Immunoelectron microscopic analysis of callose secretion and nuclear condensation associated with defense responses of plant pathogen-stressed plant cells

日本梨病型互花霉孢子在感、抗性梨叶片的胚管顶端萌发并形成附着胞。从压胞底部出现的渗透钉试图侵入易感表皮细胞的细胞壁。刺穿表皮层后沿果胶层生长为侵染菌丝，除完全破坏的细胞外，不直接侵入表皮细胞质。在真菌侵入组织之前，ak -毒素诱导的质膜修饰与胼胝质分泌相关，出现在易感叶细胞的胞间连丝上，这表明毒素来自胚芽管的假设也得到了支持。相反，在抗性叶片上接种的病原菌能够渗透到角质层中，但在渗透角质层后，真菌的入侵并不受限制。这些结果表明，对黑斑病的抗性机制在发病过程中更多地表现在表皮侵袭阶段。在细胞壁和质膜之间的空隙中发现了乳突的形成，而不考虑对病原体接种的敏感和抗性叶片，但观察到穿透钉，但不涉及乳突本身。这表明，乳突是植物对表皮损伤的非特异性反应，但不直接参与黑斑病的抗病性。免疫电镜研究表明，β- 1,3- d葡聚糖在病原菌感染的敏感和抗性叶片的乳头和毒素处理的敏感叶片的胼胝质上均有积累。结果表明，葡聚糖是由敏感叶片的高尔基体通过高尔基囊泡分泌到质膜受损部位，进行质膜修饰。活性氧（ROS）的产生是在毒素处理的敏感叶片的血浆修饰膜片段上发现的，而不是在乳头上。结果表明，ROS的产生与毒素诱导的质膜修饰有关，质膜损伤与细胞离子泄漏位点在空间上一致。在侵染过程中，病原菌接种的敏感和抗性叶片的穿透钉细胞壁上也发现了ROS的产生。活性氧可能通过细胞壁蛋白与活性氧的交叉反应导致细胞壁硬化，提示穿透钉的侵袭性增强。虽然在毒素处理和孢子接种的敏感叶片中观察到核凝聚，但核变化并未被评价为凋亡事件之一。这些变化与坏死组织的坏死一致，因为没有DNA阶梯。少

Spores of Alternaria alternata Japanese pear pathotype germinated and formed appressoria at tips of germ tubes on surfaces of the susceptible and resistant pear leaves. Penetration pegs emerged from appressorial bottom made an attempt to invade cell walls of susceptible epidermal cells. The pegs penetrated into the cuticular layers and then were grown as infection hyphae along the pectin layers, but didn't invade directly the epidermal cytoplasm except for the completely disrupted cells. AK-toxin-induced plasma membrane modifications, associated with callose secretion, appeared at plasmodesmata of susceptible leaf cells before the fungal invasion in tissues, suggesting the hypothesis that the toxin was produced from germ tubes was also supported. On the contrary, the pathogen inoculated in resistant leaves was able to penetrate into cuticles, but fungal invasion was not limited after the cuticular penetration. These results indicated that resistant mechanism to black spot disease was e … More xpressed at the stage of cuticular invasion during pathogenesis. Papilla formation was found at the spaces between cell walls and plasma membranes irrespective of susceptible and resistant leaves against pathogen inoculation, but penetration pegs were observed, but not involved in the papillae themselves. This suggests that papillae are non-specific plant responses against cuticular wounding, but didn't contribute directly to disease resistance in black spot disease. Immunoelectron microscopic study showed that β-l,3-D-glucan accumulated both at papillae in the pathogen-infected susceptible and resistant leaves, and at callose in the toxin-treated susceptible ones. The glucan was shown to be be secreted from Golgi bodies of susceptible leaves via Golgi vesicles to damaged sites of plasma membranes, plasma membrane modifications. The generation of reactive oxygen species (ROS) wsa found at the membrane fragments of plasma modifications in the toxin-treated susceptible leaves, but not at the papillae. This results showed that ROS generation was associated with the toxin-induced plasma membrane modifications which were damaged plasmalemma which were spatially consistent with cellular ion-leakage sites. ROS generation also was recognized at the cell walls of penetration pegs during infection process in the pathogen-inoculated susceptible and resistant leaves. ROS might cause hardening cell walls by cross-reaction of cell wall proteins with ROS, suggesting increased aggressiveness of penetration pegs. Although nuclear condensation was observed in the toxin-treated and spore-inoculated susceptible leaves, the nuclear changes were not evaluated as one of apoptotic events. The changes were compatible with pknosis seen in necrotic tissues because of no laddering of DNA. Less