Studies on the involvement of transposons in the pathogenicity variations of the rice blast fungus

转座子参与稻瘟病菌致病性变异的研究

• 批准号: 13660050
• 负责人: NAKAYASHIKI Hitoshi
• 金额: $ 2.11万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660050/
• 关键词: Rice blast fungus; retrotransposon; pathogenicity variations; いもち病菌; 病原性の変異; イネ

1. Deletion analysis of the MAGGYLTR promoterSeveral constructs having a deletion in the MAGGY promoter were made and examined for the promoter activity upon stress using the GUS gene as a reporter. The deletion constructs with the GUS gene were introduced into M.grisea isolate Br 48. Total protein was extracted from the M.grisea transformants after beat shock treatment (42C, 1hr), and the GUS activity was measured using MUG as a substrate. The construct that lacks up to 60 bp at 5' terminus of LTR retained the promoter activity and responded to heat shock whereas one lacking 90 bp at 5' terminus lost the promoter activity. The results indicated that some critical sepuence required for both promoter activity and stress response occur in the 60 to 90 region of MAGGYLTR.2. Analysis of the promoter activity of MAGGY during infection processes using the GFP geneTo investigate the promoter activity of MAGGY during infection processes, GFP was expressed in a M.grisea transformant under the control of the MAGGY promoter. Infection test was performed with the GUS-expressing transformants. However, no obvious upregulation of the MAGGY promoter was found during infection processes on either resistant or susceptible cultivar of rice.3. Isolation of a new LTR-retrotransposn from M.grisea.As a new candidate for a stress responsive transposable element, an LTR-retrotransposon, Pyret was isolated from M.greisea. Pyret containg gag and pol genes as do ordinary gypsy-class retrotransposons. However, this element is unique since it carries an extra domain in the gag gene, which is designated as WCCH domain according to a characteristic amino acid sequence. The WCCH domain was found only in four gypsy-class retrotransposons identified in fungal genomes so far. Therefore, the functional role of the WCCH domain is unknown at this moment.

1. MAGGYLTR启动子缺失分析利用GUS基因作为报告基因，制备了MAGGY启动子缺失的几个结构体，并检测了启动子在胁迫下的活性。将GUS基因缺失构建体导入稻瘟病菌br48中。在高温冲击（42C, 1hr）处理后，从稻瘟病菌转化体中提取总蛋白，并以MUG为底物测定GUS活性。在LTR的5′端缺失60 bp的结构保留了启动子活性并对热休克有反应，而在5′端缺失90 bp的结构则失去了启动子活性。结果表明，在maggyltr 2的60 ~ 90区存在启动子活性和胁迫应答所需的关键序列。为了研究MAGGY在感染过程中启动子的活性，我们在MAGGY启动子控制下，在稻瘟病菌转化中表达GFP。用表达gus的转化子进行感染试验。而在抗感品种侵染过程中，MAGGY启动子的表达均未见明显上调。从稻瘟病菌中分离到一个新的ltr -反转录转座子Pyret，该转座子是一种新的应激应答转座子。Pyret与普通吉普赛类反转录转座子一样含有gag和pol基因。然而，这个元件是独特的，因为它在gag基因中携带一个额外的结构域，根据一个特征氨基酸序列，它被指定为WCCH结构域。WCCH结构域仅在真菌基因组中鉴定的4个吉普赛类反转录转座子中发现。因此，WCCH结构域的功能作用目前尚不清楚。

项目成果

Ikeda et al.: "Heat shock, copper sulfate and oxidative stress activate the retrotransposon MAGGY resident in the plant pathogenic fungus Magnaporthe grisea"Molecular Genetics and Genomics. 266. 318-325 (2001)

Ikeda 等人：“热休克、硫酸铜和氧化应激激活植物病原真菌稻瘟病菌中的逆转录转座子 MAGGY”分子遗传学和基因组学。

Nakayashiki et al: "Methylation is not the main force repressing the retrotransposon MAGGY in Magnaporthe grisea"Nucleic Acids Research. 29. 1278-1284 (2001)

Nakayashiki等人：“甲基化并不是抑制稻瘟病菌中逆转录转座子MAGGY的主要力量”核酸研究。