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Sekiguchi病斑突变体中分离得到一种具有抗侵染活性的化合物，经鉴定为吲哚生物碱色胺。高浓度（>600 μg/ml）的色胺不仅抑制稻瘟病菌孢子萌发和附着胞形成，低浓度（150-300 μg/ml）的色胺也抑制洋葱细胞内侵染菌丝的形成。色胺是水稻sl突变体的正常化合物，但在水稻中进一步积累，对接种稻瘟病菌孢子产生突变型反应（关口病斑形成）。一种突变型的反应涉及光增强的抗性。色胺的积累没有诱导，但是，在水稻与野生型的反应（稻瘟病病变形成）。本研究表明，色胺在水稻sl突变体的光增强抗性中起着重要的作用。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， 关于我们 在感染稻瘟病菌的水稻s1-突变体中的应用。TDC和MAO活性在稻瘟病菌侵入水稻细胞前就已存在，并在病斑形成后仍保持较高水平。稻瘟病菌孢子萌发液也能诱导病斑的形成，使酶活性和色胺积累增加。在光照条件下，单胺氧化酶（MAO）的底物色胺和β-苯乙胺也能诱导Sekiguchi损伤，而非底物吲哚-3-丙酸、（^^+__）-苯乙胺和色氨酸在光照条件下也不能诱导Sekiguchi损伤。单胺氧化酶抑制剂甲霜灵和氨基脲以及过氧化氢清除剂抗坏血酸和过氧化氢酶均能显著抑制色胺诱导的光依赖性Sekiguchi损伤。用DAB强染色直接原位显示了Sekiguchi病斑叶片中的H_2O_2。H_2O_2处理引起黄瓜叶片的Sekiguchi损伤。关口朝日在光下，而不是在黑暗中。这种差异与光下感病叶片过氧化氢酶活性降低而黑暗下不降低有关。我们推测H_2O_2诱导的中胚叶细胞叶绿体和线粒体等细胞器的破坏可能导致TDC和MAO活性的升高和Sekiguchi病斑的发生，而野生型水稻中的S1基因产物可能对化学或环境胁迫引起的细胞器破坏起抑制作用。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