Kawanda Agricultural Research Institute, Kampala, Uganda.
Arch Insect Biochem Physiol. 2010 Feb;73(2):87-105. doi: 10.1002/arch.20342.
The general potential of plant cystatins for the development of insect-resistant transgenic plants still remains to be established given the natural ability of several insects to compensate for the loss of digestive cysteine protease activities. Here we assessed the potential of cystatins for the development of banana lines resistant to the banana weevil Cosmopolites sordidus, a major pest of banana and plantain in Africa. Protease inhibitory assays were conducted with protein and methylcoumarin (MCA) peptide substrates to measure the inhibitory efficiency of different cystatins in vitro, followed by a diet assay with cystatin-infiltrated banana stem disks to monitor the impact of two plant cystatins, oryzacystatin I (OC-I, or OsCYS1) and papaya cystatin (CpCYS1), on the overall growth rate of weevil larvae. As observed earlier for other Coleoptera, banana weevils produce a variety of proteases for dietary protein digestion, including in particular Z-Phe-Arg-MCA-hydrolyzing (cathepsin L-like) and Z-Arg-Arg-MCA-hydrolyzing (cathepsin B-like) proteases active in mildly acidic conditions. Both enzyme populations were sensitive to the cysteine protease inhibitor E-64 and to different plant cystatins including OsCYS1. In line with the broad inhibitory effects of cystatins, OsCYS1 and CpCYS1 caused an important growth delay in young larvae developing for 10 days in cystatin-infiltrated banana stem disks. These promising results, which illustrate the susceptibility of C. sordidus to plant cystatins, are discussed in the light of recent hypotheses suggesting a key role for cathepsin B-like enzymes as a determinant for resistance or susceptibility to plant cystatins in Coleoptera.
鉴于几种昆虫具有补偿消化半胱氨酸蛋白酶活性损失的天然能力，植物半胱氨酸蛋白酶抑制剂在开发抗虫转基因植物方面的普遍潜力仍有待确定。在这里，我们评估了半胱氨酸蛋白酶抑制剂在开发抗香蕉象鼻虫 Cosmopolites sordidus 的香蕉品系中的潜力，香蕉象鼻虫是非洲香蕉和大蕉的主要害虫。使用蛋白质和甲基香豆素（MCA）肽底物进行蛋白酶抑制测定，以测量不同半胱氨酸蛋白酶抑制剂在体外的抑制效率，然后用渗透有半胱氨酸蛋白酶抑制剂的香蕉茎圆盘进行饮食测定，以监测两种植物半胱氨酸蛋白酶，oryzacystatin I（OC-I，或 OsCYS1）和木瓜半胱氨酸蛋白酶（CpCYS1）对象鼻虫幼虫整体生长速度的影响。正如早些时候观察到的其他鞘翅目昆虫一样，香蕉象鼻虫产生多种蛋白酶用于膳食蛋白质消化，特别是 Z-Phe-Arg-MCA 水解（组织蛋白酶 L 样）和 Z-Arg-Arg-MCA 水解（组织蛋白酶 B 样）蛋白酶，在微酸性条件下具有活性。这两种酶群体都对半胱氨酸蛋白酶抑制剂 E-64 和包括 OsCYS1 在内的不同植物半胱氨酸蛋白酶敏感。与半胱氨酸蛋白酶的广泛抑制作用一致，OsCYS1 和 CpCYS1 导致在渗透有半胱氨酸蛋白酶抑制剂的香蕉茎圆盘内发育 10 天的幼幼虫生长严重延迟。这些有希望的结果表明，C. sordidus 易受植物半胱氨酸蛋白酶的影响，这与最近的假设一致，该假设表明组织蛋白酶 B 样酶作为鞘翅目昆虫对植物半胱氨酸蛋白酶抗性或敏感性的决定因素起着关键作用。
