Molecular characterization of a serine protease Pro1 from Plasmodiophora brassicae that stimulates resting spore germination
Molecular characterization of a serine protease Pro1 from Plasmodiophora brassicae that stimulates resting spore germination
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DOI：
10.1111/j.1364-3703.2010.00623.x
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发表时间：
2010-07-01
影响因子：
4.9
通讯作者：
Peng, Gary
中科院分区：
文献类型：
--
作者：
Feng, Jie;Hwang, Ru;Peng, Gary
油菜根结线虫病是由油菜恶性疟原虫引起的一种严重危害十字花科作物的病害。然而，甘蓝型油菜致病力的基础还不是很清楚。本研究克隆了甘蓝型油菜丝氨酸蛋白酶基因(Pro1)，并对其分子特性进行了研究。Southern分析和特异性聚合酶链式反应(PCR)扩增表明，Pro1基因是一个单拷贝基因，广泛存在于甘蓝型油菜致病菌株中。Northern分析表明，Pro1在植物侵染过程中被诱导表达，转录本的数量随着病程的不同而波动。氨基酸序列分析表明，编码蛋白(Pro1)属于S28蛋白家族，预测信号肽，理论相对分子质量为49.4 kDa。将Pro1的开放阅读框(ORF)转入巴斯德毕赤酵母中，获得异源表达的Pro1。Pro1对脱脂牛奶和N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin，具有蛋白水解性，丝氨酸蛋白酶抑制剂和螯合剂乙二胺四乙酸均能抑制其活性。Pro1的最适温度为25℃，在pH 6.0~6.4范围内表现出较高的活性。这些值与白菜疫霉菌休眠孢子在田间萌发的温度和pH条件相吻合。Pro1处理油菜根分泌物时，增强了根分泌物对油菜休眠孢子萌发的刺激作用，提示Pro1可能通过其蛋白分解活性刺激休眠孢子萌发，从而在油菜丛生孢子萌发中发挥作用。
P>Clubroot, caused by Plasmodiophora brassicae, is one of the most serious diseases of cultivated cruciferous crops in the world. However, the basis for pathogenicity in P. brassicae is not well understood. In this study, a serine protease gene (PRO1) was cloned from P. brassicae and its molecular characteristics were investigated. Southern analysis and specific polymerase chain reaction (PCR) amplification indicated that PRO1 is a single-copy gene present in a broad range of P. brassicae pathotypes. Northern analysis revealed that the expression of PRO1 was induced during plant infection, and that the quantity of transcript fluctuated according to the stage of pathogenesis. Amino acid sequence analysis suggested that the encoded protein (Pro1) belongs to the S28 family of proteases, with a predicted signal peptide and a theoretical molecular mass of 49.4 kDa. The open reading frame (ORF) of PRO1 was transferred into Pichia pastoris and Pro1 was heterologously produced. Pro1 showed proteolytic activity on skimmed milk and N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin, and the activity could be inhibited by serine protease inhibitors and the chelating agent ethylenediaminetetraacetic acid. The optimal temperature of Pro1 was 25 degrees C, and it exhibited high activity at pH 6.0-6.4. These values coincide with the temperature and pH conditions favourable for P. brassicae resting spore germination in the field. When Pro1 was used to treat canola root exudates, it enhanced the stimulating effect of the root exudates on P. brassicae resting spore germination, indicating that Pro1 may play a role during clubroot pathogenesis by stimulating resting spore germination through its proteolytic activity.