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

从水稻赤口病(Sl)突变体中分离得到一种具有抗真菌活性的化合物，经鉴定为吲哚生物碱色胺。色胺在高浓度(600μg/ml)时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(150~300μg/ml)时抑制洋葱细胞侵染菌丝的形成。色胺是水稻sl突变体的一种正常化合物，但在水稻中积累更多，对接种稻瘟病菌孢子具有突变型反应(Sekiguchi病变形成)。一种突变类型的反应与光增强抗性有关。然而，在具有野生型反应(稻瘟病形成)的水稻中，色胺的积累没有被诱导。这项研究强烈表明，色胺在水稻sl突变体的光增强抗性中可能起着重要的作用。色氨酸脱羧酶(Tdc)和单胺氧化酶(Mao)活性的增加是光依赖的，OBS…在感染稻瘟病菌的水稻sl突变体中表达更多。TDC和MAO活性在稻瘟病菌侵入水稻细胞之前就已观察到，并在关口病斑形成后仍保持在较高水平。稻瘟病菌孢子萌发液也诱导了关口病斑的形成，酶活性增加，色胺积累增加。在光照下，MAO的底物色胺和β-苯乙胺处理也能诱导关口损伤，而非底物吲哚-3-丙酸、苯乙胺和色氨酸即使在光照下也不能诱导其损伤。MAO抑制剂、甲霜灵和氨基脲、H_2O_2清除剂、抗坏血酸和过氧化氢酶可显著抑制色胺诱导的光依赖性损伤。用DAB染色直接原位显示了赤口病叶片中的H_2O_2。另一方面，H_2O_2处理诱发了青海棠叶片的关口损伤。关口朝日在灯光下，但不在黑暗下。这种差异与受感染叶片在光照条件下过氧化氢酶活性降低，而在黑暗条件下没有降低有关。我们推测，H_2O_2诱导的中粒细胞叶绿体和线粒体等细胞器的破坏可能导致TDC和MAO的高活性以及关口损伤的发生，野生型水稻的S1基因产物可能是化学或环境胁迫引起的细胞器破坏的抑制因子。较少

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