College of Forestry, Fujian Agriculture and Forestry University, Fuzhou, China.
Key Laboratory of Integrated Pest Management in Ecological Forests, Fujian Province University, Fujian Agriculture and Forestry University, Fuzhou, China.
Pest Manag Sci. 2020 Sep;76(9):3117-3126. doi: 10.1002/ps.5866. Epub 2020 May 20.
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.
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 (LC = 12.3 μg/mL) increase in toxicity to M. alternatus larvae when compared to native Cry3Aa toxin.
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. © 2020 Society of Chemical Industry.
苏云金芽孢杆菌 Cry3 毒素对几种鞘翅目幼虫具有特异性毒性。然而，由于其对松墨天牛幼虫的低毒性，Cry3A 毒素在防治松墨天牛幼虫方面没有用处。在这里，我们评估了 Cry3Aa 毒素在松墨天牛幼虫中肠的蛋白水解激活作用，并通过分子修饰提高了其毒性。
我们的结果表明，Cry3Aa 原毒素的酶解不足和 Cry3Aa 毒素在松墨天牛幼虫中肠中的非特异性酶解导致了低毒性。转录组分析、荧光底物酶活性测定以及通过质谱进行的多底物谱分析的结果表明，松墨天牛幼虫中肠的主要消化酶是胰蛋白酶样蛋白酶，其优先切割具有精氨酸和赖氨酸残基的肽。因此，在 Cry3Aa 原毒素的结构域 I 中引入了胰蛋白酶识别位点，该位点位于 α-螺旋 3 和 α-螺旋 4 之间的环区，以促进蛋白水解激活。还将 Cry3Aa 蛋白中远离螺旋片和功能区域的多个潜在胰蛋白酶切割位点突变为丙氨酸，以防止非特异性酶解。生物测定表明，与天然 Cry3Aa 毒素相比，一种修饰的 Cry3Aa-T 毒素（K65A、K70A、K231A、K468A 和 K596A）对松墨天牛幼虫的毒性增加了 9.5 倍（LC = 12.3μg/mL）。
本研究强调了一种有效提高 Cry3Aa 毒素对松墨天牛毒性的方法，该方法可能适用于管理转基因植物对其他害虫的抗性，包括农业中一些最重要的害虫。© 2020 化学工业协会。
