Urwin P E, McPherson M J, Atkinson H J
Centre for Plant Biochemistry and Biotechnology, University of Leeds, UK.
Planta. 1998 Apr;204(4):472-9. doi: 10.1007/s004250050281.
Plant defence strategies usually involve the action of several gene products. Transgenic resistance strategies are likely to have enhanced efficacy when they involve more than one transgene. Here we explore possible mechanisms for the co-delivery of multiple effectors via a single transgene. As an example we report the co-delivery of two distinct proteinase inhibitors in Arabidopsis thaliana (L.) Heynh. to examine resistance against plant parasitic nematodes. A cysteine and serine proteinase inhibitor have been joined as translational fusions by one of two peptide linkers. One linker, part of the spacer region of a plant metallothionein-like protein (PsMTa), was selected to be cleaved in planta. A second linker, derived from the fungal enzyme galactose oxidase (GO) was chosen to be refractory to cleavage in planta. Western blot analysis of cell extracts confirmed the expected pattern of predominantly single inhibitors derived from the PsMTa construct and a primarily dual inhibitor from the GO construct. Analysis of cyst and root-knot nematodes recovered from transgenic Arabidopsis expressing inhibitors as single or dual molecules revealed the uptake of inhibitors with the exception of those linked by the PsMTa linker. This unexpected result may be due to residues of the PsMTa linker interacting with cell membranes. Despite lack of ingestion, PsMTa-linked cowpea trypsin inhibitor (CpTI) affected the sexual development of the cyst nematodes, indicating an external site of action. The engineered cystatin (Oc-I delta D86) component from the PsMTa constuct had no effect, indicating that ingestion is necessary for the cystatin to be effective. The delivery of dual inhibitors linked by the GO linker showed a clear additive effect over either inhibitor delivered singly. The application of this technology to other plant pathogens is discussed.
植物防御策略通常涉及多种基因产物的作用。当转基因抗性策略涉及多个转基因时，其功效可能会增强。在此，我们探索通过单个转基因共同递送多种效应物的可能机制。作为一个例子，我们报道了在拟南芥中共同递送两种不同的蛋白酶抑制剂，以研究对植物寄生线虫的抗性。一种半胱氨酸蛋白酶抑制剂和一种丝氨酸蛋白酶抑制剂通过两种肽接头之一连接成翻译融合体。其中一个接头是植物类金属硫蛋白样蛋白（PsMTa）间隔区的一部分，被选择在植物体内可被切割。另一个接头来源于真菌酶半乳糖氧化酶（GO），被选择在植物体内不易被切割。对细胞提取物的蛋白质印迹分析证实了预期的模式，即主要从PsMTa构建体衍生出单一抑制剂，而从GO构建体主要衍生出双重抑制剂。对从表达单一或双重分子抑制剂的转基因拟南芥中回收的孢囊线虫和根结线虫的分析表明，除了与PsMTa接头连接的抑制剂外，其他抑制剂都被摄取了。这个意外的结果可能是由于PsMTa接头的残基与细胞膜相互作用。尽管没有摄取，但与PsMTa连接的豇豆胰蛋白酶抑制剂（CpTI）影响了孢囊线虫的有性发育，表明其作用位点在外部。来自PsMTa构建体的工程化半胱氨酸蛋白酶抑制剂（Oc-I delta D86）没有效果，表明半胱氨酸蛋白酶抑制剂要发挥作用需要摄取。由GO接头连接的双重抑制剂的递送对单独递送的任何一种抑制剂都显示出明显的加性效应。本文还讨论了将该技术应用于其他植物病原体的情况。
