Molecular elucidation of elicitors and suppressors in rice blast disease by using as the marker the Sekiguchi lesion formation in lesion mimic mutants

从水稻关口病（Sekiguchi disease, sl）突变体中分离到一种具有感染抑制活性的抗真菌化合物，鉴定为吲哚类生物碱色胺。色胺在高浓度（150 ~ 600 μg/ml）下不仅抑制稻瘟病菌孢子萌发和附着胞的形成，而且在低浓度（150 ~ 300 μg/ml）下抑制洋葱细胞侵染菌丝的形成。色胺是水稻sl突变体的正常化合物，但在稻瘟病菌孢子接种后产生突变型反应（关口病斑形成）的水稻中进一步积累。一种突变型的反应与光增强抗性有关。然而，在野生型反应（稻瘟病损形成）的水稻中，色胺的积累没有被诱导。本研究强烈表明，色胺可能是水稻突变体抗光增强的一个重要因素。色氨酸脱羧酶（TDC）和单胺氧化酶（MAO）活性的增加是光依赖性的，且在稻瘟病菌侵染水稻的l-突变体中表现得更为明显。TDC和MAO活性在稻瘟病菌侵入水稻细胞之前就已观察到，即使在关口病形成后仍保持较高水平。稻瘟病菌孢子萌发液也能诱导关口病的形成，酶活性和色胺积累均增加。色胺和β-苯乙胺（MAO的底物）在光照下也能诱导关口损伤，而吲哚-3-丙酸、(^^+___)-苯乙胺和色氨酸等非底物在光照下也不能诱导关口损伤。在MAO抑制剂、甲氨酰基和氨基脲、h_2o_2清除剂、抗坏血酸和过氧化氢酶存在的情况下，色胺对关口病的光依赖性诱导被显著抑制。强DAB染色直接原位显示Sekiguchi病变叶片中的H_2O_2。另一方面，H_2O_2对cv叶片产生Sekiguchi病。关口朝日下有光明，却下不了黑暗。这种差异与光照条件下侵染叶片过氧化氢酶活性降低有关，与光照条件下侵染叶片过氧化氢酶活性降低有关。我们推测h_2o_2诱导的叶肉细胞细胞器（如叶绿体和线粒体）的破坏可能导致TDC和MAO活性升高和Sekiguchi病的发生，而野生型水稻的Sl基因产物可能对化学或环境胁迫下的细胞器破坏有抑制作用。少

An antifungal compound with an infection-inhibiting activity was isolated from a rice Sekiguchi lesion (sl) mutant and identified as the indole alkaloid tryptamine. Tryptamine inhibited not only spore germination and appressorium formation of M.grisea at high concentrations (>600 μg/ml) but also the infection hypha formation in onion cells at low concentrations (150-300 μg/ml). Tryptamine is a normal compound of the rice sl mutant but accumulates further in rice with a mutant-type response (Sekiguchi lesion formation) to inoculation with M.grisea spores. A mutant type of response is involved in light-enhanced resistance. The accumulation of tryptamine was not induced, however, in rice with a wild-type response (blast lesion formation). This study strongly suggests that tryptamine plays an important role as a possible factor in light-enhanced resistance in the rice sl-mutants. Increased activities of tryptophan decarboxylase (TDC) and monoamine oxidase (MAO) were light-dependent and obs … More erved in sl-mutant of rice infected with M.grisea. TDC and MAO activites were already observed before penetration of M.grisea to rice cells and kept at high level even after Sekiguchi lesion formation. Spore germination fluid of M.grisea also induced the Sekiguchi lesion formation with increased activites of both enzymes activities and tryptamine accumulation. Sekiguchi lesion was also induced by the treatments with tryptamine and β-phenylethylamine, which are substrates for MAO, under the light, but not induced by non-substrates such as indole-3-propionic acid, (^^+___)-phenylethylamine and tryptophan even under the light. Light-dependent induction of Sekiguchi lesion by tryptamine was significantly inhibited in the presence of MAO inhibitors, metalaxyl and semicarbazide, and H_2O_2-scavengers, ascorbic acid and catalase. H_2O_2 in leaves with Sekiguchi lesion was demonstrated directly in situ by strong DAB staining. On the other hand, H_2O_2 induced Sekiguchi lesion on leaves of cv. Sekiguchi-asahi under the light, but not under the dark. This difference was associated with decrease of the catalase activity in infected leaves under the light and no decrease under the dark. We hypothesize that H_2O_2-induced breakdown of cellular organelles such as chloroplast and mitochondria in mesophtll cells may bring about high activities of TDC and MAO and development of Sekiguchi lesion, and that the Sl gene products in wild type rice may function as a suppressor of organelle breakdown cased by chemical or environmental stress. Less