatus larvae when compared to native Cry3Aa toxin. CONCLUSION: This study highlights an effective way to increase the toxicity of Cry3Aa toxin to M. alternatus, which may be suitable for managing the resistance of transgenic plants to other pests, including some of the most important pests in agriculture.