【作者】 陈晓峰；

【导师】 王宗华； 鲁国东；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 由稻瘟病菌(Magnaporthe oryzae)引起的稻瘟病是世界范围内水稻生产上的重要病害之一,严重制约着世界粮食安全保障。尽管对稻瘟病菌的生长发育和侵染致病等各个阶段都已开展了长期且较为系统的研究,但是迄今为止对其定殖和扩展的分子机制仍然知之甚少。由寄主防御反应产生的活性氧物质是稻瘟病菌定殖、扩展过程中的主要障碍之一,而NAD(H)激酶能通过调节胞内NAD(H)与NADP(H)之间的动态平衡参与活性氧胁迫的防御反应,因此,对稻瘟病菌NAD(H)激酶生物学功能的研究及其相关信号传导途径的探索将有助于为揭示该病原菌定殖、扩展的分子机制提供理论参考依据。MoPos5是稻瘟病菌三个NAD(H)激酶编码基因中的主效基因,具有NAD(H)激酶典型的保守基序和功能结构域,能够异源互补酿酒酵母POS5缺失突变的氧胁迫生长缺陷。该基因在稻瘟病菌的各个生长发育阶段和侵染致病过程中都有稳定的转录,MoPos5-GFP融合蛋白的亚细胞定位初步断定在线粒体中。MoPos5与稻瘟病菌菌丝体生长、产孢及致病性密切相关。MoPos5缺失导致稻瘟病菌菌丝体生长速度减慢,大约仅有野生型的50%,由于突变体中与胞内活性氧产生有关的MoNox1转录下调,因此推测MoPos5缺失影响了胞内NADPH的合成、打破了胞内活性氧动态平衡,最终影响菌丝体正常生长；MoPos5的缺失也导致稻瘟病菌产孢量下降约45%,主要原因在于分生孢子梗产量减少,而至于分生孢子梗产量减少的原因目前仍不知晓。虽然产孢量下降,但是产生的分生孢子形态结构正常,也能正常地萌发、产生具有功能的附着胞侵入寄主组织,可是侵入寄主组织后,缺失突变体因无法及时清除由寄主防御系统产生的活性氧物质而无法在寄主组织内进一步定殖、扩展,DAB染色可见在其侵染的叶鞘细胞中有大量活性氧物质积累,最终导致突变体致病性严重减弱。此外,MoPos5也是稻瘟病菌利用亚麻酸作为碳源时所必需的,因其催化产生的NADPH参与了过氧化物酶体中偶数位不饱和肪酸β-氧化。MoPos5的过量表达对稻瘟病菌的生长发育、细胞分化及致病性并无明显影响,只是产孢量略微有所增加、MoNox1的转录水平略微上调。稻瘟病菌中的另两个NAD(H)激酶编码基因MoNADK2和MoNADK3在Mopos5缺失突变体中表达上调,表明三个NAD(H)激酶间在一定程度上存在功能互补关系,MoNADK2和MoNADK3可能部分弥补着MoPos5缺失造成的功能缺陷,比如维持胞内必需的NADPH水平。借助酵母双杂交系统,共获得了9个MoPos5的互作蛋白,其中6个是通过酵母双杂交筛选稻瘟病菌Guy11菌丝体阶段cDNA文库获得的,另3个则属于Pos5p互作蛋白在稻瘟病菌中的同源蛋白。通过生物信息学预测分析,并结合相关同源蛋白的研究报道,MoPos5的互作蛋白很可能参与调控了细胞骨架建成、细胞运动、细胞分裂、细胞老化与死亡、线粒体功能维持、胁迫防御反应、物质与能量代谢和信号传导等多种多样的生物学进程。综上所述,NAD(H)激酶MoPos5可能通过多种多样的效应蛋白调节着胞内外的活性氧物质水平,从而调控了稻瘟病菌的生长发育及其在寄主组织内定殖、扩展。更多还原

【Abstract】 Rice blast disease, caused by Magnaporthe oryzae, is one of the most destructive and prevalent diseases in global rice production, which severely restricts the food security worldwide. Although in the past decades of years, systematic research on the growth, development and pathogenicity of this fungus has been carrying out effectively, the mechanisms of colonization and expansion are still not well-understood. Host-driven ROS seem to be the first major barrier for the colonization and expansion of pathogens, while NAD(H) kinases play an important role in ROS scavenging system through regulating the fine intracellular balance between NAD(P) and NADP(H). Therefore, functional analysis of a NAD(H) kinase MoPos5in M. oryzae and exploration of the signal pathways which it is involved in will be conductive to further understanding of the molecular mechanisms of colonization and expansion of this fungus in rice plants.MoPos5, containing typical conserved motifs and domains of NAD(H) kinase, can functionally complement the growth deficiency of the POS5deletion mutant of Saccharomyces cerevisiae under oxidative stress. During the whole life and infection cycle of M. oryzae, MoPos5always kept in a relatively stable transcription level, and its subcellular localization was roughly considered to be mitochondria with the assistance of Janus Green B staining.MoPos5was proved to be closely related to the mycelia growth, conidiation and pathogenicity of M. oryzae. The deletion mutants showed obviously mycelia growth deficiency compared to the wild type strain. Due to the downregulation of MoNoxl in deletion mutants, which is involved in intracellular ROS generation, it was indicated that disfunction of MoPos5might cause negative effect on intracellular ROS scavenging systems, which then brought about cytotoxicity and the final growth defects. The conidiations of deletion mutants were also deficient, and it had been demonstrated that the decreased production of conidiophores was the primary cause. However, the conidia produced by the deletion mutants possessed normal morphology, and they could also germinate to form effective appressoria just like the wild type strain. Nevertheless, after penetration into host tissues, the mutants could not fulfill colonization and expansion successfully, which were proved by the microscopic observation and biochemical staining for the ROS accumulation around the infection site, so the pathogenicity of mutants fatally reduced. Besides, MoPos5showed to be essential for utilizing linolenic acid as carbon source, which might be connected with its NADPH generation that was necessary for β-oxidation of linolenic acid. Moreover, overexpression of MoPos5had nothing to do with fungal growth, development and pathogenicity, except for slight increase of conidiation and upregulation of MoNoxl transcription.Other two NAD(H) kinase genes MoNADK2and MoNADK3were both upregulated in MoPos5deletion mutants, which indicated that there was a certain degree of functional redundancy among those three NAD(H) kinases in M. oryzae, and MoNADK2and MoNADK3might partially make up for the deficiencies caused by MoPos5deletion, such as supplying an essential amount of NADPH for survival.Drawing support from yeast two-hybrid system, nine MoPos5interaction proteins were obtained, six of them were gained from screening the mycelia cDNA library of Guy11, and the other three were the homologous of Pos5p interactions proteins in M. oryzae. According to bioinformatical predictions and relevant research papers, the MoPos5interaction proteins were likely to participate in various biological processes, including cytoskeleton morphogenesis, cell movement, cell division, cell ageing and death, maintainance of mitochondrial function, defense response to environmental stress, material and energy metabolism and signal transduction.In conclusion, it appeared more likely that NAD(H) kinase MoPos5regulated both intracellular and extracellular ROS levels through diverse effectors, and was thereby directly related to the growth, conidiation, colonization and expansion in host tissues of rice blast fungus.更多还原