Comprehensive analysis of Cry1Ac protoxin activation mediated by midgut proteases in susceptible and resistant Plutella xylostella (L.)
Comprehensive analysis of Cry1Ac protoxin activation mediated by midgut proteases in susceptible and resistant Plutella xylostella (L.)
复制标题
易感和抗性小菜蛾中肠蛋白酶介导的Cry1Ac原毒素激活的综合分析
DOI：
10.1016/j.pestbp.2019.10.006
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发表时间：
2020-02-01
影响因子：
4.7
通讯作者：
Zhang, Youjun
中科院分区：
文献类型：
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作者：
Guo, Zhaojiang;Gong, Lijun;Zhang, Youjun
由苏云金芽孢杆菌（Bacillus thuringiensis，Bt）产生的杀虫毒素已广泛应用于叶面喷洒和转基因作物中防治农业害虫。然而，昆虫对Cry毒素的抗性的快速进化需要阐明Cry抗性所涉及的分子机制。已经描述了两种提出的模型来解释Cry蛋白的毒性，经典模型指出Cry原毒素被中肠蛋白酶激活，导致激活的毒素结合受体并在中肠细胞中形成孔，触发幼虫死亡，新提出的Bt Cry毒素作用模式的双重模型表明，原毒素和活化毒素可能具有不同的作用机制，因为几种抗性毒素菌株对活化的Cry毒素仍然对相同的Cry-原毒素敏感。中肠蛋白酶激活原毒素是两种模型中的关键步骤。在此，我们评估了敏感小菜蛾（Plutella xylostella（L.）菌株（DBM 1AcS）和具有高场进化的Cry 1Ac抗性的近等基因菌株（NIL-R）。先前的工作表明，NIL-R中的Cry 1Ac抗性与由于增强的MAPK信号通路和ABCC 2受体的下调而减少的与中肠受体的结合相关。然而，减少中肠胰蛋白酶水平和改变中肠蛋白酶基因转录中也观察到Cry 1Ac耐药现场分离菌株，是NIL-R菌株的父母。因此，我们分析了DBM 1Ac-S和NIL-R菌株中的中肠蛋白酶活性。酶活性检测结果表明，敏感菌株和抗性菌株的酪蛋白分解蛋白酶、胰蛋白酶和胰凝乳蛋白酶活性差异不显著。此外，用不同的胰蛋白酶或胰凝乳蛋白酶抑制剂，如N α-甲苯磺酰基-L-赖氨酸氯甲基酮（TLCK）或N α-甲苯磺酰基-L-苯丙氨酸氯甲基酮（TPCK）处理不会影响DBM 1Ac-S和NIL-R幼虫对Cry 1Ac原毒素的敏感性。生物测定结果表明，NIL-R幼虫对CrylAc原毒素和胰蛋白酶激活毒素的抗性水平相似。两者合计，我们的研究结果表明，高水平的字段演变的Cry 1Ac抗性在NIL-R菌株是独立的Cry 1Ac原毒素激活和特定的原毒素的作用机制。这一发现将加强我们对不同昆虫Bt抗性的复杂机制基础的全面理解。
Insecticidal Cry toxins produced by Bacillus thuringiensis (Bt) have been widely used to control agricultural pests in both foliage sprays and transgenic crops. Nevertheless, rapid evolution of insect resistance to Cry toxins requires elucidation of the molecular mechanisms involved in Cry resistance. Two proposed models have been described to explain the toxicity of Cry proteins, the classic model states that Cry protoxin is activated by midgut proteases resulting in activated toxin that binds to receptors and forms a pore in the midgut cells triggering larval death, and the newly proposed dual model of the mode of action of Bt Cry toxins states that protoxin and activated toxins may have different mechanisms of action since several resistant strains to activated Cry toxins are still susceptible to the same Cry-protoxin. Protoxin activation by midgut proteases is a key step in both models. Herein, we evaluated Cry1Ac protoxin activation in a susceptible Plutella xylostella (L.) strain (DBM1AcS) and in the near-isogenic strain (NIL-R) with high field-evolved Cry1Ac resistance. Previous work showed that Cry1Ac resistance in NIL-R correlates with reduced binding to midgut receptors due to enhanced MAPK signaling pathway and down regulation of ABCC2 receptor. However, reduced midgut trypsin levels and altered midgut protease gene transcription were also observed in the Cry1Ac-resistant field isolated strain that is parent of the NIL-R strain. Therefore, we analyzed the midgut protease activities in both DBM1Ac-S and NIL-R strains. Detection of enzymatic activities showed that caseinolytic protease, trypsin and chymotrypsin activities were not significantly different between the susceptible and resistant strains. Furthermore, treatment with different trypsin or chymotrypsin inhibitors, such as N alpha-tosyl-L-lysine chloromethyl ketone (TLCK) or N alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not affect the susceptibility to Cry1Ac protoxin of the DBM1Ac-S and NIL-R larvae. Bioassay results indicated that the NIL-R larvae showed similar resistant levels to both CrylAc protoxin and trypsin-activated toxin. Taken together, our results demonstrated that high-level field-evolved Cry1Ac resistance in the NIL-R strain is independent of Cry1Ac protoxin activation and the specific protoxin mechanism of action. This discovery will strengthen our comprehensive understanding of the complex mechanistic basis of Bt resistance in different insects.