A study on the role of catalase from Magnaporthe grisea and an inhibitor of catalase from rice cells in the rice-blast

为了建立一种新的分析水稻细胞间相互作用的分子模式的实验系统。将悬浮培养的水稻细胞与稻瘟病菌不同田间分离物的分生孢子悬浮液共培养，研究了水稻对H_2O_2的产生和防御相关基因的表达。与不亲和真菌共培养时，H_2O_2水平高于与亲和真菌共培养。这种差异不是由于基因间的关系，而是由于在分生孢子悬浮液中发现的过氧化氢酶活性，其水平是每个菌株所特有的。在叶鞘测定中，接种亲和孢子和不亲和孢子都可以去除过氧化氢酶活性，从而显著降低侵染率。在洗涤后的孢子悬浮液中添加过氧化氢酶可提高侵染效率。孢子悬浮液中过氧化氢酶的去除或添加与二氨基苯甲胺染色观察到的侵染叶片细胞中积累的H_2O_2水平有关。在叶片试验中，接种洗涤的亲和孢子在5d时形成的病斑比添加过氧化氢酶的洗涤的孢子小。这些结果表明，真菌过氧化氢酶是稻瘟病菌的致病相关因子。在该真菌的培养滤液中检测到相似的过氧化氢酶活性，并通过凝胶过滤、DEAE-纤维素柱层析和SDS-聚丙烯酰胺凝胶电泳法从培养滤液中部分纯化了该酶。对该蛋白用酶消化后的内源氨基酸序列分析表明，过氧化氢降解活性是由真核微生物特有的过氧化氢酶/过氧化物酶携带的。在稻瘟病菌中，这种酶是由单拷贝基因编码的。

In order to develop a novel experimental system to analyze the molecular mode of interaction between rice cells (Oryza sativa cv. Nipponbare) and rice blast fungus (Magnaporthe grisea), suspension-cultured cells of rice were co-cultured with conidia suspension of various field isolates of the rice blast fungus and analyzed defense responses by rice including the production of H_2O_2 and expression of defense-related genes. The level of H_2O_2 was higher when co-cultured with incompatible fungal isolates than with compatible ones. The difference was due not to the gene-for-gene relationship but to the activity of catalase found in the conidia suspension, the level of which was specific to each isolates. In the leaf sheath assay for up to 48 h, inoculation with both compatible and incompatible spores washed to remove catalase activity resulted in a significant reduction in invasion. The addition of catalase to the washed spore suspension increased the efficiency of invasion. The removal or addition of catalase in the spore suspension was related to the level of accumulated H_2O_2 in the infected leaf cells as observed by staining with diaminobenthidine. In the leaf blade assay, inoculation of washed compatible spores formed smaller lesions than washed spores with added catalase at 5 days. These results suggested that fungal catalase is a pathogenesis-related factor in the rice blast fungus. Similar catalase activities were detected in the culture filtrate of the fungus, and the enzyme was partially purified from the culture filtrate by gel filtration, DEAE-cellulose column chromatography and SDS-polyacrylamide gel electrophoresis. Analysis of the endogenous amino acid sequences after digestion of the protein with protease showed that the H_2O_2-degrading activity is carried by a catalase/peroxidase that is specifically found in eukaryotic microbes. In Magnaporthe grisea, a single-copied gene encodes the enzyme.