Takemoto Daigo, Jones David A
Plant Pathology Laboratory, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601, Japan.
Methods Mol Biol. 2014;1127:91-101. doi: 10.1007/978-1-62703-986-4_7.
Plant pathogens, including fungi, oomycetes, bacteria, aphids, and nematodes, produce a variety of effector proteins to counter plant disease resistance mechanisms. After delivery into the cytosol of the plant cell, effectors may target proteins localized to different compartments within the plant cell. Plants, in turn, have evolved disease resistance (R) proteins to recognize the action of effectors. Elucidation of the subcellular localization of pathogen effectors, the plant proteins they target, and plant disease resistance proteins is essential to fully understand their interactions during pathogen challenge. In recent years, expression of fluorescent protein fusions has been widely used to determine the subcellular localization of plant proteins and pathogen effectors. Use of fluorescent proteins enables researchers to monitor the dynamic behavior of proteins in living cells. Among various methods available for the introduction of genes into plant cells, particle bombardment-mediated transient expression is the most rapid method suitable for both the identification of localization signals in proteins of interest and their dissection via amino acid substitutions generated using site-directed mutagenesis. This chapter describes a rapid procedure for particle bombardment-mediated transient expression in leaf epidermal cells. This method is also applicable to detection of pathogen effector protease activities directed against target proteins in the plant cell and analysis of protease recognition sites within these target proteins.
包括真菌、卵菌、细菌、蚜虫和线虫在内的植物病原体可产生多种效应蛋白，以对抗植物的抗病机制。效应蛋白被递送到植物细胞的细胞质后，可能会作用于定位于植物细胞内不同区室的蛋白质。反过来，植物也进化出了抗病（R）蛋白来识别效应蛋白的作用。阐明病原体效应蛋白、它们所作用的植物蛋白以及植物抗病蛋白的亚细胞定位，对于全面了解病原体攻击过程中它们之间的相互作用至关重要。近年来，荧光蛋白融合表达已被广泛用于确定植物蛋白和病原体效应蛋白的亚细胞定位。使用荧光蛋白可使研究人员监测活细胞中蛋白质的动态行为。在可用于将基因导入植物细胞的各种方法中，粒子轰击介导的瞬时表达是最快速的方法，适用于鉴定目标蛋白中的定位信号以及通过定点诱变产生的氨基酸替换对其进行剖析。本章介绍了一种在叶片表皮细胞中进行粒子轰击介导的瞬时表达的快速方法。该方法也适用于检测针对植物细胞中目标蛋白的病原体效应蛋白酶活性，以及分析这些目标蛋白内的蛋白酶识别位点。
