MOLECULAR AND CELL BIOLOGICAL RESERCH FOR REGURATION OF CYTOSKELETAL REORGANIZATION WHICH IS INVOLVED IN NONHOST RESISTANCE EXPRESSION

本研究旨在分析植物非寄主抗性的分子基础细胞骨架调控，为植物非寄主抗性增强分子育种提供基础知识。用罗丹明-phalloidin染色，荧光显微镜下观察接种了非病原体Erisiphe pisi的大麦胚芽鞘细胞的细胞骨架组织变化。肌动蛋白微丝聚集在非病原体的穿透点下。当用肌动蛋白聚合抑制剂（细胞松弛素）处理球囊细胞时，单病原体能够成功地渗透到非宿主大麦细胞中。同时，肌动蛋白抑制剂抑制细胞器极化和防御相关反应。以小麦和烟草为研究对象，分别接种非致病菌E.pisi和炭疽菌（Colletotrichum lagenarium），观察其肌动蛋白骨架的重组和细胞松弛素的作用。这些结果表明，肌动蛋白细胞骨架参与非寄主抗性的机制在许多植物物种中普遍存在。由于细胞骨架的重排对抗性的表达至关重要，我们试图从大麦cDNA文库中克隆肌动蛋白异构体基因，以期在阐明细胞骨架重组调控方面开辟一个突破。对3个主要的肌动蛋白异构体基因Actl、2和3进行了克隆和测序。接种E.pisi后，大麦胚芽组织中Act3的表达水平特异性升高。Act3似乎参与了植物细胞的极化，因为该基因也在富含根毛的年轻根组织中强烈表达。因此，Act3基因的产物与非寄主抗性的表达密切相关。

The aim of the research is analysis of the cytoskeletal regulation, which seems to be a molecular basis of plant nonhost resistance, and obtaining basic knowledge for molecular breeding of a plant which is enhanced nonhost resistance. To investigate a change of cytoskeletal organization, barley coleoptile cells which were inoculated with a nonpathogen, Erisiphe pisi, were stained with rhodamine-phalloidin and observed with fluorescent microscope. Actin microfilaments gathered beneath penetration sites of the nonpathogen. When coleoptile cells were treated with actin polymerization inhibitor, cytochalasin, the monpathogen became able to penetnate successfully into nonhost, barley, cells. At the same time, polarization of organelles and defense related responses were suppressed by treatment of the actin inhibitor. The other plants, such as wheat and tobacco, were inculated with nonpathogens, E.pisi and Colletotrichum lagenarium, and reorganization of the actin cytoskeleton and the effects of cytochalasin were investigated. These results showed that the mechanism of nonhost resistance involving the actin cytoskeleton generally exist in many plant species. Because rearrangement of the cytoskeleton is important for the expression of the resistance, we tried to clone actin isoform genes from a barley cDNA libary in order to open a breakthrough of elucidating a regulation of cytoskeletal reorganization. Three major actin isoform genes, Actl, 2 and 3, were cloned and sequenced. When barley coleoptiles were inoculated with E.pisi, the expression level of Act3 was increased specifically. Act3 seems to be involved in making polarization in plant cells, because the gene also strongly expresses in young root tissue, which abundantly contain apical-growing root hairs. Therefore, it is suggested that product of Act3 gene closely related to the expression of nonhost resistance.